Podocyte-specific Nup160 knockout mice develop nephrotic syndrome and glomerulosclerosis.

More than 60 monogenic genes mutated in steroid-resistant nephrotic syndrome (SRNS) have been identified. Our previous study found that mutations in nucleoporin 160 kD (NUP160) are implicated in SRNS. The NUP160 gene encodes a component of the nuclear pore complex. Recently, two siblings with homozygous NUP160 mutations presented with SRNS and a nervous system disorder. However, replication of nephrotic syndrome (NS)-associated phenotypes in a mammalian model following loss of Nup160 is needed to prove that NUP160 mutations cause SRNS. Here, we generated a podocyte-specific Nup160 knockout (Nup160podKO) mouse model using CRISPR/Cas9 and Cre/loxP technologies. We investigated NS-associated phenotypes in these Nup160podKO mice. We verified efficient abrogation of Nup160 in Nup160podKO mice at both the DNA and protein levels. We showed that Nup160podKO mice develop typical signs of NS. Nup160podKO mice exhibited progression of proteinuria to average albumin/creatinine ratio (ACR) levels of 15.06 ± 2.71 mg/mg at 26 weeks, and had lower serum albumin levels of 13.13 ± 1.34 g/l at 30 weeks. Littermate control mice had urinary ACR mean values of 0.03 mg/mg and serum albumin values of 22.89 ± 0.34 g/l at the corresponding ages. Further, Nup160podKO mice exhibited glomerulosclerosis compared with littermate control mice. Podocyte-specific Nup160 knockout in mice led to NS and glomerulosclerosis. Thus, our findings strongly support that mutations in NUP160 cause SRNS. The newly generated Nup160podKO mice are a reliable mammalian model for future study of the pathogenesis of NUP160-associated SRNS.

Evaluating the efficacy and safety of pozelimab in patients with CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy disease: an open-label phase 2 and 3 study.

BACKGROUND: CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (CHAPLE) is an ultra-rare genetic disorder characterised by intestinal lymphatic damage, lymphangiectasia, and protein-losing enteropathy caused by overactivation of the complement system. We assessed the efficacy and safety of pozelimab, an antibody blocking complement component 5. METHODS: This open-label, single-arm, historically controlled, multicentre phase 2 and 3 study evaluated ten patients with CHAPLE disease. This study was conducted at three hospitals in Thailand, Türkiye, and the USA. Patients aged 1 year or older with a clinical diagnosis of CHAPLE disease and a CD55 loss-of-function variant identified by genetic analysis and confirmed by flow cytometry or western blot of CD55 from peripheral blood cells were eligible for this study. Patients received a single intravenous loading dose of pozelimab 30 mg per kg of bodyweight, followed by a once-per-week subcutaneous dose over the treatment period based on bodyweight at a concentration of 200 mg/mL as either a single injection (<40 kg bodyweight) or two injections (≥40 kg bodyweight). The primary endpoint was proportion of patients with serum albumin normalisation with an improvement in active clinical outcomes and no worsening in inactive clinical outcomes (frequency of problematic abdominal pain, bowel movement frequency, facial oedema severity, and peripheral oedema severity) at week 24 compared with baseline, assessed in the full analysis set. This study is registered with ClinicalTrials.gov (NCT04209634) and is active but not recruiting. FINDINGS: 11 patients were recruited between Jan 27, 2020, and May 12, 2021, ten of which were enrolled in the study and included in the analysis populations. The efficacy data corresponded to all patients completing the week 48 assessment and having at least 52 weeks of treatment exposure, and the safety data included an additional 90 days of follow-up and corresponded to all patients having at least 72 weeks of treatment. Patients were predominantly paediatric (with a median age of 8·5 years), and originated from Türkiye, Syria, Thailand, and Bolivia. Patients had markedly low weight-for-age and stature-for-age at baseline, and mean albumin at baseline was 2·2 g/dL, which was considerably less than the local laboratory reference range. After pozelimab treatment, all ten patients had serum albumin normalisation and improvement with no worsening in clinical outcomes. There was a complete inhibition of the total complement activity. Nine patients had adverse events; two were severe events, and one patient had an adverse event considered related to pozelimab. INTERPRETATION: Pozelimab inhibits complement overactivation and resolves the clinical and laboratory manifestations of CHAPLE disease. Pozelimab is the only currently approved therapeutic drug for patients with this life-threatening, ultra-rare condition. In patients with protein-losing enteropathy where known causes have been excluded, testing for a CD55 deficiency should be contemplated. A diagnosis of CHAPLE disease should lead to early consideration of treatment with pozelimab. FUNDING: Regeneron Pharmaceuticals and the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Determinants of clinical response to empirical antibiotic treatment in patients with cirrhosis and bacterial and fungal infections-Results from the ICA "Global Study" (EABCIR-Global Study).

