Internalization of transferrin-tagged Myxococcus xanthus encapsulins into mesenchymal stem cells.

Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of genetic and chemical engineering may produce novel carriers for precise targeting different cellular proteins, which is important for both therapy and diagnosis of various pathologies. Here we present the novel nanocontainers based on vectorized genetically encoded Myxococcus xanthus (Mx) encapsulin, confining a fluorescent photoactivatable mCherry (PAmCherry) protein. The shells of such encapsulins were modified using chemical conjugation of human transferrin (Tf) prelabeled with a fluorescein-6 (FAM) maleimide acting as a vector. We demonstrate that the vectorized encapsulin specifically binds to transferrin receptors (TfRs) on the membranes of mesenchymal stromal/stem cells (MSCs) followed by internalization into cells. Two spectrally separated fluorescent signals from Tf-FAM and PAmCherry are clearly distinguishable and co-localized. It is shown that Tf-tagged Mx encapsulins are internalized by MSCs much more efficiently than by fibroblasts. It has been also found that unlabeled Tf effectively competes with the conjugated Mx-Tf-FAM formulations. That indicates the conjugate internalization into cells by Tf-TfR endocytosis pathway. The developed nanoplatform can be used as an alternative to conventional nanocarriers for targeted delivery of, e.g., genetic material to MSCs.

Iron parameters analysis in dogs with myxomatous mitral valve disease.

BACKGROUND: Myxomatous mitral valve disease (MMVD) is the most common acquired cardiovascular disease in small breed dogs. In contrast to human patients with heart failure (HF), iron deficiency (ID) prevalence in dogs with MMVD is weakly known. The study aimed to assess the usability of ID markers in serum and reticulocyte parameters from whole blood of dogs with MMVD to evaluate early ID symptoms. RESULTS: Sixty-eight dogs (43 male and 25 female) were included in the study. MMVD dogs were assigned according to the 2019 ACVIM guidelines for groups B1 (n = 9), B2 (n = 10), C (n = 27) and D (n = 10). Groups were also combined into B1 and B2 as non-symptomatic HF and C with D as symptomatic HF. Healthy controls were 12 dogs. Serum iron concentration below the reference range in dogs with MMVD was 12.5%. Other ID indices, such as %SAT, UIBC, and TIBC were similar in the MMVD groups and healthy controls (p > 0.05 for all parameters). Statistical comparison between control group and 4 groups of different stages of MMVD showed that significant differences occur only in serum transferrin. The assessment of ferritin and soluble transferrin receptors using Western Blotting did not show differences between control (n = 7) and MMVD (n = 33) dogs. Study has shown positive correlation between ID parameters and echocardiographic indices such as LA/Ao and LVIDdN, and some biochemical parameters. A significant increase in reticulocytes percentage, assessed manually, was observed in the HF group of animals (p = 0.027) compared to the control group. CONCLUSIONS: Studies have shown that ID parameters in serum are not significantly different in dogs with MMVD compared to healthy dogs. However, there is a clear correlation between atrial size and normalised left ventricular size to body size and some biochemical parameters, including ID parameters and therefore the severity of MMVD.

Transferrin-conjugated magnetic nanoparticles for the isolation of brain-derived blood exosomal MicroRNAs: A novel approach for Parkinson's disease diagnosis.

BACKGROUND: Brain-derived exosomes circulate in the bloodstream and other bodily fluids, serving as potential indicators of neurological disease progression. These exosomes present a promising avenue for the early and precise diagnosis of neurodegenerative conditions. Notably, miRNAs found in plasma extracellular vesicles (EVs) offer distinct diagnostic benefits due to their stability, abundance, and resistance to breakdown. RESULTS: In this study, we introduce a method using transferrin conjugated magnetic nanoparticles (TMNs) to isolate these exosomes from the plasma of patients with neurological disorders. This TMNs technique is both quick (<35 min) and cost-effective, requiring no high-priced ingredients or elaborate equipment for EV extraction. Our method successfully isolated EVs from 33 human plasma samples, including those from patients with Parkinson's disease (PD), Multiple Sclerosis (MS), and Dementia. Using quantitative polymerase chain reaction (PCR) analysis, we evaluated the potential of 8 exosomal miRNA profiles as biomarker candidates. Six exosomal miRNA biomarkers (miR-195-5p, miR-495-3p, miR-23b-3P, miR-30c-2-3p, miR-323a-3p, and miR-27a-3p) were consistently linked with all stages of PD. SIGNIFICANCE: The TMNs method provides a practical, cost-efficient way to isolate EVs from biological samples, paving the way for non-invasive neurological diagnoses. Furthermore, the identified miRNA biomarkers in these exosomes may emerge as innovative tools for precise diagnosis in neurological disorders including PD.

