Tailoring lipid nanoparticles for T-cell targeting in allergic asthma: Insights into efficacy and specificity.

Asthma impacts over 300 million patients globally, with significant health implications, especially in cases of its allergic subtype. The disease is characterized by a complex interplay of airway inflammation and immune responses, often mediated by Th2 cell-related cytokines. In this study, we engineered lipid nanoparticles (LNPs) to specifically deliver therapeutic siRNA via the transferrin receptor to T cells. Strain-promoted azide-alkyne cycloaddition (SPAAC) was employed for the conjugation of transferrin ligands to PEGylated lipids in the LNPs, with the goal of enhancing cellular uptake and gene knockdown. The obtained LNPs exhibited characteristics that make them suitable for pulmonary delivery. Using methods such as nanoparticle tracking analysis (NTA) and enzyme-linked immunosorbent assay (ELISA), we determined the average number of transferrin molecules bound to individual LNPs. Additionally, we found that cellular uptake was ligand-dependent, achieving a GATA3 knockdown of more than 50% in relevant in vitro and ex vivo models. Notably, our findings highlight the limitations inherent to modifying the surface of LNPs, particularly with regard to their targeting capabilities. This work paves the way for future research aimed at optimizing targeted LNPs for the treatment of immunologic diseases such as allergic asthma.

Protein Coronas Derived from Mucus Act as Both Spear and Shield to Regulate Transferrin Functionalized Nanoparticle Transcellular Transport in Enterocytes.

The epithelial mucosa is a key biological barrier faced by gastrointestinal, intraoral, intranasal, ocular, and vaginal drug delivery. Ligand-modified nanoparticles demonstrate excellent ability on this process, but their efficacy is diminished by the formation of protein coronas (PCs) when they interact with biological matrices. PCs are broadly implicated in affecting the fate of NPs in vivo and in vitro, yet few studies have investigated PCs formed during interactions of NPs with the epithelial mucosa, especially mucus. In this study, we constructed transferrin modified NPs (Tf-NPs) as a model and explored the mechanisms and effects that epithelial mucosa had on PCs formation and the subsequent impact on the transcellular transport of Tf-NPs. In mucus-secreting cells, Tf-NPs adsorbed more proteins from the mucus layers, which masked, displaced, and dampened the active targeting effects of Tf-NPs, thereby weakening endocytosis and transcellular transport efficiencies. In mucus-free cells, Tf-NPs adsorbed more proteins during intracellular trafficking, which enhanced transcytosis related functions. Inspired by soft coronas and artificial biomimetic membranes, we used mucin as an "active PC" to precoat Tf-NPs (M@Tf-NPs), which limited the negative impacts of "passive PCs" formed during interface with the epithelial mucosa and improved favorable routes of endocytosis. M@Tf-NPs adsorbed more proteins associated with endoplasmic reticulum-Golgi functions, prompting enhanced intracellular transport and exocytosis. In summary, mucus shielded against the absorption of Tf-NPs, but also could be employed as a spear to break through the epithelial mucosa barrier. These findings offer a theoretical foundation and design platform to enhance the efficiency of oral-administered nanomedicines.

CORONAVIRUS SARS-COV-2 MODIFIES ANTITUMOR REDOX STATUS OF BLOOD AND INTERCELLULAR MATRIX IN METASTATIC COLORECTAL CANCER PATIENTS (A PILOT STUDY).

BACKGROUND: The current studies demonstrate that SARS-CoV-2 infection results in increasing complications incidence and the total risk of death in cancer patients. SARS-CoV-2 infection triggers oxidative stress representing one of the major factors of the inflammation contributing to the complicated course of the diseases including cancer. AIM: To assess the effect of hypoxia caused by SARS-CoV-2 infection on the redox status of blood in patients with metastatic colorectal cancer (mCRC). MATERIALS AND METHODS: 10 patients with SARS-CoV-2, 11 mCRC patients with metachronous liver disease, and 14 mCRC patients with preceding SARS-CoV-2 infection were included in the study. The data on blood biochemistry (C-reactive protein, ferritin, transferrin, and free iron) were analyzed. The levels of superoxide radicals (ROS) in blood cells were determined by electron paramagnetic resonance (EPR) using the spin trap technique. The metalloproteinase activity was measured by polyacrylamide gel zymography with the addition of gelatin as a substrate. RESULTS: In mCRC patients with prior SARS-CoV-2 infection, a 1.26-fold increase in ROS-generating activity of blood neutrophils was observed compared to mCRC patients with no history of SARS-CoV-2 infection. The blood content of C-reactive protein, transferrin, and free iron in mCRC patients with prior SARS-CoV-2 infection increased by 2, 6, and 1.4 times, respectively. The total activity of gelatinases in platelets and neutrophils in the blood of mCRC patients with prior SARS-CoV-2 infection was 1.4 and 1.2 times higher compared to mCRC patients with no history of SARS-CoV-2 infection. CONCLUSION: mCRC patients with prior COVID-19 have a higher risk of exacerbation of inflammatory reactions. SARS-CoV-2 infection results in redox dіsbalance, which may contribute to the unfavorable course of the disease.