BACKGROUND: The administration of an appropriate empirical antibiotic treatment is essential in cirrhosis and severe bacterial infections. We aimed to investigate the predictors of clinical response of empirical antibiotic treatment in a prospective cohort of patients with cirrhosis and bacterial and fungal infections included in the International Club of Ascites "Global Study." METHODS: Patients hospitalized with cirrhosis and bacterial/fungal infection were prospectively enrolled at 46 centers. Clinical response to antibiotic treatment was defined according to changes in markers of infection/inflammation, vital signs, improvement of organ failure, and results of cultures. RESULTS: From October 2015 to September 2016, 1302 patients were included at 46 centers. A clinical response was achieved in only 61% of cases. Independent predictors of lack of clinical response to empirical treatment were C-reactive protein (OR = 1.16; 95% CI = 1.02-1.31), blood leukocyte count (OR = 1.39;95% CI = 1.09-1.77), serum albumin (OR = 0.70; 95% CI = 0.55-0.88), nosocomial infections (OR = 1.96; 95% CI = 1.20-2.38), pneumonia (OR = 1.75; 95% CI = 1.22-2.53), and ineffective treatment according to antibiotic susceptibility test (OR = 5.32; 95% CI = 3.47-8.57). Patients with a lack of clinical response to first-line antibiotic treatment had a significantly lower resolution rate of infections (55% vs. 96%; p < 0.001), a higher incidence of second infections (29% vs. 15%; p < 0.001), shock (35% vs. 7%; p < 0.001) and new organ failures (52% vs. 19 %; p < 0.001) than responders. Clinical response to empirical treatment was an independent predictor of 28-day survival ( subdistribution = 0.20; 95% CI = 0.14-0.27). CONCLUSIONS: Four out of 10 patients with cirrhosis do not respond to the first-line antibiotic therapy, leading to lower resolution of infections and higher mortality. Broader-spectrum antibiotics and strategies targeting systemic inflammation may improve prognosis in patients with a high degree of inflammation, low serum albumin levels, and severe liver impairment.

Serum albumin exposure enhances cell invasiveness and paclitaxel resistance in human neuroblastoma cells, with attenuation by valeriana-type iridoid glycosides.

Neuroblastoma, a prevalent extracranial solid tumor in children, arises from undifferentiated nerve cells. While tumor vasculature, often characterized by increased permeability, influences metastasis and recurrence, the direct impact of blood-borne molecules on tumor progression remains unclear. In the present study, we focused on the effect of exposure to albumin, one of the most abundant proteins in the serum, on human neuroblastoma cells. Albumin exposure elevated oxidative stress and led to mitochondria dysfunction via the activation of TGFß and PI3K pathways, accompanied by an increase in the metastatic and invasive properties of neuroblastoma cells. Proteins relevant to the induction of autophagy were upregulated in response to prolonged albumin exposure. Additionally, pre-exposure to albumin before treatment resulted in increased resistance to paclitaxel. Two valeriana-type iridoid glycosides, patrisophoroside and patrinalloside, recently isolated from Nardostachys jatamansi significantly mitigated the effect of albumin on oxidative stress, cell invasiveness, and chemoresistance. These findings illuminate the potential role of blood-borne molecules, such as albumin, in the progression and metastasis of neuroblastoma, as well as the possible therapeutic implications of valeriana-type iridoid glycosides in anti-cancer treatment.

Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

Synergy of a STING agonist and an IL-2 superkine in cancer immunotherapy against MHC I-deficient and MHC I+ tumors.

Cyclic dinucleotides (CDN) and Toll-like receptor (TLR) ligands mobilize antitumor responses by natural killer (NK) cells and T cells, potentially serving as complementary therapies to immune checkpoint therapy. In the clinic thus far, however, CDN therapy targeting stimulator of interferon genes (STING) protein has yielded mixed results, perhaps because it initiates responses potently but does not provide signals to sustain activation and proliferation of activated cytotoxic lymphocytes. To improve efficacy, we combined CDN with a half life-extended interleukin-2 (IL-2) superkine, H9-MSA (mouse serum albumin). CDN/H9-MSA therapy induced dramatic long-term remissions of the most difficult to treat major histocompatibility complex class I (MHC I)­deficient and MHC I+ tumor transplant models. H9-MSA combined with CpG oligonucleotide also induced potent responses. Mechanistically, tumor elimination required CD8 T cells and not NK cells in the case of MHC I+ tumors and NK cells but not CD8 T cells in the case of MHC-deficient tumors. Furthermore, combination therapy resulted in more prolonged and more intense NK cell activation, cytotoxicity, and expression of cytotoxic effector molecules in comparison with monotherapy. Remarkably, in a primary autochthonous sarcoma model that is refractory to PD-1 checkpoint therapy, the combination of CDN/H9-MSA with checkpoint therapy yielded long-term remissions in the majority of the animals, mediated by T cells and NK cells. This combination therapy has the potential to activate responses in tumors resistant to current therapies and prevent MHC I loss accompanying acquired resistance of tumors to checkpoint therapy.

Evidence for a semisolid phase state of aerosols and droplets relevant to the airborne and surface survival of pathogens.

The phase state of respiratory aerosols and droplets has been linked to the humidity-dependent survival of pathogens such as SARS-CoV-2. To inform strategies to mitigate the spread of infectious disease, it is thus necessary to understand the humidity-dependent phase changes associated with the particles in which pathogens are suspended. Here, we study phase changes of levitated aerosols and droplets composed of model respiratory compounds (salt and protein) and growth media (organic-inorganic mixtures commonly used in studies of pathogen survival) with decreasing relative humidity (RH). Efflorescence was suppressed in many particle compositions and thus unlikely to fully account for the humidity-dependent survival of viruses. Rather, we identify organic-based, semisolid phase states that form under equilibrium conditions at intermediate RH (45 to 80%). A higher-protein content causes particles to exist in a semisolid state under a wider range of RH conditions. Diffusion and, thus, disinfection kinetics are expected to be inhibited in these semisolid states. These observations suggest that organic-based, semisolid states are an important consideration to account for the recovery of virus viability at low RH observed in previous studies. We propose a mechanism in which the semisolid phase shields pathogens from inactivation by hindering the diffusion of solutes. This suggests that the exogenous lifetime of pathogens will depend, in part, on the organic composition of the carrier respiratory particle and thus its origin in the respiratory tract. Furthermore, this work highlights the importance of accounting for spatial heterogeneities and time-dependent changes in the properties of aerosols and droplets undergoing evaporation in studies of pathogen viability.

Plasmonic Nanotrenches with 1 nm Nanogaps for Surface-Enhanced Raman Scattering-Based Screening of His-Tagged Proteins.

This study represents a highly sensitive and selective approach to protein screening using surface-enhanced Raman scattering (SERS) facilitated by octahedral Au nanotrenches (OANTs). OANTs are a novel class of nanoparticles characterized by narrow, trench-like excavations indented into the eight facets of a Au octahedron. This unique configuration maximizes electromagnetic near-field focusing as the gap distance decreases to ∼1 nm. Owing to geometrical characteristics of the OANTs, near-field focusing can be maximized through the confinement and reflectance of light trapped within the trenches. We used Ni ions and molecular linkers to confer selective binding affinity for His-tagged proteins on the surfaces of the OANTs for SERS-based protein screening. Remarkably, SERS-based protein screening with the surface-modified OANTs yielded outstanding screening capabilities: 100% sensitivity and 100% selectivity in distinguishing His-tagged human serum albumin (HSA) from native HSA. This highlights the significantly enhanced protein screening capabilities achieved through the synergistic combination of SERS and the OANTs.