Cross-sectional and longitudinal associations of Iron biomarkers and cardiovascular risk factors in pre- and postmenopausal women: leveraging repeated measurements to address natural variability.

BACKGROUND: The association between iron biomarkers and cardiovascular disease risk factors (CVD-RFs) remains unclear. We aimed to (1) evaluate the cross-sectional and longitudinal associations between iron biomarkers (serum ferritin, transferrin saturation (TSAT), transferrin) and CVD-RFs among women, and (2) explore if these associations were modified by menopausal status. METHOD: Cross-sectional and longitudinal analyses including 2542 and 1482 women from CoLaus cohort, respectively. Multiple linear regression and multilevel mixed models were used to analyse the associations between Iron biomarkers and CVD-RFs. Variability of outcomes and iron markers between surveys was accessed using intraclass correlation (ICC). RESULTS: After multivariable adjustment, elevated serum ferritin levels were associated with increased insulin and glucose levels, while higher transferrin levels were linked to elevated glucose, insulin and total cholesterol, and systolic and diastolic blood pressure (p < 0.05). No association was observed between CVD-RFs and TSAT (p > 0.05). Iron biomarkers demonstrated low reliability across reproductive stages but exhibited stronger associations in the perimenopausal group. In longitudinal analysis, we found association only for transferrin with lower glucose levels [ß = - 0.59, 95% CI (- 1.10, - 0.08), p = 0.02] and lower diastolic blood pressure [ß = - 7.81, 95% CI (- 15.9, - 0.56), p = 0.04]. CONCLUSION: In cross-sectional analysis, transferrin was associated with several CVD-RFs, and the associations did not change according to menopausal status. Conversely, in the longitudinal analyses, changes in transferrin were associated only with lower glucose and diastolic blood pressure levels. These differences might stem from the substantial longitudinal variation of iron biomarkers, underscoring the need for multiple iron measurements in longitudinal analyses.

Serum iron level is independently associated with sarcopenia: a retrospective study.

Sarcopenia greatly reduces the quality of life of the elderly, and iron metabolism plays an important role in muscle loss. This study aimed to investigate the association between iron status and sarcopenia. A total of 286 adult patients hospitalized between 2019 and 2021 were included in this study, of which 117 were diagnosed with sarcopenia. Serum iron, total iron binding capacity (TIBC), transferrin, and transferrin saturation levels were compared between groups with and without sarcopenia and were included in the logistic analyses, with significant variables further included in the logistic regression model for the prediction of sarcopenia. Serum iron, TIBC, and transferrin levels decreased significantly in the sarcopenia group (p < 0.05), and were negatively associated with handgrip strength, relative skeletal muscle index, and multiple test performances (p < 0.05). Multivariate logistic analysis showed that sex, age, body mass index (BMI), and serum iron level were independent risk factors for sarcopenia. In the final logistic regression model, male sex (odds ratio [OR] 3.65, 95% confidence interval [CI] 1.67-7.98), age > 65 years (OR 5.40, 95% CI 2.25-12.95), BMI < 24 kg/m2 (OR 0.17, 95% CI 0.08-0.36), and serum iron < 10.95 µmol/L (OR 0.39, 95% CI 0.16-0.93) were included. Our study supported the impact of iron metabolism on muscle strength and performance.

Unveiling the Molecular Interactions Between Human Transferrin and Limonene: Natural Compounds in Alzheimer's Disease Therapeutics.

Background: Neurodegeneration is a term describing an irreversible process of neuronal damage. In recent decades, research efforts have been directed towards deepening our knowledge of numerous neurodegenerative disorders, with a particular focus on conditions such as Alzheimer's disease (AD). Human transferrin (htf) is a key player in maintaining iron homeostasis within brain cells. Any disturbance in this equilibrium gives rise to the emergence of neurodegenerative diseases and associated pathologies, particularly AD. Limonene, a natural compound found in citrus fruits and various plants, has shown potential neuroprotective properties. Objective: In this study, our goal was to unravel the binding of limonene with htf, with the intention of comprehending the interaction mechanism of limonene with htf. Methods: Binding was scrutinized using fluorescence quenching and UV-Vis spectroscopic analyses. The binding mechanism of limonene was further investigated at the atomic level through molecular docking and extensive 200 ns molecular dynamic simulation (MD) studies. Results: Molecular docking uncovered that limonene interacted extensively with the deep cavity located within the htf binding pocket. MD results indicated that binding of limonene to htf did not induce substantial structural alterations, ultimately forming stable complex. The findings from fluorescence binding indicated a pronounced interaction between limonene and htf, limonene binds to htf with a binding constant (K) of 0.1×105 M-1. UV spectroscopy also advocated stable htf-limonene complex formation. Conclusions: The study deciphered the binding mechanism of limonene with htf, providing a platform to use limonene in AD therapeutics in context of iron homeostasis.

