Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

Feature Selection for the Interpretation of Antioxidant Mechanisms in Plant Phenolics.

Antioxidants, represented by plant phenolics, protect living tissues by scavenging reactive oxygen species through diverse reaction mechanisms. Research on antioxidants is often individualized, for example, focusing on the evaluation of their activity against a single reactive oxygen species or examining the antioxidant properties of compounds with similar structures. In this study, multivariate analysis was used to comprehensively examine antioxidant properties. Eighteen features were selected to explain the results of the antioxidant capacity tests. These selected features were then evaluated by supervised learning, using the results of the antioxidant capacity assays. Dimension-reduction techniques were also used to represent the compound space with antioxidants as a two-dimensional distribution. A small amount of data obtained from several assays provided us with comprehensive information on the relationships between the structures and activities of antioxidants.

Prediction and Chemical Interpretation of Singlet-Oxygen-Scavenging Activity of Small Molecule Compounds by Using Machine Learning.

A chemically explainable machine learning model was constructed with a small dataset to quantitatively predict the singlet-oxygen-scavenging ability. In this model, ensemble learning based on decision trees resulted in high accuracy. For explanatory variables, molecular descriptors by computational chemistry and Morgan fingerprints were used for achieving high accuracy and simple prediction. The singlet-oxygen-scavenging mechanism was explained by the feature importance obtained from machine learning outputs. The results are consistent with conventional chemical knowledge. The use of machine learning and reduction in the number of measurements for screening high-antioxidant-capacity compounds can considerably improve prediction accuracy and efficiency.

Effects of change in the formulation of lanthanum carbonate on laboratory parameters.

Lanthanum carbonate (LC) is available in the two formulations of chewable tablets and granules. In this study, we changed the formulation of LC from chewable tablet to granules, and compared the laboratory parameters for 3 months before and after changing formulation in 58 hemodialysis (HD) patients. We also surveyed patients about their preferences for the two formulations. The mean serum phosphorus (P) levels decreased significantly (P < 0.01) from 6.7 mg/dL to 6.4 mg/dL after the change. The levels for serum albumin and geriatric nutritional risk index increased significantly (P < 0.01). Serum calcium levels also increased significantly (P < 0.01), while serum intact parathyroid hormone levels decreased significantly (P < 0.01). In the survey, approximately half of the patients responded that the granules were easier to take than the chewable tablets. These findings suggest that changing the formulation of LC to granules may reduce serum P levels of the HD patients in clinical practices.

A comparison of systemic inflammation-based prognostic scores in patients on regular hemodialysis.

BACKGROUND/AIMS: Systemic inflammation-based prognostic scores have prognostic power in patients with cancer, independently of tumor stage and site. Although inflammatory status is associated with mortality in hemodialysis (HD) patients, it remains to be determined as to whether these composite scores are useful in predicting clinical outcomes. METHODS: We calculated the 6 prognostic scores [Glasgow prognostic score (GPS), modified GPS (mGPS), neutrophil-lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), prognostic index (PI) and prognostic nutritional index (PNI), which have been established as a useful scoring system in cancer patients. We enrolled 339 patients on regular HD (age: 64 ± 13 years; time on HD: 129 ± 114 months; males/females = 253/85) and followed them for 42 months. The area under the receiver-operating characteristics curve was used to determine which scoring system was more predictive of mortality. RESULTS: Elevated GPS, mGPS, NLR, PLR, PI and PNI were all associated with total mortality, independent of covariates. If GPS was raised, mGPS, NLR, PLR and PI were also predictive of all-cause mortality and/or hospitalization. GPS and PNI were associated with poor nutritional status. Using overall mortality as an endpoint, the area under the curve (AUC) was significant for a GPS of 0.701 (95% CI: 0.637-0.765; p < 0.01) and for a PNI of 0.616 (95% CI: 0.553-0.768; p = 0.01). However, AUC for hypoalbuminemia (<3.5 g/dl) was comparable to that of GPS (0.695, 95% CI: 0.632-0.759; p < 0.01). CONCLUSION: GPS, based on serum albumin and highly sensitive C-reactive protein, has the most prognostic power for mortality prediction among the prognostic scores in HD patients. However, as the determination of serum albumin reflects mortality similarly to GPS, other composite combinations are needed to provide additional clinical utility beyond that of albumin alone in HD patients.

Association of HCV core antigen seropositivity with long-term mortality in patients on regular hemodialysis.

