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ABSTRACTLipid nanoparticles (LNPs) coated with functional and biocompatible polymers have been widely used as carriers to deliver oligonucleotide and messenger RNA therapeutics to treat diseases. Poly(ethylene glycol) (PEG) is a representative material used for the surface coating, but the PEG surface-coated LNPs often have reduced cellular uptake efficiency and pharmacological activity. Here, we demonstrate the effect of pH-responsive ethylenediamine-based polycarboxybetaines with different molecular weights as an alternative structural component to PEG for the coating of LNPs. We found that appropriate tuning of the molecular weight around polycarboxybetaine-modified LNP, which incorporated small interfering RNA, could enhance the cellular uptake and membrane fusion potential in cancerous pH condition, thereby facilitating the gene silencing effect. This study demonstrates the importance of the design and molecular length of polymers on the LNP surface to provide effective drug delivery to cancer cells.

Abstract Hyperphosphatemia is a major risk for poor prognosis in patients with end-stage renal disease. However, the molecular mechanism behind this link remains elusive. We and others have demonstrated that serum phosphorus levels correlate positively with circulating levels of calciprotein particles (CPPs). CPPs are colloidal mineral-protein complexes containing insoluble calcium-phosphate precipitates and have been reported to induce calcification in cultured vascular smooth muscle cells and inflammatory responses in cultured macrophages. Hence, we hypothesize that CPPs may be responsible for disorders associated with hyperphosphatemia. Using hyperphosphatemic miniature pigs receiving hemodialysis, here we show that removal of CPPs from the blood with a newly developed CPP adsorption column improves survival and alleviates complications including coronary artery calcification, vascular endothelial dysfunction, metastatic pulmonary calcification, left ventricular hypertrophy, and chronic inflammation. The present study identifies CPPs as an effective therapeutic target and justifies clinical trials to determine whether the CPP adsorption column may be useful as a medical device for improving clinical outcomes of hemodialysis patients.

Abstract Nano‐sized contrast agents (NCAs) hold potential for highly specific tumor contrast enhancement during magnetic resonance imaging. Given the quantity of contrast agents loaded into a single nano‐carrier and the anticipated relaxation effects, the current molecular design approaches its limits. In this study, a novel molecular mechanism to augment the relaxation of NCAs is introduced and demonstrated. NCA formation is driven by the intramolecular self‐folding of a single polymer chain that possesses systematically arranged hydrophilic and hydrophobic segments in water. Utilizing this self‐folding molecular design, the relaxivity value can be elevated with minimal loading of gadolinium complexes, enabling sharp tumor imaging. Furthermore, the study reveals that this NCA can selectively accumulate into tumor tissues, offering effective anti‐tumor results through gadolinium neutron capture therapy. The efficacy and versatility of this self‐folding molecular design underscore its promise for cancer diagnosis and treatment.

Abstract Background Choline, as a neurotransmitter acetylcholine precursor, is reportedly associated with cognitive function. Although there are several cohort and animal studies on choline-containing foods and cognitive function, only a few interventional studies were reported. Egg yolk is a rich source of different choline-containing chemical forms, such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and α-glycerophosphocholine (α-GPC). This study aimed to investigate the effect of consuming 300 mg of egg yolk choline per day on cognitive function of Japanese adults. Methods A 12-week, randomized, double-blind, placebo-controlled, parallel-group study was conducted in 41 middle-aged and elderly males and females (43.9% female) aged ≥ 60 years and ≤ 80 years without dementia. Participants were randomly assigned to placebo and choline groups. The choline group received a supplement containing egg yolk choline (300 mg/day), and the placebo group received an egg yolk supplement free from choline for 12 weeks. Assessments of Cognitrax, Trail Making Tests (TMT) part A and B, the MOS 36-Item Short-Form Health Survey (SF-36), the Simplified Japanese Version of the WHO-Five Well-Being Index (WHO-5), and plasma choline levels were performed before and 6 and 12 weeks after supplement intake. In the present study, 19 subjects (9 in the placebo group and 10 in the choline group) were excluded due to the violation of the discontinuation criteria or participant compliance, and 41 subjects were analyzed. Results The change amount of verbal memory scores and verbal memory test-correct hit (delay) was significantly higher in the choline group than in the placebo group at baseline-6 and baseline-12 weeks. The plasma free choline level was significantly higher in the choline group compared with the placebo group at 6 weeks. Conversely, the choline group showed significantly lower Cognitrax processing speed scores, symbol digit coding testing correct responses, and SF-36 physical quality of life summary scores compared to the placebo group at 6 weeks. Conclusions The results suggested that continued 300 mg/day intake of egg yolk choline improved verbal memory, which is a part of cognitive functions. To confirm the observed effects of egg yolk choline, more well-designed and large-scale studies are warranted. Trial registration Study protocols were pre-registered in the Clinical Trials Registration System (UMIN-CTR) (UMIN 000045050).