Efficacy and Safety of Oral Supplementation with Liposomal Iron in Non-Dialysis Chronic Kidney Disease Patients with Iron Deficiency.

INTRODUCTION: Iron deficiency is common in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD). Oral iron supplementation is recommended in these patients, but it is associated with a higher incidence of gastrointestinal adverse reactions. Liposomal iron therapy has been proposed as a new iron formulation, improving iron bioavailability with less side effects; however, few data are available in patients with NDD-CKD. METHODS: We designed a single-arm pilot study to evaluate the efficacy of liposomal iron administered for six months in correcting iron deficiency (defined as serum ferritin < 100 ng/mL and/or transferrin saturation < 20%) in patients with NDD-CKD stages 1-5. The primary endpoints were the achievement of serum ferritin ≥ 100 ng/mL and transferrin saturation ≥ 20%. Secondary outcomes were hemoglobin (Hb) changes and the safety of liposomal iron. RESULTS: The efficacy population included 34/38 patients, who completed at least one visit after baseline. Liposomal iron increased the achievement of transferrin saturation targets from 11.8% at baseline to 50.0% at month 6 (p = 0.002), while no significant correction of serum ferritin (p = 0.214) and Hb was found (p = 0.465). When patients were stratified by anemia (Hb < 12 g/dL in women and Hb < 13 g/dL in men), a significant improvement of transferrin saturation was observed only in anemic patients (from 13.3 ± 5.8% to 20.2 ± 8.1%, p = 0.012). Hb values slightly increased at month 6 only in anemic patients (+0.60 g/dL, 95%CI -0.27 to +1.48), but not in those without anemia (+0.08 g/dL, 95%CI -0.73 to +0.88). In patients taking at least one dose of liposomal iron (safety population, n = 38), the study drug was discontinued in eight patients due to death (n = 2), a switch to intravenous iron (n = 2), and the occurrence of side effects (n = 4). CONCLUSIONS: The use of liposomal iron in patients with NDD-CKD is associated with a partial correction of transferrin saturation, with no significant effect on iron storage and Hb levels.

Drosophila Evi5 is a critical regulator of intracellular iron transport via transferrin and ferritin interactions.

Vesicular transport is essential for delivering cargo to intracellular destinations. Evi5 is a Rab11-GTPase-activating protein involved in endosome recycling. In humans, Evi5 is a high-risk locus for multiple sclerosis, a debilitating disease that also presents with excess iron in the CNS. In insects, the prothoracic gland (PG) requires entry of extracellular iron to synthesize steroidogenic enzyme cofactors. The mechanism of peripheral iron uptake in insect cells remains controversial. We show that Evi5-depletion in the Drosophila PG affected vesicle morphology and density, blocked endosome recycling and impaired trafficking of transferrin-1, thus disrupting heme synthesis due to reduced cellular iron concentrations. We show that ferritin delivers iron to the PG as well, and interacts physically with Evi5. Further, ferritin-injection rescued developmental delays associated with Evi5-depletion. To summarize, our findings show that Evi5 is critical for intracellular iron trafficking via transferrin-1 and ferritin, and implicate altered iron homeostasis in the etiology of multiple sclerosis.

Glycosylation and Characterization of Human Transferrin in an End-Stage Kidney Disease.

Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the peritoneum for solute transfer but is associated with complications including protein loss, including transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes.

Integration of metalloproteome and immunoproteome reveals a tight link of iron-related proteins with COVID-19 pathogenesis and immunity.