Aggregation of Apo/Glycated Human Serum Albumins and Aptamer-Saturated Graphene Quantum Dot: A Simulation Study.

Human serum albumin (HSA) is a protein carrier that transports a wide range of drugs and nutrients. The amount of glycated HSA (GHSA) is used as a diabetes biomarker. To quantify the GHSA amount, the fluorescent graphene-based aptasensor has been a successful method. In aptasensors, the key mechanism is the adsorption/desorption of albumin from the aptamer-graphene complex. Recently, the graphene quantum dot (GQD) has been reported to be an aptamer sorbent. Due to its comparable size to aptamers, it is attractive enough to explore the possibility of GQD as a part of an albumin aptasensor. Therefore, molecular dynamics (MD) simulations were performed here to reveal the binding mechanism of albumin to an aptamer-GQD complex in molecular detail. GQD saturated by albumin-selective aptamers (GQDA) is studied, and GHSA and HSA are studied in comparison to understand the effect of glycation. Fast and spontaneous albumin-GQDA binding was observed. While no specific GQDA-binding site on both albumins was found, the residues used for binding were confined to domains I and III for HSA and domains II and III for GHSA. Albumins were found to bind preferably to aptamers rather than to GQD. Lysines and arginines were the main contributors to binding. We also found the dissociation of GLC from all GHSA trajectories, which highlights the role of GQDA in interfering with the ligand binding affinity in Sudlow site I. The binding of GQDA appears to impair albumin structure and function. The insights obtained here will be useful for the future design of diabetes aptasensors.

Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

Plasma albumin, bilirubin, and uric acid and the subsequent risk of cancer: a case-cohort study in the Japan Public Health Center-based Prospective Study.

Several epidemiologic studies have investigated the circulating levels of albumin, bilirubin, and uric acid (UA) in relation to cancer risk; however, they have provided equivocal evidence. In this prospective case-cohort study, we measured the plasma levels of albumin, bilirubin, and UA and investigated their association with cancer incidence in 3584 case patients and 4270 randomly selected participants with a median follow-up of 15.8 years. The adjusted hazard ratios (HRs) and 95% CIs of total cancer for the highest quartile (Q4) versus lowest quartile (Q1) was 0.77 (95% CI, 0.67-0.90; P <.001 for trend) for albumin. This association was attenuated after excluding liver cancer cases with lower plasma albumin levels. Plasma bilirubin levels were positively related to liver cancer but inversely to total cancer after excluding liver cancer with, for Q4 versus Q1, an adjusted HR of 0.86 (95% CI, 0.74-0.99; P = .015 for trend). Plasma UA levels were not dose-responsively associated with total cancer risk. Higher plasma bilirubin levels were associated with a decreased risk of total cancer after excluding liver cancer, which is likely attributed to the antioxidant properties of bilirubin.

Impact of hypoalbuminemia on clinical outcomes among patients with obesity treated with ceftriaxone.

The use of ceftriaxone, a highly protein-bound drug, in the setting of hypoalbuminemia may result in suboptimal drug exposure. Patients with obesity also exhibit higher absolute drug clearance. We aimed to evaluate the impact of hypoalbuminemia on clinical success among hospitalized adults with obesity who were treated with ceftriaxone. This retrospective review included adult inpatients with weight >100 kg or body mass index >40 kg/m2 who received ceftriaxone 2 g intravenously every 12 hours for at least 72 hours. The primary outcome was clinical success, a composite of clinical cure and microbiologic cure. Secondary outcomes included clinical cure, microbiologic cure, length of stay, ICU length of stay, mortality, 30-day readmission, and adverse events. In all, 137 patients were included, 34 of whom had a serum albumin of ≤2.5 g/dL. In a propensity-score-weighted analysis, clinical success was significantly more common among those without hypoalbuminemia (91.2%) as compared to those with hypoalbuminemia (77.8%) (P = 0.038). Death within 30 days (13.7% vs 0%, P < 0.001) and 30-day readmission (31.6% vs 12.0%, P = 0.008) were more common in the hypoalbuminemia group. In a univariate analysis, serum albumin and indication for ceftriaxone use were found to be predictors of clinical success. Hypoalbuminemia was associated with a lower rate of clinical success among patients with obesity who were treated with ceftriaxone 2 g every 12 hours.