ABSTRACTSustainable social activity is a major goal in an aging society, although this is limited by loss of athleticism, with osteoporosis-related fractures being the most common cause of long-term behavioral restrictions in older people. Therefore, the development of therapeutics that shorten the duration of fracture therapy is essential to improve the quality of life and social activity of older individuals. In this study, we developed a polyethylene glycol-modified alendronate (PEG-ALN) that can efficiently deliver the active ingredient (ALN) to fracture sites. PEG-ALN released ALN in response to an acidic pH and was systemically administered to mice in a fracture model. PEG-ALN exhibited selective accumulation at the fracture site and significantly accelerated bone healing compared to free ALN. This study highlights the utility of a simple polymer modification of ALN as a systemically injectable medicine for patients with bone fractures.

Abstract Calcium phosphate forms particles under excessive urinary excretion of phosphate in the kidney. While the formation of calcium phosphate particles (CaPs) has been implicated in the damage to renal tubular cells and renal dysfunction, clarifying the ultrastructural information and the elemental composition of the small CaPs in the wide areas of kidney tissue has been technically difficult. This study introduces correlative and sequential light as well as electron microscopic CaP observation in the kidney tissue by combining fluorescent staining for CaPs and energy-dispersive X-ray spectroscopy (EDS) in scanning electron microscopy (SEM) on resin sections prepared using high-pressure freezing and freeze substitution. CaPs formed in mouse kidneys under long-term feeding of a high-phosphate diet were clearly visualized on resin sections by fluorescence-conjugated alendronate derivatives and toluidine blue metachromasia. These CaPs were verified by correlative observation with EDS. Furthermore, small CaPs formed in the kidney under short-term feeding were detected using fluorescent probes. The elemental composition of the particles, including calcium and magnesium, was identified following EDS analyses. These results suggest that the correlative microscopy approach is helpful for observing in situ distribution and elemental composition of CaPs in the kidney and contributing to studies regarding CaP formation-associated pathophysiology.

Abstract Calciprotein particles (CPPs) are colloids composed of solid-phase calcium-phosphate and serum protein fetuin-A. CPPs form a polydispersed system with different particle size and density. CPPs with specific physical properties can induce calcification and innate immune responses in cultured cells. In hemodialysis patients, blood CPP levels were reported to correlate with vascular calcification and inflammation. However, little is known about relation between these disorders and physical properties of CPPs. Here, we show that the association between physical properties of plasma CPPs and serum levels of inflammatory cytokines/chemokines in 78 hemodialysis out-patients by cross-sectional study. Patients with cardiovascular disease (CVD) had significantly higher high density CPP (H-CPP) levels than patients without CVD but not low density CPP (L-CPP). Seven cytokines/chemokines (EGF, eotaxin, IL-8, IP-10, MCP-1, MIP-1, MIP-1β and TNFα) were detectable in the serum samples from > 95% of the patients. In multivariate regression analysis, H-CPP was positively associated with eotaxin after adjusting for age, gender, smoking, serum phosphate and FGF23. L-CPP was negatively associated with IL-8 after adjusting for age, gender, serum albumin, phosphate and FGF23. High H-CPP levels were associated with pro-inflammatory response, whereas L-CPPs were associated with anti-inflammatory response. CPPs with different physical properties may impact differently on pathophysiology in HD patients.

Abstract Calciprotein particles (CPPs) are circulating colloidal mineral-protein complexes containing crystalline and/or non-crystalline (amorphous) calcium-phosphate (CaPi). Serum CPP levels correlate with vascular stiffness and calcification in patients with chronic kidney disease (CKD). In vitro studies showed that CPPs containing crystalline CaPi were more arteriosclerogenic and inflammogenic than CPPs without containing crystalline CaPi. Thus, we hypothesized that not only the quantity but also the quality of CPPs (the phase of CaPi) might affect clinical outcomes. To test this hypothesis, we quantified amorphous CaPi ratio defined as the ratio of the amorphous CaPi amount to the total CaPi amount in serum CPPs from 183 hemodialysis patients and explored its possible correlation with serum parameters associated with prognosis of hemodialysis patients. Multivariate analysis revealed that the amorphous CaPi ratio correlated positively with hemoglobin and negatively with fibroblast growth factor-21 (FGF21), which remained significant after adjusting for the total CaPi amount. Because low hemoglobin and high FGF21 are associated with increased mortality, the present study warrants further studies to determine whether low amorphous CaPi ratio in circulating CPPs may be associated with poor prognosis in hemodialysis patients.