Protein-Decorated Microbubbles for Ultrasound-Mediated Cell Surface Manipulation.

Delivering cargo to the cell membranes of specific cell types in the body is a major challenge for a range of treatments, including immunotherapy. This study investigates employing protein-decorated microbubbles (MBs) and ultrasound (US) to "tag" cellular membranes of interest with a specific protein. Phospholipid-coated MBs were produced and functionalized with a model protein using a metallochelating complex through an NTA(Ni) and histidine residue interaction. Successful "tagging" of the cellular membrane was observed using microscopy in adherent cells and was promoted by US exposure. Further modification of the MB surface to enable selective binding to target cells was then achieved by functionalizing the MBs with a targeting protein (transferrin) that specifically binds to a receptor on the target cell membrane. Attachment and subsequent transfer of material from MBs functionalized with transferrin to the target cells significantly increased, even in the absence of US. This work demonstrates the potential of these MBs as a platform for the noninvasive delivery of proteins to the surface of specific cell types.

Increased IL-6 Levels and the Upregulation of Iron Regulatory Biomarkers Contribute to the Progression of Japanese Encephalitis Virus Infection's Pathogenesis.

Integrated analysis of iron regulatory biomarkers and inflammatory response could be an important strategy for Japanese encephalitis viral (JEV) infection disease management. In the present study, the inflammatory response was assessed by measuring serum Interleukin-6 (IL-6) levels using ELISA, and the transcription levels of iron homeostasis regulators were analyzed via RT-PCR. Furthermore, inter-individual variation in the transferrin gene was analyzed by PCR-RFLP and their association with clinical symptoms, susceptibility, severity, and outcomes was assessed through binary logistic regression and classification and regression tree (CART) analysis. Our findings revealed elevated levels of IL-6 in serum as well as increased expression of hepcidin (HAMP), transferrin (TF), and transferrin receptor-1 (TFR1) mRNA in JEV infection cases. Moreover, we found a genetic variation in TF (rs4481157) associated with clinical symptoms of meningoencephalitis. CART analysis indicates that individuals with the wild-type TF genotype are more susceptible to moderate JEV infection, while those with the homozygous type are in the high-risk group to develop a severe JEV condition. In summary, the study highlights that JEV infection induces alteration in both IL-6 levels and iron regulatory processes, which play pivotal roles in the development of JEV disease pathologies.

Controlled SPION-Exosomes Loaded with Quercetin Preserves Pancreatic Beta Cell Survival and Function in Type 2 Diabetes Mellitus.