Increasing clinical data show that the imbalance of host metallome is closely associated with different kinds of disease, however, the intrinsic mechanisms of action of metals in immunity and pathogenesis of disease remain largely undefined. There is lack of multiplexed profiling system to integrate the metalloproteome-immunoproteome information at systemic level for exploring the roles of metals in immunity and disease pathogenesis. In this study, we build up a metal-coding assisted multiplexed proteome assay platform for serum metalloproteomic and immunoproteomic profiling. By taking COVID-19 as a showcase, we unbiasedly uncovered the most evident modulation of iron-related proteins, i.e., Ft and Tf, in serum of severe COVID-19 patients, and the value of Ft/Tf could work as a robust biomarker for COVID-19 severity stratification, which overtakes the well-established clinical risk factors (cytokines). We further uncovered a tight association of transferrin with inflammation mediator IL-10 in COVID-19 patients, which was proved to be mainly governed by the monocyte/macrophage of liver, shedding light on new pathophysiological and immune regulatory mechanisms of COVID-19 disease. We finally validated the beneficial effects of iron chelators as anti-viral agents in SARS-CoV-2-infected K18-hACE2 mice through modulation of iron dyshomeostasis and alleviating inflammation response. Our findings highlight the critical role of liver-mediated iron dysregulation in COVID-19 disease severity, providing solid evidence on the involvement of iron-related proteins in COVID-19 pathophysiology and immunity.

Challenges for heat stress: Intestinal culturable bacteria of Lohmann Brown chickens.

This study aimed to assess how heat stress, specifically within the range of 35-38 °C, affects the populations of culturable intestinal lactobacilli, enterococci, and Escherichia coli, as well as the expression of Heat Shock Proteins (HSP70), in Lohmann Brown chickens. It also explored the influence of the chickens' blood transferrin and ceruloplasmin genotypes on these responses. Thirty chickens underwent eight hours of heat stress, maintained at an average temperature of 37 °C and a relative humidity of 75-80%, with continuous access to food and water. Behavioral monitoring was conducted throughout to prevent excessive heat-related mortality. The Lohmann Brown chickens from the Yerevan "Arax" poultry farm were initially classified based on their blood transferrin and ceruloplasmin genotypes to investigate potential correlations between intestinal bacterial composition and variations in these polymorphisms. A significant correlation was found between heat stress and the abundance of culturable enterococci within the intestinal microbiota, regardless of chicken TfAB, TfBC, CpAB, CpCC and TfAB, TfBC, CpAB, CpCD genotypes. Heat stress led to nearly double the HSP70 levels in chicken blood, along with a reduction in the culturable enterococci population by at least 10,000-fold in the intestinal microbiota. These findings are significant for targeted management strategies to mitigate heat stress in chicken populations.

Transferrin levels are associated with malnutrition markers in hemodialysis patients in KwaZulu-Natal, South Africa.

INTRODUCTION: Malnutrition is a global phenomenon and may be contributing to the increasing size of the hemodialysis (HD) population in South Africa and is affecting morbidity and clinical outcomes. Our study assessed whether transferrin could be a possible marker for malnutrition in the HD population. METHODS: Clinical parameters (including skinfold thickness and mid-upper arm circumference [MUAC]) and laboratory markers (including transferrin and hemoglobin) were measured during a six-month period in a sample of 59 HD patients. RESULTS: Linear regression analysis showed that MUAC (p = 0.027) as well as skinfold thickness (p = 0.021) had a significant association with transferrin levels within the HD participants. There was no significant association between transferrin levels or MUAC with hemoglobin levels (p = 0.075). Furthermore, the study found that decreased transferrin levels (< 2.15 g/dL to 3.80 g/dL) were closely related to malnutrition in the malnutrition distribution groups within the study, with 97.7% of HD participants being classified in one of the malnutrition groups. CONCLUSION: Thus, transferrin levels are a valuable marker for malnutrition within the HD patient population and can be included along with clinical assessment parameters such as MUAC and skinfold thickness as primary indicators for malnutrition.

Effects of Cucurbita Moschata squash (Butternut) seed paste in improving zinc and iron status in children attending Early Childhood Development centres in Limpopo province, South Africa.