Goal-directed Perioperative Albumin Substitution Versus Standard of Care to Reduce Postoperative Complications: A Randomized Clinical Trial (SuperAdd Trial).

OBJECTIVE: To investigate whether goal-directed albumin substitution during surgery and postanesthesia care to maintain a serum albumin concentration >30 g/L can reduce postoperative complications. BACKGROUND: Hypoalbuminemia is associated with numerous postoperative complications. Since albumin has important physiological functions, substitution of patients with hypoalbuminemia is worth considering. METHODS: We conducted a single-center, randomized, controlled, outcome assessor-blinded clinical trial in adult patients, American Society of Anesthesiologists physical status classification 3 to 4 or undergoing high-risk surgery. Patients, whose serum albumin concentration dropped <30 g/L were randomly assigned to goal-directed albumin substitution maintaining serum concentration >30 g/L or to standard care until discharge from the postanesthesia intermediate care unit. Standard of care allowed albumin substitution in hemodynamic instable patients with serum concentration <20 g/L, only. Primary outcome was the incidence of postoperative complications ≥2 according to the Clavien-Dindo Classification in at least 1 of 9 domains (pulmonary, infectious, cardiovascular, neurological, renal, gastrointestinal, wound, pain, and hematological) until postoperative day 15. RESULTS: Of 2509 included patients, 600 (23.9%) developed serum albumin concentrations <30 g/L. Human albumin 60 g (40-80 g) was substituted to 299 (99.7%) patients in the intervention group and to 54 (18.0%) in the standard care group. At least 1 postoperative complication classified as Clavien-Dindo Classification ≥2 occurred in 254 of 300 patients (84.7%) in the intervention group and in 262 of 300 (87.3%) in the standard treatment group (risk difference -2.7%, 95% CI, -8.3% to 2.9%). CONCLUSION: Maintaining serum albumin concentration of >30 g/L perioperatively cannot generally be recommended in high-risk noncardiac surgery patients.

A Randomized Trial of Albumin Infusions in Hospitalized Patients with Cirrhosis.

BACKGROUND: Infection and increased systemic inflammation cause organ dysfunction and death in patients with decompensated cirrhosis. Preclinical studies provide support for an antiinflammatory role of albumin, but confirmatory large-scale clinical trials are lacking. Whether targeting a serum albumin level of 30 g per liter or greater in these patients with repeated daily infusions of 20% human albumin solution, as compared with standard care, would reduce the incidences of infection, kidney dysfunction, and death is unknown. METHODS: We conducted a randomized, multicenter, open-label, parallel-group trial involving hospitalized patients with decompensated cirrhosis who had a serum albumin level of less than 30 g per liter at enrollment. Patients were randomly assigned to receive either targeted 20% human albumin solution for up to 14 days or until discharge, whichever came first, or standard care. Treatment commenced within 3 days after admission. The composite primary end point was new infection, kidney dysfunction, or death between days 3 and 15 after the initiation of treatment. RESULTS: A total of 777 patients underwent randomization, and alcohol was reported to be a cause of cirrhosis in most of these patients. A median total infusion of albumin of 200 g (interquartile range, 140 to 280) per patient was administered to the targeted albumin group (increasing the albumin level to ≥30 g per liter), as compared with a median of 20 g (interquartile range, 0 to 120) per patient administered to the standard-care group (adjusted mean difference, 143 g; 95% confidence interval [CI], 127 to 158.2). The percentage of patients with a primary end-point event did not differ significantly between the targeted albumin group (113 of 380 patients [29.7%]) and the standard-care group (120 of 397 patients [30.2%]) (adjusted odds ratio, 0.98; 95% CI, 0.71 to 1.33; P = 0.87). A time-to-event analysis in which data were censored at the time of discharge or at day 15 also showed no significant between-group difference (hazard ratio, 1.04; 95% CI, 0.81 to 1.35). More severe or life-threatening serious adverse events occurred in the albumin group than in the standard-care group. CONCLUSIONS: In patients hospitalized with decompensated cirrhosis, albumin infusions to increase the albumin level to a target of 30 g per liter or more was not more beneficial than the current standard care in the United Kingdom. (Funded by the Health Innovation Challenge Fund; ATTIRE EudraCT number, 2014-002300-24; ISRCT number, N14174793.).