Anti-hepatitis C virus (HCV) antibody seropositivity is independently associated with poor prognosis in hemodialysis (HD) patients. However, anti-HCV antibody cannot distinguish between patients with active infection and those who have recovered from infection. We therefore aimed in this study to examine the association of HCV core antigen (HCVcAg) seropositivity with mortality in HD patients. We first measured serum HCVcAg using an immunoradiometric assay and anti-HCV antibody in 405 patients on regular HD, and followed them for 104 months. There were 82 patients (20.2%) who had been positive for anti-HCV antibodies; 57 (69.5%) of these were positive for HCVcAg. During the follow-up, 29 patients were excluded, so we tested the association of HCVcAg seropositivity with all-cause, cardiovascular (CV) and non-CV mortalities in 376 patients. A total of 209 patients (55.6%) had expired during the observational period, 92 out of them due to CV causes. After adjusting for comorbid parameters, HCVcAg was independently associated with overall mortality (HR 1.61, 95% CI 1.05-2.47, p < 0.05). HCV infection was significantly related to liver disease-related mortality. Past HCV infection also contributed to CV mortality (HR 2.63, 95% CI 1.27-5.45, p < 0.01). In contrast, anti-HCV antibody and HCVcAg seropositivities did not associate with infectious disease-related and cancer-related (expect for hepatocellular carcinoma) mortality. It follows from these findings that HCVcAg serology is associated with all-cause and CV mortality in HD patients.

Influence of the assay for measuring serum albumin on corrected total calcium in chronic hemodialysis patients.

The Japanese Society for Dialysis Therapy guideline for secondary hyperparathyroidism recommends the use of albumin-corrected serum Ca as a therapeutic target in chronic hemodialysis patients; however, the assay used for albumin measurement may affect the corrected Ca level. In this study, we examined the impact of the albumin assay on corrected Ca levels in hemodialysis patients. We measured serum albumin using bromocresol green (BCG) and modified bromocresol purple (BCP) assays, and corrected Ca for albumin using Payne's formula in 422 hemodialysis patients (age 66±13 years; time on hemodialysis 116±111 months). Serum albumin values were 3.7±0.4 (1.4-4.6) g/dL by BCG and 3.3±0.4 (1.0-4.3) g/dL by modified BCP, with the differences between the two assays ranging from 0.0 to 0.6 with a mean of 0.35±0.09 g/dL. Serum C-reactive protein and globulin values were significantly higher in patients with differences in albumin greater than 0.5 g/dL (P<0.01). Based on the BCG method, 71 patients (16.8%) were classified with hypocalcemia, 51 (12.1%) with hypercalcemia, and 300 (70.0%) as normocalcemic. In contrast, when using modified BCP, 33 patients (7.9%) were labeled as hypocalcemic, while 92 (21.8%) were hypercalcemic. Depending on the use of either BCG or modified BCP, a discrepancy of classification was observed in 79 patients (18.7%): 41 patients were re-classified from normocalcemic to hypercalcemic, and 38 patients were re-classified from hypocalcemic to normocalcemic by selecting the modified BCP assay. These findings suggest that the type of assay used for albumin measurement has an impact on albumin-corrected Ca levels.

Modulatory role of dopamine D2 receptors and fundamental role of L-type Ca2+ channels in the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway of anesthetized rats.

We have previously found that the induction of long-term potentiation in the synaptic pathway from the basolateral amygdala to the dentate gyrus (BLA-DG LTP) is regulated by L-type Ca(2+) channels, dopamine D(2) receptors and GABAergic inhibition. In the present study, we investigated possible relations among the three mechanisms by using anesthetized rats. Blockade of GABAergic inhibition with picrotoxin abolished both the inhibitory effect of the dopamine D(2) receptor antagonist chlorpromazine and the promoting effect of the dopamine D(2) receptor agonist quinpirole on the induction of BLA-DG LTP. However, the inhibitory effect of the L-type Ca(2+) channel blocker verapamil on BLA-DG LTP was not affected by picrotoxin. These results suggest that the role of dopamine D(2) receptors in the induction of BLA-DG LTP is modulatory and depends on GABAergic inhibition, whereas the role of L-type Ca(2+) channels is fundamental.

Basolateral amygdala D1- and D2-dopaminergic system promotes the formation of long-term potentiation in the dentate gyrus of anesthetized rats.