HDL are dynamic transporters of diverse molecular cargo and play critical roles in lipid metabolism and inflammation. We have previously reported that HDL transport both host and nonhost small RNAs (sRNA) based on quantitative PCR and sRNA sequencing approaches; however, these methods require RNA isolation steps which have potential biases and may not isolate certain forms of RNA molecules from samples. HDL have also been reported to accept functional sRNAs from donor macrophages and deliver them to recipient endothelial cells; however, using PCR to trace HDL-sRNA intercellular communication has major limitations. The present study aims to overcome these technical barriers and further understand the pathways involved in HDL-mediated bidirectional flux of sRNAs between immune cells. To overcome these technical limitations, SYTO RNASelect, a lipid-penetrating RNA dye, was used to quantify a) overall HDL-sRNA content, b) bidirectional flux of sRNAs between HDL and immune cells, c) HDL-mediated intercellular communication between immune cells, and d) HDL-mediated RNA export changes in disease. Live cell imaging and loss-of-function assays indicate that the endo-lysosomal system plays a critical role in macrophage storage and export of HDL-sRNAs. These results identify HDL as a substantive mediator of intercellular communication between immune cells and demonstrate the importance of endocytosis for recipient cells of HDL-sRNAs. Utilizing a lipid-penetrating RNA-specific fluorescence dye, we were able to both quantify the absolute concentration of sRNAs transported by HDL and characterize HDL-mediated intercellular RNA transport between immune cells.

Many nanoparticles are stabilised by chemical functionalisation with poly(ethylene glycol) (PEG). Here, the authors report on the functionalisation of gold nanoparticles by the physical absorption of cyclic PEG and demonstrate superior stabilisation against heating, freezing and lyophilisation.

Abstract Background Car accidents due to unexpected forward or backward runaway by older drivers are a serious social problem. Although the cause of these accidents is often attributed to stepping on the accelerator instead of the brake, it is difficult to induce such pedal application errors systematically with usual drive simulators. We developed a simple personal computer system that induces the pedal errors, and investigate the effects of age on the error behaviors. Methods The system consisted of a laptop computer and a three-pedal foot mouse. It measured response time, accuracy, and flexibility of pedal operation to visual stimuli. The system displayed two open circles on the computer display, lighting one of the circles in a random order and interval. Subjects were instructed to press the foot pedal with their right foot as quickly as possible when the circle was lit; the ipsilateral pedal to the lit circle in a parallel mode and the contralateral pedal in a cross mode. When the correct pedal was pressed, the light went off immediately, but when the wrong pedal was pressed, the buzzer sounded and the light remained on until the correct pedal was pressed. During a 6-min trial, the mode was switched between parallel and cross every 2 min. During the cross mode, a cross mark appears on the display. The pedal responses were evaluated in 52 subjects divided into young (20–29 years), middle-aged (30–64 years), and older (65–84 years) groups. Additionally, the repeatability of the pedal response characteristic indicators was examined in 14 subjects who performed this test twice. Results The mean response time was 95 ms (17%) longer in the older group than in the young group. More characteristically, however, the older group showed 2.1 times more frequent pedal errors, fell into long hesitations (response freezing > 3 s) 16 times more often, and took 1.8 times longer period to correct the wrong pedal than the young groups. The indicators of pedal response characteristics showed within-individual repeatability to the extent that can identify the age-dependent changes. Conclusions Hesitations and extended error correction time can be associated with increased crash risk due to unexpected runaway by older drivers. The system we have developed may help to uncover and evaluate physiological characteristics related to crash risk in the elderly population.

Immune-regulating activity of Cyclopia subternata and C. genistoides extracts (honeybush tea) in vitro, using a murine cell culture, merited investigation of their immune-regulating functions in vivo. Oral administration of the extracts and ovalbumin (OVA) to OVA specific-T cell receptor transgenic mice (DO11.10) significantly (P

Abstract Calciprotein particles (CPP) are solid-phase calcium-phosphate bound to serum protein fetuin-A and dispersed as colloids in the blood. Recent clinical studies indicated that serum CPP levels were increased with decline of renal function and associated with inflammation and vascular calcification. However, CPP assays used in these studies measured only a part of CPP over a certain particle size and density. Here we show that such CPP are mostly artifacts generated during processing of serum samples in vitro. The native CPP in fresh plasma are smaller in size and lower in density than those artifactual CPP, composed of fetuin-A carrying amorphous and/or crystalline calcium-phosphate, and increased primarily with serum phosphate levels. We have identified several physicochemical factors that promote aggregation/dissolution of CPP and transition of the calcium-phosphate from the amorphous phase to the crystalline phase in vitro, including addition of anti-coagulants, composition of buffer for sample dilution, the number of freeze-thaw cycles, the speed for sample freezing, and how many hours the samples were left at what temperature. Therefore, it is of critical importance to standardize these factors during sample preparation in clinical studies on CPP and to investigate the biological activity of the native CPP.