Cucurbita moschata (Butternut squash) seeds are a rich source of nutrition containing nutrients including iron, zinc, copper, calcium, potassium, and phosphorus. The aim of this study was to determine if Cucurbita Moschata squash seed paste improves zinc and iron status, anthropometric status, and dietary intake in preschool children. A pretest-posttest control group trial using cluster randomisation was conducted over 6 months. Four preschools were randomly assigned to receive 100 g of intervention or 100 g of a placebo as the control to enhance iron and zinc status. A total of 276 preschool children were recruited from eight government registered Early Childhood Development centres in Limpopo province, South Africa. The control group consumed Cucurbita moschata flesh twice-weekly, while the intervention group consumed Cucurbita moschata seed paste twice-weekly during a six-month period. Iron (serum iron, transferrin, transferrin saturation, ferritin) and zinc (serum zinc) status and anthropometric indices such as weight, height and mid upper arm circumference for children were evaluated at baseline and the endpoint. Iron and zinc-rich food consumption was measured using a 24-hour dietary recall and food record during the study, and dietary intake was estimated using a food frequency questionnaire which was conducted at the beginning and endpoint. The intervention group significantly improved in the mean serum iron 0.23 µg/dL (95% CI: 0.11;0.33); ferritin 0.21µg/dL (95% CI: 0.13;0.39), transferrin saturation 0.33% (0.23;0.74) and zinc 0.16 µmol/dl (95% CI: 0.13;0.25) at the end of the study. In addition, the intervention group exhibited greater mean weight for age of 0.13 z-score (95% CI: 0.28; 0.34) and weight for height of 0.04 z-score (95% CI: 0.12,0.05), as well as the consumption of iron (p < 0,001), zinc (p < 0,001), and vitamin C (p < 0.001). At the end of the trial, fiber (p < 0.001), riboflavin (p = 0.001), vitamin B6 (p < 0.001), and vitamin B12 (p < 0.001) were significantly higher in the control group. Thus, the inclusion of intervention in the diet of children in an impoverished area of South Africa improved the iron and zinc status of these children. This supplement could be a cost effective and sustainable approach to improve nutrient status in rural South Africa. Trial registration: Pan African Clinical Trial Registry (PACTR202308740458863).

A strategy for oral delivery of FGF21 for mitigating inflammation and multi-organ damage in sepsis.

Fibroblast growth factor 21 (FGF21) shows great therapeutic potential in metabolic, neurodegenerative and inflammatory diseases. However, current FGF21 administration predominantly relies on injection rather than oral ingestion due to its limited stability and activity post-gastrointestinal transit, thereby hindering its clinical utility. Milk-derived exosomes (mEx) have emerged as a promising vehicle for oral drug delivery due to their ability to maintain structural integrity in the gastrointestinal milieu. To address the challenge associated with oral delivery of FGF21, we encapsulated FGF21 within mEx (mEx@FGF21) to protect its activity post-oral administration. Additionally, we modified the surface of mEx@FGF21 by introducing transferrin (TF) to enhance intestinal absorption and transport, designated TF-mEx@FGF21. In vitro results demonstrated that the surface modification of TF promoted FGF21 internalization by intestinal epithelial cells. Orally administered TF-mEx@FGF21 showed promising therapeutic effects in septic mice. This study represents a practicable strategy for advancing the clinical application of oral FGF21 delivery.

Proteomic Analysis of Egg Yolk Proteins During Embryonic Development in Wanxi White Goose.

To investigate the alterations of yolk protein during embryonic development in Wanxi white goose, the egg yolk protein composition at days 0, 4, 7, 14, 18, and 25 of incubation (D0, D4, D7, D14, D18, and D25) was analyzed by two-dimensional gel electrophoresis combined with mass spectrometry. A total of 65 spots representing 11 proteins with significant abundance changes were detected. Apolipoprotein B-100, vitellogenin-1, vitellogenin-2-like, riboflavin-binding protein, and serotransferrin mainly participated in nutrient (lipid, riboflavin, and iron ion) transport, and vitellogenin-2-like showed a lower abundance after D14. Ovomucoid-like were involved in endopeptidase inhibitory activity and immunoglobulin binding and exhibited a higher expression after D18, suggesting a potential role in promoting the absorption of immunoglobulin and providing passive immune protection for goose embryos after D18. Furthermore, myosin-9 and actin (ACTB) were involved in the tight junction pathway, potentially contributing to barrier integrity. Serum albumin mainly participated in cytolysis and toxic substance binding. Therefore, the high expression of serum albumin, myosin-9, and ACTB throughout the incubation might protect the developing embryo. Apolipoprotein B-100, vitellogenin-1, vitellogenin-2-like, riboflavin-binding protein, and serotransferrin might play a crucial role in providing nutrition for embryonic development, and VTG-2-like was preferentially degraded/absorbed.

Correlation between ferroptosis and adriamycin resistance in breast cancer regulated by transferrin receptor and its molecular mechanism.