We have previously found that the induction of hippocampal long-term potentiation (LTP) is modulated by neuron activities in the basolateral amygdala (BLA). However, little is known about what neurotransmitter system in the BLA contributes to modulation of hippocampal LTP. In the present study, we investigated possible involvement of BLA dopaminergic system in the induction of LTP at the perforant path (PP)-dentate gyrus (DG) granule cell synapses of anesthetized rats. The induction of PP-DG LTP was significantly attenuated by intra-BLA injection of the D(1) receptor antagonist SCH23390 (2 or 6 nmol) or the D(2) receptor antagonists, chlorpromazine (30 or 100 nmol) or haloperidol (4.4 or 13.3 nmol). The effects of SCH23390 and haloperidol were abolished by concomitant intra-BLA injection of the D(1) receptor agonist SKF38393 (17 nmol) and the D(2) receptor agonist quinpirole (3 nmol), respectively. Furthermore, lesioning with 6-hydroxydopamine of the ventral tegmental area, the origin of the dopaminergic system projecting to the BLA, resulted in attenuated PP-DG LTP, which was restored by intra-BLA injection of SKF38393 or quipirole. These results suggest that the induction of PP-DG LTP is promoted by the BLA dopaminergic system via both D(1) and D(2) receptors.

A helix-to-coil transition at the epsilon-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis.

Processing of amyloid precursor protein (APP) by gamma-secretase is the last step in the formation of the Abeta peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618-660), we show that the TM domain forms an alpha-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the epsilon-cleavage site. This helix-to-coil transition is required for gamma-secretase processing; mutations that extend the TM alpha-helix inhibit epsilon cleavage, leading to a low production of Abeta peptides and an accumulation of the alpha- and beta-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM alpha-helix.

Stimulation of basolateral amygdaloid serotonin 5-HT(2C) receptors promotes the induction of long-term potentiation in the dentate gyrus of anesthetized rats.

We have previously found that the induction of hippocampal long-term potentiation (LTP) is modulated by neuron activities in the basolateral amygdala (BLA). However, little is known about what neurotransmitter system in the BLA contributes to modulation of hippocampal LTP. In the present study, we investigated possible involvement of BLA serotonergic system in the induction of LTP at the perforant path (PP)-dentate gyrus (DG) granule cell synapses of anesthetized rats. The induction of PP-DG LTP was significantly inhibited by intra-BLA injection of the 5-HT(2) receptor antagonist cinanserin (25-50nmol), but not by intra-BLA injection of the 5-HT(1,7) receptor antagonist methiothepin (50nmol), the 5-HT(3) receptor antagonist ondansetron (50nmol) or the 5-HT(4) receptor antagonist RS23597-190 (100nmol). In addition, intra-BLA injection of the 5-HT(2C) receptor agonist MK212 (50nmol) facilitated the induction of PP-DG LTP. These results suggest that the induction of PP-DG LTP is promoted by activation of 5-HT(2C) receptors in the BLA.

Involvement of dopamine D2 receptors in the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway of anesthetized rats.

We have previously found that synaptic pathway from the basolateral amygdala (BLA) to the dentate gyrus (DG) displays N-methyl-D-aspartate (NMDA) receptor-independent form of long-term potentiation (LTP), which should be a valuable model for elucidating neural mechanisms linking emotion and memory. To explore its cellular mechanisms, we investigated possible involvement of the beta-adrenergic, muscarinic cholinergic and dopaminergic systems on LTP in this pathway of anesthetized rats. The induction of BLA-DG LTP was not affected by administration of the beta-adrenoceptor antagonist propranolol (50-150nmol, i.c.v.), the muscarinic receptor antagonist scopolamine (2-6mg/kg, i.p.), the cholinesterase inhibitor physostigmine (50 nmol, i.c.v.) or the dopamine D(1) receptor antagonist SCH23390 (100nmol, i.c.v.), but significantly inhibited by the dopamine D2 receptor antagonists, chlorpromazine (15nmol, i.c.v.) and haloperidol (0.15-0.5mg/kg, i.p.), and significantly promoted by the dopamine D2 receptor agonist quinpirole (78nmol, i.c.v.). Furthermore, lesioning with 6-hydroxydopamine of the ventral tegmental area (VTA), the origin of mesolimbic dopaminergic neurons, resulted in attenuated BLA-DG LTP. These results suggest that the D2-dopaminergic system, but not the beta-adrenergic, muscarinic or D1-dopaminergic system, is involved in the induction of BLA-DG LTP. In addition, inhibition of BLA-DG LTP by haloperidol or VTA lesion was abolished by blockade of GABAergic inhibition with picrotoxin. It is probable that the D2-dopaminergic system promotes the induction of BLA-DG LTP by suppressing GABAergic inhibition.