Introduction: Quercetin has an ideal therapeutic effect on islet function improvement in type 2 diabetes mellitus (T2DM). However, the therapeutic benefit of quercetin is hindered by its poor bioavailability and limited concentration in pancreatic islets. In this study, superparamagnetic iron oxide nanoparticle (SPION)-modified exosomes were prepared to load quercetin, hoping to endow quercetin with enhanced water solubility and active targeting capacity with the help of magnetic force (MF). Methods: Transferrin-modified SPIONs (Tf-SPIONs) were synthesized by exploiting N-hydroxysuccinimidyl (NHS) conjugation chemistry, and quercetin-loaded exosomes (Qu-exosomes) were acquired by electroporation. Tf-SPION-modified quercetin-loaded exosomes (Qu-exosome-SPIONs) were generated by the self-assembly of transferrin (Tf) and the transferrin receptor (TfR). The solubility of quercetin was determined by high-performance liquid chromatography (HPLC) analysis. The pancreatic islet targeting capacity and insulin secretagogue and antiapoptotic activities of Qu-exosome-SPIONs/MF were evaluated both in vitro and in vivo. Results: The Qu-exosome-SPIONs were well constructed and harvested by magnetic separation with a uniform size and shape in a diameter of approximately 86.2 nm. The water solubility of quercetin increased 1.97-fold when loaded into the SPION-modified exosomes. The application of SPIONs/MF endowed the Qu-exosomes with favorable targeting capacity. In vitro studies showed that Qu-exosome-SPIONs/MF more effectively inhibited or attenuated ß cell apoptosis and promoted insulin secretion in response to elevated glucose (GLC) compared with quercetin or Qu-exosome-SPIONs. In vivo studies demonstrated that Qu-exosome-SPIONs/MF displayed an ideal pancreatic islet targeting capacity, thereby leading to the restoration of islet function. Conclusion: The Qu-exosome-SPIONs/MF nano-delivery system significantly enhanced the quercetin concentration in pancreatic islets and thereby improved pancreatic islet protection.

The Effect of a Six-Week Nordic Walking Training Cycle on Oxidative Damage of Macromolecules and Iron Metabolism in Older Patients with Multiple Myeloma in Remission-Randomized Clinical Trial.

Multiple myeloma (MM) is an incurable hematologic malignancy originating from clonal plasma cell proliferation within the bone marrow, predominantly affecting older individuals. While anemia serves as a diagnostic criterion for MM, it often ameliorates upon achieving disease remission. Iron metabolism parameters have emerged as potential prognostic indicators in MM. Notably, physical exercise has been established to influence iron metabolism. This study aimed to assess alterations in serum iron, ferritin, and transferrin concentrations, as well as leukocyte gene expression, in MM patients undergoing a six-week cycle of Nordic walking training. Thirty patients divided into an exercise group (NW, n = 15, mean age 63.1 ± 8.4 years) and a control group (CG, n = 15, mean age: 63.5 ± 3.6 years) completed the study protocol. Blood samples were collected at baseline, after three and six weeks of training, and after nine weeks. Serum ferritin, transferrin, and iron concentrations were measured, along with the leukocyte expression of genes. Additionally, serum oxidative damage marker levels were determined. Following the Nordic walking training cycle, a declining trend in serum ferritin concentrations was observed. Intracellular mRNA levels of genes associated with iron metabolism were positively influenced by the training regimen, indicating the potential impact of this physical activity on gene expression and ferritin concentrations. Although positive trends were noted, extended training periods might be requisite for significant changes. To conclude, moderate-intensity exercise induces favorable shifts in the analyzed parameters among MM patients, potentially influencing disease progression. Consequently, Nordic walking training is a safe recommendation for MM patients, though sustained training beyond six weeks could be necessary for notable effects on iron metabolism factors.

Rosiglitazone inhibits acyl-CoA synthetase long-chain family number 4 and improves secondary brain injury in a rat model of surgical brain injury.

Ferroptosis is a recently discovered non-apoptotic form of cellular death. Acyl-CoA synthetase long-chain family number 4 (ACSL4) is necessary for iron-dependent cellular death, and reactive oxygen species (ROS) produced by ACSL4 are the executioners of ferroptosis. Rosiglitazone improves ferroptosis by inhibiting ACSL4. There is no research indicating whether ACSL4 plays a role in cell death after surgical brain injury (SBI). This study aimed to investigate the role of ACSL4 in SBI via the ferroptosis pathway. Ninety male Sprague-Dawley rats were examined using a model of SBI. Subsequently, the inhibitory effect of rosiglitazone on ACSL4 was assessed via western blot, real-time polymerase chain reaction (PCR), immunofluorescence, fluoro-jade C staining, Perl's staining, ROS assay, and neurological scoring. The results showed that compared with the Sham group, the protein levels of ACSL4 and transferrin were significantly increased after SBI. Administration of rosiglitazone significantly reduced neuronal necrosis, iron deposition, brain water content and ROS in brain tissue and ameliorated neurological deficits at 48 h after SBI, which was concomitant with decreased transferrin expression. These findings demonstrate that SBI-induced upregulation of ACSL4 may be partly mediated by the ferroptosis pathway, which can be reversed by rosiglitazone administration.

Elevated expression of immune and DNA repair genes in mated queens and kings of the Reticulitermes chinensis termites.