Breast cancer is the most prevalent malignant tumor in women. Adriamycin (ADR) is a primary chemotherapy drug, but resistance limits its effectiveness. Ferroptosis, a newly identified cell death mechanism, involves the transferrin receptor (TFRC), closely linked with tumor cells. This study aimed to explore TFRC and ferroptosis's role in breast cancer drug resistance. Bioinformatics analysis showed that TFRC was significantly downregulated in drug-resistant cell lines, and patients with low TFRC expression might demonstrate a poor chemotherapeutic response to standard treatment. High expression of TFRC was positively correlated with most of the ferroptosis-related driver genes. The research findings indicate that ferroptosis markers were higher in breast cancer tissues than in normal ones. In chemotherapy-sensitive cases, Ferrous ion (Fe2+ ) and malondialdehyde (MDA) levels were higher than in resistant cases (all p < .05). TFRC expression was higher in breast cancer than in normal tissue, especially in the sensitive group (all p < .05). Cytological experiments showed increased hydrogen peroxide (H2 O2 ) after ADR treatment in both sensitive and resistant cells, with varying MDA changes (all p < .05). Elevating TFRC increased Fe2+ and MDA in ADR-resistant cells, enhancing their sensitivity to ADR. However, TFRC upregulation combined with ADR increased proliferation and invasiveness in resistant cell lines (all p < .05). In conclusion, ADR resistance to breast cancer is related to the regulation of iron ion-mediated ferroptosis by TFRC. Upregulation of TFRC in ADR-resistant breast cancer cells activates ferroptosis and reverses ADR chemotherapy resistance of breast cancer.

Exploring the Specific Role of Iron Center in the Catalytic Activity of Human Serum Transferrin: CTAB-Induced Conformational Changes and Sequestration by Mixed Micelles.

Conformational changes play a seminal role in modulating the activity of proteins. This concept becomes all the more relevant in the context of metalloproteins, owing to the formation of specific conformation(s) induced by internal perturbations (like a change in pH, ligand binding, or receptor binding), which may carry out the binding and release of the metal ion/ions from the metal binding center of the protein. Herein, we investigated the conformational changes of an iron-binding protein, monoferric human serum transferrin (Fe-hTF), using several spectroscopic approaches. We could reversibly tune the cetyltrimethylammonium bromide (CTAB)-induced conformation of the protein, exploiting the concept of mixed micelles formed by three sequestrating agents: (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) hydrate (CHAPS) and two bile salts, namely, sodium cholate (NaC) and sodium deoxycholate (NaDC). The formation of mixed micelles between CTAB and these reagents (CHAPS/NaC/NaDC) results in the sequestration of CTAB molecules from the protein environment and aids the protein in reattaining its native-like structure. However, the guanidinium hydrochloride-induced denatured Fe-hTF did not acquire its native-like structure using these sequestrating agents, which substantiates the exclusive role of mixed micelles in the present study. Apart from this, we found that the conformation of transferrin (adopted in the presence of CTAB) displays pronounced esterase-like activity toward the para-nitrophenyl acetate (PNPA) substrate as compared to native transferrin. We also outlined the impact of the iron center and amino acids surrounding the iron center on the effective catalytic activity in the CTAB medium. We estimated ∼3 times higher specific catalytic efficiency for the iron-depleted Apo-hTF compared to the fully iron-saturated Fe2-hTF in the presence of CTAB.

Myricetin Oligomer Triggers Multi-Receptor Mediated Penetration and Autophagic Restoration of Blood-Brain Barrier for Ischemic Stroke Treatment.

Restoration of blood-brain barrier (BBB) dysfunction, which drives worse outcomes of ischemic stroke, is a potential target for therapeutic opportunities, whereas a sealed BBB blocks the therapeutics entrance into the brain, making the BBB protection strategy paradoxical. Post ischemic stroke, hypoxia/hypoglycemia provokes the up-regulation of transmembrane glucose transporters and iron transporters due to multiple metabolic disorders, especially in brain endothelial cells. Herein, we develop a myricetin oligomer-derived nanostructure doped with Ce to bypass the BBB which is cointermediated by glucose transporters and iron transporters such as glucose transporters 1 (GLUT1), sodium/glucose cotransporters 1 (SGLT1), and transferrin(Tf) reporter (TfR). Moreover, it exhibits BBB restoration capacity by regulating the expression of tight junctions (TJs) through the activation of protective autophagy. The myricetin oligomers scaffold not only acts as targeting moiety but is the prominent active entity that inherits all diverse pharmacological activities of myricetin. The suppression of oxidative damage, M1 microglia activation, and inflammatory factors makes it a multitasking nanoagent with a single component as the scaffold, targeting domain and curative components.

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.