Important role for fibronectin-EIIIA during renal tubular repair and cellular recovery in uranyl acetate-induced acute renal failure of rats.

The present study was designed to identify the source and kinetics of an alternatively spliced "embryonic" cellular fibronectin EIIIA (cFn-EIIIA) in relation to regenerating renal tubules in uranyl acetate (UA)-induced acute renal failure (ARF) in rats. Damage of the proximal tubules was found as early as day 2 after induction of ARF, peaked at day 5, and was almost substituted by epithelial relining by day 7. Immunohistochemistry showed de novo deposition of cFn-EIIIA in peritubular regions as early as day 2, then on the tubular basement membrane (TBM) after day 4. beta1 Integrin, the receptor for Fn, was mainly found at the basal side of tubules in the normal control and increased in the interstitium after induction of ARF, but the staining pattern gradually returned to the control after day 7. Immunoelectron microscopy revealed that cFn-EIIIA was produced initially by the peritubular endothelium and later by fibroblastic cells and was deposited to the TBM, on which regenerating tubules proliferated, probably with cFn-EIIIA production. beta1 Integrin was expressed in cFn-EIIIA-producing cells, especially in regenerating tubular cells, suggesting that cFn-EIIIA signal transduction affects regenerating tubules. Transforming growth factor (TGF)-beta1 was found in some damaged proximal tubules and interstitial cells after induction of ARF and later in the regenerating tubules. CFn-EIIIA and beta1 integrin mRNA levels were upregulated as early as day 2. TGF-beta1 mRNA level significantly increased after day 3, suggesting a modulatory role for TGF-beta1 on cFn-EIIIA production, but not by day 2. Our data suggest that cFn-EIIIA production by the endothelium during the very early response to tubular injury and by fibroblastic cells and regenerating tubules may play an important role in the cellular recovery of UA-induced ARF in rats.

A mechanism for the development of subepithelial deposits in a patient with type III membranoproliferative glomerulonephritis.

We report on a patient with membranoproliferative glomerulonephritis (MPGN) type III, whose repeated renal biopsies show evolution from a type I-like pattern to a type III pattern of immune complex formation over a 1-year period. Based on the development of experimental in situ immune complex glomerulonephritis, we discuss possible mechanisms for the formation of subepithelial deposits in type III MPGN with reference to an experimental in situ immune complex model of glomerulonephritis.

Relation of distal nephron changes to proximal tubular damage in uranyl acetate-induced acute renal failure in rats.

AIMS: To elucidate the pathophysiological roles of the changes of distal nephron in uranyl acetate (UA)-induced acute renal failure (ARF), we investigated the relation of changes of constituent molecules in distal nephron to proximal tubular damage and repair in UA-treated rats. METHODS: ARF was induced in rats by intravenous injection of UA, and all rats received bromodeoxyuridine (BrdU) intraperitoneally 1 h before sacrifice. RESULTS: Proximal tubular damage with necrosis appeared as early as day 2, mainly in the outer stripe of outer medulla and reached a peak level at day 5. Slight cellular damage was evident in the distal nephron as early as day 3, reaching a peak level around day 9. Immunoreactive BrdU- or vimentin-positive regenerating proximal tubules (PT) appeared at day 2 and regenerating PT relining was almost completed by day 7. Immunostaining for EGF, which was constitutively expressed in the thick ascending limb (TAL) and distal convoluted tubule (DCT), diminished significantly as early as day 2, when PT regeneration became evident, and remained below normal levels until day 21. In contrast, slight immunoreactivity for EGF was observed in regenerated PT accompanying brush-border formation mainly after day 9, suggesting newly expressed EGF might contribute to PT maturation. Lectin staining or immunostaining for representative constituent molecules of the thin descending limb, TAL, DCT and collecting duct demonstrated marked and transient reduction after day 5. CONCLUSIONS: EGF was not associated with regenerating PT, but may be involved in the maturation of PT. Transient reduction in expression of constituent molecules of the distal nephron following the reduction in EGF could reflect dedifferentiation or phenotypic simplification during regenerative repair of PT in UA-induced ARF in rats.

Mechanisms and kinetics of Bowman's epithelial-myofibroblast transdifferentiation in the formation of glomerular crescents.