Studies have identified that mating induces a series of physiological changes in animals. In this period, males tending to invest more energy, immune peptides, and other substances to reduce the cost of living for females. This results in lower survival rates in later life than females. Meanwhile, both males and females shorten lifespans due to reproduction. However, the reasons why termites' queens and kings are both extremely long-lived and highly fecund are unclear. Therefore, this study aimed to examine the effects of mating on the expression of immune and DNA repair genes for lifespan extension in termite queens and kings. Here, we reported that mated queens show relatively higher expression of immune genes (phenoloxidase, denfensin, termicin, transferrin), antioxidant genes (CAT, SOD), detoxification genes (GST, CYP450) than virgin queens in the Reticulitermes chinensis. In addition, mated kings also highly expressed these genes, except for termicin, transferrin, GST, and CYP450. After mating, both queens and kings significantly upregulated the expression of DNA repair genes (MLH1, BRCA1, XRCC3, RAD54-like). Mismatch repair genes (MMR) MSH2, MSH4, MSH6 were considerably increased in mated queens, while MSH4, MSH5, MSH6 were upregulated in mated kings. Our results suggest that mating increases the expression of immune and DNA repair genes in the termite queens and kings, and thus possibly improving their survival during reproductive span due to the omnipresent pathogens.

Modulation of Cellular Senescence in HEK293 and HepG2 Cells by Ultrafiltrates UPla and ULu Is Partly Mediated by Modulation of Mitochondrial Homeostasis under Oxidative Stress.

Protein probes, including ultrafiltrates from the placenta (UPla) and lung (ULu) of postnatal rabbits, were investigated in premature senescent HEK293 and HepG2 cells to explore whether they could modulate cellular senescence. Tris-Tricine-PAGE, gene ontology (GO), and LC-MS/MS analysis were applied to describe the characteristics of the ultrafiltrates. HEK293 and HepG2 cells (both under 25 passages) exposed to a sub-toxic concentration of hydrogen peroxide (H2O2, 300 µM) became senescent; UPla (10 µg/mL), ULu (10 µg/mL), as well as positive controls lipoic acid (10 µg/mL) and transferrin (10 µg/mL) were added along with H2O2 to the cells. Cell morphology; cellular proliferation; senescence-associated beta-galactosidase (SA-ß-X-gal) activity; expression of senescence biomarkers including p16 INK4A (p16), p21 Waf1/Cip1 (p21), HMGB1, MMP-3, TNF-α, IL-6, lamin B1, and phospho-histone H2A.X (γ-H2AX); senescence-related gene expression; reactive oxygen species (ROS) levels; and mitochondrial fission were examined. Tris-Tricine-PAGE revealed prominent detectable bands between 10 and 100 kDa. LC-MS/MS identified 150-180 proteins and peptides in the protein probes, and GO analysis demonstrated a distinct enrichment of proteins associated with "extracellular space" and "proteasome core complex". UPla and ULu modulated senescent cell morphology, improved cell proliferation, and decreased beta-galactosidase activity, intracellular and mitochondrial ROS production, and mitochondrial fission caused by H2O2. The results from this study demonstrated that UPla and Ulu, as well as lipoic acid and transferrin, could protect HEK293 and HepG2 cells from H2O2-induced oxidative damage via protecting mitochondrial homeostasis and thus have the potential to be explored in anti-aging therapies.

Standardized Preclinical In Vitro Blood-Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways.

The presence of the blood-brain barrier (BBB) creates a nigh-on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a "Trojan Horse" strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB-penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that help delineate the ability of modified large bivalent IgG antibodies conjugated to the transferrin receptor binder scFv8D3 to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administration of bivalent antibodies into the endothelial monolayer, a highly sensitive enzyme-linked immunosorbent assay (ELISA) is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis, respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of receptors and proteins that are likely involved in the transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed that transcytosis of the transferrin-receptor-targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB-penetrating capabilities of transferrin-receptor-targeting antibodies. We believe that the In-Cell BBB-Trans assay can be used as a powerful, preclinical screening platform for therapeutic neurological pathologies.

Understanding the pH-dependent interaction of anthocyanin with two food-derived transferrins.