Fluorimetric Tool to Discriminate Glomerular and Tubular Injuries In Vivo.

The etiology and pathological complexity of acute kidney injury (AKI) pose great challenges for early diagnosis, typing, and personalized treatment. It is an important reason for poor prognosis and high mortality of AKI. In order to provide a relatively noninvasive diagnostic and typing method for AKI, we proposed the pathological changes of albumin permeability after glomerular injury and reabsorption efficiency after tubular injury as potential entry points. Thus, a renal tubule labeling fluorescent dye which features albumin concentration-related fluorescence intensity was used to fit these pathological changes. Utilizing this fluorescence assay, we realized urinary tract obstruction imaging as early as 12 h after morbidity. For glomerular and tubular injury discrimination, compared to a healthy control, membranous nephropathy as a representative glomerular injury resulted in enhanced fluorescence intensity of the kidney due to increased albumin penetration, while renal tubular injury caused insufficient dye reabsorption to exhibit weakened fluorescence intensity. The significant differences demonstrated the feasibility of this approach for fluorescence imaging-based AKI typing in vivo.

A Rationally Designed Prodrug for the Fluorogenic Labeling of Albumin and Theranostic Effects on Drug-Induced Liver Injury.

The development of small-molecular fluorogenic tools for the chemo-selective labeling of proteins in live cells is important for the evaluation of intracellular redox homeostasis. Dynamic imaging of human serum albumin (HSA), an antioxidant protein under oxidative stress with concomitant release of antioxidant drugs to maintain redox homeostasis, affords potential opportunities for disease diagnosis and treatment. In this work, we developed a nonfluorogenic prodrug named TPA-NAC, by introducing N-acetyl-l-cysteine (NAC) into a conjugated acceptor skeleton. Through combined thiol and amino addition, coupling with HSA results in fluorescence turn-on and drug release. It was reasoned that the restricted intramolecular motion of the probe under an HSA microenvironment after covalent bonding inhibited the nonradiative transitions. Furthermore, the biocompatibility and photochemical properties of TPA-NAC enabled it to image exogenous and endogenous HSA in living cells in a wash-free manner. Additionally, the released drug evoked upregulation of superoxide dismutase (SOD), which synergistically eliminated reactive oxygen species in a drug-induced liver injury model. This study provides insights into the design of new theranostic fluorescent prodrugs for chemo-selective protein labeling and disease treatments.

Significant Association of Serum Albumin With the Severity of Coronary Microvascular Dysfunction Using Dynamic CZT-SPECT.

OBJECTIVE: Both low serum albumin (SA) concentration and coronary microvascular dysfunction (CMD) are risk factors for the development of heart failure (HF). We hypothesized that SA concentration is associated with myocardial flow reserve (MFR) and implicated in pathophysiological mechanism of HF. METHODS: We retrospectively studied 454 patients undergoing dynamic cardiac cadmium-zinc-telluride myocardial perfusion imaging from April 2018 to February 2020. The population was categorized into three groups according to SA level (g/dL): Group 1: >4, Group 2: 3.5-4, and Group 3: <3.5. Myocardial blood flow (MBF) and myocardial flow reserve (MFR, defined as stress/rest MBF ratio) were compared. RESULTS: The mean age of the whole cohort was 66.2 years, and 65.2% were men. As SA decreased, stress MBF (mL min-1 g-1) and MFR decreased (MBF: 3.29 ± 1.03, MFR: 3.46 ± 1.33 in Group 1, MBF: 2.95 ± 1.13, MFR: 2.51 ± 0.93 in Group 2, and MBF: 2.64 ± 1.16, MFR: 1.90 ± 0.50 in Group 3), whereas rest MBF (mL min-1 g-1) increased (MBF: 1.05 ± 0.42 in Group 1, 1.27 ± 0.56 in Group 2, and 1.41 ± 0.61 in Group 3). After adjusting for covariates, compared with Group 1, the odds ratios for impaired MFR (defined as MFR < 2.5) were 3.57 (95% CI: 2.32-5.48) for Group 2 and 34.9 (95% CI: 13.23-92.14) for Group 3. The results would be similar if only regional MFR were assessed. The risk prediction for CMD using SA was acceptable, with an AUC of 0.76. CONCLUSION: Low SA concentration was associated with the severity of CMD in both global and regional MFR as well as MBF.