BACKGROUND: We investigated the mechanisms and kinetics of Bowman's epithelial-myofibroblast transdifferentiation in the formation of glomerular crescents. METHODS: Crescentic glomerulonephritis was induced by i.v. injection of rabbit anti-rat glomerular basement membrane antiserum in WKY rats. RESULTS: Cellular crescents (83.5% of glomeruli) were first observed at day 7 after disease induction. Immunostaining of alpha-smooth muscle actin (alpha-SMA), as a marker for the myofibroblast phenotype, was found in some periglomerular regions as early as day 3, when it was also seen in parietal epithelial cells (PEC) of Bowman's capsule at day 5 and in crescent formation at day 7. Proliferation marker Ki67-positive PEC was found at day 3, and double Ki67- and alpha-SMA-positive PEC could be seen at day 5. The migratory figure of PEC with the expression of alpha-SMA was found by immunoelectron microscopy. At day 7, some crescent cells were stained positive for PEC marker, protein gene product 9.5, in association with alpha-SMA or Ki67. Expression of transforming growth factor (TGF)-beta receptor types I and II, as well as platelet-derived growth factor (PDGF) receptor beta and PDGF-B increased in PEC as early as day 3. At day 5 marked deposition of cellular and common fibronectin, but not other extracellular matrix components examined was found in Bowman's spaces where ED 1-positive macrophages infiltrated. CONCLUSIONS: PEC may be stimulated to proliferate and/or transdifferentiate into myofibroblast phenotype possibly by action of TGF-beta and PDGF and/or binding of fibronectin to PEC, then migrate and/or proliferate, participating in glomerular crescents.

Recovery from hemodialysis therapy in a patient with renal cholesterol crystal embolism.

The prognosis of renal cholesterol crystal embolism (CCE) is poor, and many patients progressively develop to the end-stage of chronic renal failure. We herein experienced a 66-year-old male patient who recovered from hemodialysis (HD) shortly after an amputation of inflammatory toes. The patient complained of painful digital cyanosis at bilateral toes and livedo reticularis at right lower leg 4 weeks following aortic angiography. Laboratory examinations revealed eosinophilia and overt proteinuria (3.0 g/day). His serum creatinine level increased from 2.18 to 8.57 mg/dl over 6 weeks, and HD treatment was started. Treatment with simvastatin (5 mg/day) did not reverse renal failure and hypereosinophilia, but the amputation of right gangrene toes promptly increased urine output and eosinophilia completely disappeared concomitantly with a decline of C-reactive protein from 9.7 to 0.7 mg/dl. Serum creatinine level was also reduced to 3.46 mg/dl, and he eventually stopped HD totally after 32 sessions. This case suggested that the surgical amputation promptly recovered renal function. Reversal of inflammation may be more effective than lipid-lowering therapy for renal failure in our patient.

Mycophenolate mofetil inhibits regenerative repair in uranyl acetate-induced acute renal failure by reduced interstitial cellular response.

We recently reported that transient appearance of interstitial myofibroblasts and infiltrating macrophages might play a role in cellular recovery in uranyl acetate (UA)-induced acute renal failure (ARF). Here we tested the effects of mycophenolate mofetil (MMF), which attenuates infiltration of lymphocytes, macrophages, and myofibroblasts, but does not suppress epithelial regeneration, on renal tissue repair. Rats treated with MMF (20 mg/kg/day) or vehicle were sacrificed at 2, 5, and 7 days after induction of ARF by injection of 5 mg/kg UA. Renal tissues were immunostained for bromodeoxyuridine (BrdU) and Ki67, alpha-smooth muscle actin (alpha-SMA), ED1, and CD43. The expression levels of alpha-SMA mRNA were examined by reverse transcription-polymerase chain reaction. Body weight loss or serum albumin levels were similar in MMF and vehicle rats during the experiment. In vehicle group, serum creatinine (Scr) significantly increased after day 5, but proximal tubular (PT) damage score increased as early as day 2 after UA injection. BrdU- or Ki67-positive regenerating tubular cells, ED1-positive macrophages and alpha-SMA-positive myofibroblasts significantly increased in the interstitium after day 5. In MMF-treated rats, Scr and PT damage score significantly increased at day 7 and the number of regenerating PT were significantly reduced compared with vehicle-treated rats at days 5 and 7. The numbers of macrophages and myofibroblasts and the expression of alpha-SMA mRNA were significantly lower in MMF than in vehicle rats at day 5, indicating that reduced interstitial cellular response is linked to the inhibition of regenerative repair. CD43-positive lymphocytes were significantly reduced in MMF group than in vehicle group at day 7, suggesting that lymphocyte infiltration does not seem to contribute to early regenerative response of proximal tubules. The transient appearance of myofibroblasts and macrophages in the interstitium may promote regenerative repair in UA-induced ARF in rats.