The potential binding of cyanidin-3-O-glucoside (C3G) to bovine lactoferrin (BLF) and ovotransferrin (OTF) at pH 3, 5, and 7 was investigated for the first time. Multiple spectroscopic techniques demonstrated pH-dependent alterations in the conformational characteristics of BLF and OTF upon complexation with C3G. Fluorescence quenching assays showed that their highest binding affinity was at pH 7. Hydrophobic interactions and hydrogen bonds were found to be crucial in molecular dynamics simulations but with significantly lower probabilities of formation at pH 3 (p < 0.05). At pH 7, electrostatic attraction can occur for the negatively charged forms of C3G, and the well-maintained native structures of BLF and OTF may be favorable for stabilizing the C3G binding sites. This study sheds light on the stronger interaction of C3G with BLF/OTF at pH 7, which may have implications for future applications such as anthocyanin stabilization or the development of functional food ingredients.

Regulation of transferrin receptor trafficking by optineurin and its disease-associated mutants.

Transferrin receptor (TFRC) is a transmembrane protein that plays a crucial role in mediating homeostasis of iron in the cell. The binding of transferrin (that is bound to iron) to TFRC at the cell membrane generally starts endocytosis of TFRC-transferrin complex, which leads to formation of vesicles that are positive for TFRC. These vesicles travel to the early endosomes and later to the endocytic recycling compartment. Release of iron occurs in the early endosomes because of acidic pH. Major fraction of the transferrin and TFRC is transported back to the cell membrane; however, a minor fraction of it is transported to lysosomes through the process of autophagy. Optineurin (OPTN) is a multi-functional adaptor protein that plays a pivotal role in the control of TFRC trafficking, recycling and autophagy dependent degradation. Optineurin also plays a role in cargo-selective and non-selective autophagy. Here, we review our understanding of the function of OPTN in regulating TFRC trafficking, recycling and autophagy dependent degradation. We also discuss the mechanisms by which certain disease-associated mutations of OPTN alter these processes.

Oxidative stress and hepcidin expression in pediatric sickle cell anemia with iron overload.

BACKGROUND: Blood transfusion (BT) is essential in treating sickle cell disease (SCD); however, it leads to iron overload (IO) and oxidative stress. We studied the relationship between oxidative stress, iron status parameters, hepcidin mRNA gene expression, and IO in SCD patients. METHODS: We classified all SCD patients (n = 90) into two groups: Group I, 45 children (s.ferritin ≥ 938 ng/mL) and Group II, 45 children (s.ferritin < 938 ng/mL). A total of 55 children, age and sex matched, participated as a control group. Malondialdehyde (MDA), nitrite, s.iron, s.total iron-binding capacity (sTIBC), transferrin saturation %, s.ferritin, s.hepcidin, and hepcidin mRNA gene expression were assessed. RESULTS: Among SCD BT-dependent patients (>3 times/year), 63% were from Group I and 37% from Group II, p < .01. The two patient groups had significantly lower s.hepcidin and hepcidin gene expression than controls ( p < .001). TIBC, s.iron, s.ferritin, transferrin saturation %, ferritin/hepcidin ratio, and MDA levels were higher among SCD patients than controls ( p < .001). Group I had higher mean level of ferritin/hepcidin ratio and MDA than Group II ( p < .01). The higher level of MDA and increased frequency of BT were the significant predicting risk factors for IO ( p < .05). A receiver-operating characteristic curve indicates that MDA is the outstanding significant biomarker for high level of s.ferritin with subsequent IO progression. CONCLUSION: MDA may serve as a biomarker of oxidative stress and IO in SCD patients. This result paid attention for urgent initiation of antioxidant and chelation therapy on detecting increased MDA level.

Identification of subclinical iron deficiency using new erythrocytes, leukocytes, and reticulocytes parameters during nonsevere acute infection in pediatric outpatients.