Abstract The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43–related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts.

The effect of dietary milk-derived sphingomyelin concentrate (SPC) on atopic-like dermatitis was investigated. Atopic-like dermatitis was induced by sensitization to and repeated challenges with picryl chloride in male NC/Nga mice. Mice were fed an SPC-containing diet for 12 weeks, beginning 2 weeks before sensitization. The effect of SPC was assessed by monitoring clinical scores, serum immunoglobulin E levels, scratching behaviours, and in vitro cytokine productivities by splenocytes and mesenteric lymph node cells. Skin lesions were histologically investigated. Scratching behaviours were improved in the SPC group compared with the control group. The clinical score was ameliorated, and interleukin-6 productivity by splenocytes was significantly reduced in the SPC group. The numbers of infiltrated and degranulated mast cells in skin lesions were significantly lower in the SPC group than in the control group. These results indicate that dietary SPC significantly ameliorates atopic-like dermatitis in NC/Nga mice by suppressing systemic and peripheral inflammation.

Several drugs targeting the pathogenesis of aortic aneurysm have shown efficacy in model systems but not in clinical trials, potentially owing to the lack of targeted drug delivery. Here, we designed a novel drug delivery system using nanoparticles to target the disrupted aortic aneurysm micro-structure. We generated poly(ethylene glycol)-shelled nanoparticles incorporating rapamycin that exhibited uniform diameter and long-term stability. When injected intravenously into a rat model in which abdominal aortic aneurysm (AAA) had been induced by infusing elastase, labeled rapamycin nanoparticles specifically accumulated in the AAA. Microscopic analysis revealed that rapamycin nanoparticles were mainly distributed in the media and adventitia where the wall structures were damaged. Co-localization of rapamycin nanoparticles with macrophages was also noted. Rapamycin nanoparticles injected during the process of AAA formation evinced significant suppression of AAA formation and mural inflammation at 7 days after elastase infusion, as compared with rapamycin treatment alone. Correspondingly, the activities of matrix metalloproteinases and the expression of inflammatory cytokines were significantly suppressed by rapamycin nanoparticle treatment. Our findings suggest that the nanoparticle-based delivery system achieves specific delivery of rapamycin to the rat AAA and might contribute to establishing a drug therapy approach targeting aortic aneurysm.

Fibroblast growth factor-1 (FGF-1) is secreted by astrocytes and stimulates apolipoprotein E (apoE)-HDL biogenesis by an autocrine mechanism to help in recovery from brain injury. In apoE-deficient mouse astrocytes, FGF-1 stimulated cholesterol biosynthesis without enhancing its release, indicating a signaling pathway independent of apoE biosynthesis upregulation. SU5402, an inhibitor of FGF receptor, inhibited FGF-1-induced phosphorylation of MEK, ERK, and Akt, as well as all the apoE-HDL biogenesis-related events in rat astrocytes. LY294002, an inhibitor of phosphatidylinositide 3-OH kinase (PI3K) and of Akt phosphorylation, inhibited apoE-HDL secretion but not cholesterol biosynthesis, whereas U0126, an inhibitor of MEK and of ERK phosphorylation, inhibited cholesterol biosynthesis but not apoE-HDL secretion. Increase of apoE-mRNA by FGF-1 was not influenced by either inhibitor. When rat apoE/pcDNA3.his was transfected to transformed rat astrocyte GA-1 cells that otherwise do not synthesize apoE (GA-1/25), FGF-1 did not influence apoE-mRNA, but did increase the apoE secretion and Akt phosphorylation that were suppressed by LY294002. Lipid biosynthesis was increased by FGF-1 in GA-1/25 cells and suppressed by U0126. FGF-1 upregulates apoE-HDL biogenesis by three independent signaling pathways. The PI3K/Akt pathway upregulates secretion of apoE/apoE-HDL, the MEK/ERK pathway stimulates cholesterol biosynthesis, and an unknown pathway enhances apoE transcription.