A Versatile Platform to Generate Prodrugs with Rapid and Precise Albumin Hitchhiking and High Cargo Loading for Tumor-Targeted Chemotherapy.

Due to its tumor homing and long serum half-life, albumin is an ideal drug carrier for chemotherapy. For endogenous albumin hitchhiking with high cargo loading, a trimeric albumin-binding domain (ABD), i.e., ABD-Tri is designed by fusing an ABD with high specificity and affinity for albumin to a self-trimerizing domain (Tri) with an additional cysteine residue. ABD-Tri is highly (40 mg L-1) expressed as soluble and trimeric proteins in Escherichia coli (E. coli). Once mixed together, ABD-Tri rapidly and specifically forms a stable complex with albumin under physiological conditions without obviously changing its receptor- and cell-binding and tumor-homing properties. Maleimide-modified prodrugs are highly effectively conjugated to ABD-Tri to produce homogenous ABD-Tri-prodrugs with triple cargo loading under physiological conditions by thiol-maleimide click chemistry. Unlike the maleimide moiety, which can only mediate time- and concentration-dependent albumin binding, ABD-Tri mediated fast (within several minutes) albumin binding of drugs even at extremely low concentrations (µg mL-1). Compared to maleimide-modified prodrugs, ABD-Tri-prodrugs exhibit better tumor homing and greater in vivo antitumor effect, indicating that conjugation of chemical drug to ABD-Tri outperforms maleimide modification for endogenous albumin hitchhiking. The results demonstrate that ABD-Tri may serve as a novel platform to produce albumin-binding prodrugs with high cargo-loading capacity for tumor-targeted chemotherapy.

Immunological Quantitation of the Glycation Site Lysine-414 in Serum Albumin in Human Plasma Samples by Indirect ELISA Using Highly Specific Monoclonal Antibodies.

Diabetes mellitus, a metabolic disorder that is characterized by elevated blood glucose levels, is common throughout the world and its prevalence is steadily increasing. Early diagnosis and treatment are important to prevent acute complications and life-threatening long-term organ damage. Glycation sites in human serum albumin (HSA) are considered to be promising biomarkers of systemic glycemic status. This work aimed to develop a sensitive and clinically applicable ELISA for the quantification of glycation site Lys414 in HSA (HSAK414 ). The monoclonal antibodies (mAbs) were generated by immunizing mice with a glycated peptide. The established indirect ELISA based on mAb 50D8 (IgG1 isotype) yielded a limit of detection of 0.39 nmol/g HSA for HSAK414 with a linear dynamic range from 0.50 to 6.25 nmol/g glycated HSA. The inter- and intra-day assays with coefficients of variation less than 20 % indicated good assay performance and precision. Assay evaluation was based on plasma samples from diabetic and non-diabetic subjects with known HSAK414 glycation levels previously determined by LC-MS. Both data sets correlated very well. In conclusion, the generated mAb 50D8 and the established ELISA could be a valuable tool for the rapid quantitation of glycation site HSAK414 in plasma samples to evaluate its clinical relevance.

Conjugating Hemoglobin and Albumin by Strain-Promoted Azide- Alkyne Cycloaddition.

A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted alkyne-azide cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each ß-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from unreacted hemoglobin. The oxygen binding properties of the two-protein bioconjugate demonstrate oxygen affinity and cooperativity that are suitable for use in an acellular oxygen carrier.