BACKGROUND: This study aims to investigate the diagnostic utility of new erythrocytes, leukocytes, and reticulocytes parameters for the identification of subclinical iron deficiency (ID) in children under 6 years with nonsevere acute infection in pediatric outpatients. METHODS: The study included 102 children with acute infections and 31 true ID. Traditional and new hematology parameters were measured in a Sysmex-XN®, along with C-reactive protein level, and iron parameters. Participants' ID were categorized as: the ferritin < 100 ng/mL, transferrin saturation < 20% was defined as "subclinical or functional ID (FID) in Group 1"; ferritin < 30 ng/mL, transferrin saturation < 20%, as "absolute-ID (AID)" in Group 2; ferritin < 12 ng/mL without anemia and infection, as "true ID" in Group 3. RESULTS: The frequencies of FID and AID among the 102 children with acute infection were 24% and 76%, respectively. Compared with the Group 2 patients, Group 1 had a significantly higher mean percentage of hypochromic erythrocytes (Hypo-He), and significantly lower levels of hemoglobin (Hb) and Hb content of reticulocytes (RET-He) (p < 0.05 for all). Compared with Group 2 and Group 3 patients, Group 1 had a significantly higher mean percentage of immature reticulocyte fraction (IRF) and immature granulocyte (IG) values (p < 0.05 for all). The RET-He, IRF%, Hypo-He%, and IG% cut-off values for predicting FID during infection were 27.0 pg, 10.6%, 2.5%, and 0.35% respectively. DISCUSSION: The RET-He, Hypo-He, IRF, and IG may be useful parameters for identifying subclinical ID in small children with nonsevere acute infection in pediatric outpatients.

Novel pathogenic variants in CUBN uncouple proteinuria from renal function.

BACKGROUND: Proteinuria is an unfavorable clinical condition highly associated with a risk of renal and cardiovascular disease in chronic kidney disease (CKD). However, whether all proteinuria forms are linked to renal impairment are still unclear. Cubilin is an endocytic receptor highly expressed in renal proximal tubules mediating uptake of albumin, transferrin and α1-microglobulin. METHODS: Exome sequencing method initially identified candidate genes. With the application of exome sequencing combined with Sanger sequencing, we further focused on CUBN through bioinformatics analysis. The pathogenic effects of the potentially causative variants were verified utilizing complementary analysis of clinical data and systematic characterization of the variants' expression and function with clinical samples and in vitro experiments in HEK293T cell lines along with in vivo experiments in mice. RESULTS: In this study, we identified four novel variants locating after the vitamin B12 (vitB12)-binding domain of Cubilin (encoded by CUBN, NM_001081.3: c.4397G > A (p.C1466Y), c.6796C > T (p.R2266X), c.6821 + 3A > G and c.5153_5154delCT (p.S1718X)) in two families. Moreover, the variants severely affected the expression and function of Cubilin in renal proximal tubules and caused albuminuria, increasing levels in urine transferrin and α1-microglobulin, but without progressive glomerular filtration barrier (GFB) impairment, vitB12 deficiencies or abnormal blood levels of HDL and albumin. Further mechanistic insights showed that the variants after the vitB12-binding domain of CUBN merely disrupted the association with Amnionless (AMN) that exhibited aberrant localization in cell cytoplasm rather than membrane. CONCLUSIONS: Here, our findings suggested that different mutation types after the vitB12-binding domain of CUBN uncouple proteinuria from glomerular filtration barrier, that may be an unexpectedly common benign condition in humans and may not require any proteinuria-lowering treatment or renal biopsy.

Inhibitors of RNA and protein synthesis cause Glut4 translocation and increase glucose uptake in adipocytes.

Insulin stimulates glucose uptake in adipocytes by triggering translocation of glucose transporter 4-containg vesicles to the plasma membrane. Under basal conditions, these vesicles (IRVs for insulin-responsive vesicles) are retained inside the cell via a "static" or "dynamic" mechanism. We have found that inhibitors of RNA and protein synthesis, actinomycin D and emetine, stimulate Glut4 translocation and glucose uptake in adipocytes without engaging conventional signaling proteins, such as Akt, TBC1D4, or TUG. Actinomycin D does not significantly affect endocytosis of Glut4 or recycling of transferrin, suggesting that it specifically increases exocytosis of the IRVs. Thus, the intracellular retention of the IRVs in adipocytes requires continuous RNA and protein biosynthesis de novo. These results point out to the existence of a short-lived inhibitor of IRV translocation thus supporting the "static" model.

High-Level Expression of Cell-Surface Signaling System Hxu Enhances Pseudomonas aeruginosa Bloodstream Infection.