Introduction of ligands into 100 nm scaled hollow capsules has great potential for diagnostic and therapeutic applications in drug delivery systems. Polyethylene glycol-conjugated (PEGylated) polyion complex vesicles (PICsomes) are promising hollow nano-capsules that can survive for long periods in the blood circulation and can be used to deliver water-soluble macromolecules to target tissues. In this study, cyclic RGD (cRGD) peptide, which is specifically recognized by αVβ3 and αvβ5 integrins that are expressed at high levels in the neovascular system, was conjugated onto the distal end of PEG strands on PICsomes for active neovascular targeting. Density-tunable cRGD-conjugation was achieved using PICsomes with definite fraction of end-functionalized PEG, to substitute 20, 40, and 100% of PEG distal end of the PICsomes to cRGD moieties. Compared with control-PICsomes without cRGD, cRGD-PICsomes exhibited increased uptake into human umbilical vein endothelial cells. Intravital confocal laser scanning microscopy revealed that the 40%-cRGD-PICsomes accumulated mainly in the tumor neovasculature and remained in the perivascular region even after 24 h. Furthermore, we prepared superparamagnetic iron oxide (SPIO)-loaded cRGD-PICsomes for magnetic resonance imaging (MRI) and successfully visualized the neovasculature in an orthotopic glioblastoma model, which suggests that SPIO-loaded cRGD-PICsomes might be useful as a MRI contrast reagent for imaging of the tumor microenvironment, including neovascular regions that overexpress αVβ3 integrins.

Single-stranded DNA binding proteins (SSBs) regulate multiple DNA transactions, including replication, transcription, and repair. We recently identified SSB1 as a novel protein critical for the initiation of ATM signaling and DNA double-strand break repair by homologous recombination. Here we report that germline Ssb1(-/-) embryos die at birth from respiratory failure due to severe rib cage malformation and impaired alveolar development, coupled with additional skeletal defects. Unexpectedly, Ssb1(-/-) fibroblasts did not exhibit defects in Atm signaling or γ-H2ax focus kinetics in response to ionizing radiation (IR), and B-cell specific deletion of Ssb1 did not affect class-switch recombination in vitro. However, conditional deletion of Ssb1 in adult mice led to increased cancer susceptibility with broad tumour spectrum, impaired male fertility with testicular degeneration, and increased radiosensitivity and IR-induced chromosome breaks in vivo. Collectively, these results demonstrate essential roles of Ssb1 in embryogenesis, spermatogenesis, and genome stability in vivo.

Flagellum-mediated motility of Trypanosoma brucei is considered to be essential for the parasite to complete stage development in the tsetse fly vector, while the mechanism by which flagellum-mediated motility is controlled are not fully understood. We thus compared T. brucei whole gene products (amino acid sequence) with Caenorhabditis elegans UNC (uncoordinated) proteins, in order to find uncharacterized motility-related T. brucei genes. Through in silico analysis, we found 88 gene products which were highly similar to C. elegans UNC proteins and categorized them as TbCEUN (T. brucei gene products which have high similarity to C. elegansUNC proteins). Approximately two thirds of the 88 TbCEUN gene products were kinesin-related molecules. A gene product highly similar to C. elegans UNC119 protein was designated as TbUNC119. RNAi-mediated depletion of TbUNC119 showed no apparent phenotype. However, knock-down analysis of both TbUNC119 and its binding protein (TbUNC119BP) which was found by yeast two-hybrid analysis showed characteristic phenotypes, including reduced motility, morphological change (extended cell shape), and cellular apoptosis. Based on the observed phenotypes, possible function of the TbUNC119 and TbUNC119BP is discussed.

The anti-allergic effects of extracts prepared from two species of honeybush, Cyclopia genistoides and Cyclopia subternata, were demonstrated in vivo in a murine allergy model for inhaled antigen induced with ovalbumin (OVA) inhalation to mimic pollen allergy. Intake of the extracts increased the production of OVA-specific immunoglobulin (Ig) E (IgE), IgG1, and IgG2a antibodies in serum and significantly suppressed anaphylactic reaction-induced body temperature decline. Moreover, the extracts significantly inhibited antigen-antibodyinduced degranulation in RBL-2H3 cells. They also inhibited body temperature decline when the allergic mice were given them after antigen sensitization, indicating that anti-degranulation activity is the major mechanism underlying the anti-allergic effect of Cyclopia extracts. Despite their qualitative and quantitative differences in phenolic composition, the two extracts exhibited similar effects, suggesting that several active compounds might be involved in the activity. Therefore, oral administration of either Cyclopia extract potentially exerts a systemic anti-allergic effect, supporting the increased consumption of honeybush tea for general wellness and improved quality of life. [ABSTRACT FROM AUTHOR]