Bloodstream infections (BSIs) caused by Pseudomonas aeruginosa are associated with a high mortality rate in the clinic. However, the fitness mechanisms responsible for the evolution of virulence factors that facilitate the dissemination of P. aeruginosa to the bloodstream are poorly understood. In this study, a transcriptomic analysis of the BSI-associated P. aeruginosa clinical isolates showed a high-level expression of cell-surface signaling (CSS) system Hxu. Whole-genome sequencing and comparative genomics of these isolates showed that a mutation in rnfE gene was responsible for the elevated expression of the Hxu-CSS pathway. Most importantly, deletion of the hxuIRA gene cluster in a laboratory strain PAO1 reduced its BSI capability while overexpression of the HxuIRA pathway promoted BSI in a murine sepsis model. We further demonstrated that multiple components in the blood plasma, including heme, hemoglobin, the heme-scavenging proteins haptoglobin, and hemopexin, as well as the iron-delivery protein transferrin, could activate the Hxu system. Together, these studies suggested that the Hxu-CSS system was an important signal transduction pathway contributing to the adaptive pathogenesis of P. aeruginosa in BSI.

Differential effects of high-altitude exposure on markers of oxidative stress, antioxidant capacity, and iron profiles.

High-altitude (HA) exposure may stimulate significant physiological and molecular changes, resulting in HA-related illnesses. HA may impact oxidative stress, antioxidant capacity, and iron homeostasis, yet it is unclear how both repeated exposure and HA acclimatization may modulate such effects. Therefore, we assessed the effects of weeklong repeated daily HA exposure (2,900-5,050 m) in altitude-naïve individuals (n = 21 individuals, 13 females, mean ± SD, 25.3 ± 3.7 yr) to mirror the working schedule of HA workers (n = 19 individuals, all males, 41.1 ± 9.4 yr) at the Atacama Large Millimeter Array (ALMA) Observatory (San Pedro de Atacama, Chile). Markers of oxidative stress, antioxidant capacity, and iron homeostasis were measured in blood plasma. Levels of protein oxidation (P < 0.001) and catalase activity (P = 0.023) increased and serum iron (P < 0.001), serum ferritin (P < 0.001), and transferrin saturation (P < 0.001) levels decreased with HA exposure in both groups. HA workers had lower levels of oxidative stress, and higher levels of antioxidant capacity, iron supply, and hemoglobin concentration as compared with altitude-naïve individuals. On a second week of daily HA exposure, changes in levels of protein oxidation, glutathione peroxidase, and nitric oxide metabolites were lower as compared with the first week in altitude-naïve individuals. These results indicate that repeated exposure to HA may significantly alter oxidative stress and iron homeostasis, and the degree of such changes may be dependent on if HA is visited naïvely or routinely. Further studies are required to fully elucidate differences in HA-induced changes in oxidative stress and iron homeostasis profiles among visitors of HA.

Self-recovery study of fluoride-induced ferroptosis in the liver of zebrafish (Danio rerio).

Ferroptosis plays a key role in fluorosis in aquatic organisms, but whether it is involved in fluoride-induced liver damage remains unclear. Previous studies have indicated that fluoride toxicity has the reversible tendency, but the mechanism of self-recovery after fluorosis in aquatic animals has not been elucidated. In this study, adult zebrafish and embryos were exposed to 0, 20, 40, 80 mg/L of fluoride for 30, 60 and 90 d and 3, 4 and 5 d post-fertilization (dpf), respectively. After 90 d, adult zebrafish were transferred to clean water for self-recovery of 30 d. The results showed that fluoride induced the prominent histopathologial changes in liver of adults, and the developmental delay and dark liver area in larvae. Fluoride significantly increased the iron overload, while decreased the expression levels of transferrin (tf), transferrin receptor (tfr), ferroportin (fpn), membrane iron transporter (fpn), and ferritin heavy chain (fth) in adults and larvae. Fluoride also induced the oxidative stress in adults and larvae by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), while decreasing the glutathione (GSH) content and the levels of glutathione peroxidase 4 (gpx4) and solute carrier family 7 member 11 (slc7a11). Self-recovery relieved fluoride-induced ferroptosis by reducing the histopathological damage and oxidative stress, reversing the expression levels of fth and slc7a11, Fe2+ metabolism and GSH synthesis. Lipid peroxidation and Fe2+ metabolism may be the key factor in alleviating effects of self-recovery on fluoride toxicity. Moreover, males are more sensitive than females. Our results provide a theoretical basis for studying the alleviating effects of self-recovery on fluoride toxicity and the underlying mechanism of its protective effect.