Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema.

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.

Engineering Interconnected Open-Porous Particles via Microfluidics Using Bijel Droplets as Structural Templates.

Designing porous structures is key in materials science, particularly for separation, catalysis, and cell culture systems. Bicontinuous interfacially jammed emulsion gels represent a unique class of soft matter formed by kinetically arresting the separation of the spinodal decomposition phase, which is stabilized by colloidal particles with neutral wetting. This study introduces a microfluidic technique to create highly interconnected open-porous particles using bijel droplets stabilized with hexadecyltrimethylammonium bromide (CTAB)-modified silica particles. Monodisperse droplets comprising a hydrophobic monomer, water, ethanol, silica particles, and CTAB were initially formed in the microfluidic device. The diffusion of ethanol from these droplets into the continuous cyclohexane phase triggered spinodal decomposition within the droplets. The phase-separated structure within the droplets was stabilized by the CTAB-modified silica particles, and subsequent photopolymerization yielded microparticles with highly interconnected, open pores. Moreover, the influence of the ratio of the CTAB and silica particles, fluid composition, and microchannel direction on the final structure of the microparticles was explored. Our findings indicated that the phase-separated structure of the particles transitioned from oil-in-water to water-in-oil as the CTAB/silica ratio was increased. At intermediate CTAB/silica ratios, microparticles with bicontinuous structures were formed. Regardless of the fluid composition, the pore size of the particles increased with time after phase separation. However, this coarsening was arrested 15 s after droplet formation in the CTAB-modified silica particles, accompanied by a change in the particle shape from spherical to ellipsoidal. In situ observations of the bijel droplet formation revealed that the particle shape deformation is caused by the rolling of elastic bijel droplets at the bottom of the microchannel. As such, the channel setup was altered from horizontal to vertical to prevent the deformation of bijel droplets, resulting in spherical particles with open pores.

A combined computational-biophysical approach to understanding fatty acid binding to FABP7.

Members of the fatty acid binding protein (FABP) family function as intracellular transporters of long-chain fatty acids and other hydrophobic molecules to different cellular compartments. Brain FABP (FABP7) exhibits ligand-directed differences in cellular transport. For example, when FABP7 binds to docosahexaenoic acid (DHA), the complex relocates to the nucleus and influences transcriptional activity, whereas FABP7 bound with monosaturated fatty acids remains in the cytosol. Preferential binding of FABP7 to polyunsaturated fatty acids like DHA has been previously observed and is thought to play a role in differential localization. However, we find that at 37°C, FABP7 does not display strong selectivity, suggesting that the conformational ensemble of FABP7 and its perturbation upon binding may be important. We use molecular dynamics simulations, NMR, and a variety of biophysical techniques to better understand the conformational ensemble of FABP7, how it is perturbed by fatty acid binding, and how this may be related to ligand-directed transport. We find that FABP7 has high degree of conformational heterogeneity that is substantially reduced upon ligand binding. We also observe substantial heterogeneity in ligand binding poses, which is consistent with our finding that ligand binding is resistant to mutations in key polar residues in the binding pocket. Our NMR experiments show that DHA binding leads to chemical shift perturbations in residues near the nuclear localization signal, which may point toward a mechanism of differential transport.

A multisynaptic pathway from the ventral midbrain toward spinal motoneurons in monkeys.

Motivation boosts motor performance. Activity of the ventral midbrain (VM), consisting of the ventral tegmental area (VTA), the substantia nigra pars compacta (SNc) and the retrorubral field (RRF), plays an important role in processing motivation. However, little is known about the neural substrate bridging the VM and the spinal motor output. We hypothesized that the VM might exert a modulatory influence over the descending motor pathways. By retrograde transneuronal labelling with rabies virus, we demonstrated the existence of multisynaptic projections from the VM to the cervical enlargement in monkeys. The distribution pattern of spinal projection neurons in the VM exhibited a caudorostral gradient, in that the RRF and the caudal part of the SNc contained more retrogradely labelled neurons than the VTA and the rostral part of the SNc. Electrical stimulation of the VM induced muscle responses in the contralateral forelimb with a delay of a few milliseconds following the responses of the ipsilateral primary motor cortex (M1). The magnitude and number of evoked muscle responses were associated with the stimulus intensity and number of pulses. The muscle responses were diminished during M1 inactivation. Thus, the present study has identified a multisynaptic VM-spinal pathway that is mediated, at least in part, by the M1 and might play a pivotal role in modulatory control of the spinal motor output. KEY POINTS: Motivation to obtain reward is thought to boost motor performance, and activity in the ventral midbrain is important to the motivational process. Little is known about a neural substrate bridging the ventral midbrain and the spinal motor output. Retrograde trans-synaptic experiments revealed that the ventral midbrain projects multisynaptically to the spinal cord in macaque monkeys. Ventral midbrain activation by electrical stimulation generated cortical activity in the motor cortex and forelimb muscle activity. A multisynaptic ventral midbrain-spinal pathway most probably plays a pivotal role in modulatory control of the spinal motor output.

Synthesis of Tetrahydrofuro[2,3-d]oxazoles and Oxazoles by Hypervalent Iodine (III)-Promoted [2 + 2 + 1] Annulation.

Reaction of a hypervalent iodine reagent with bistriflimide efficiently promotes three-component regioselective cyclization of tetrahydrofuro[2,3-d]oxazoles and oxazoles from homopropargyl alcohols bearing a phenyl group, with different substituents on the aryl alkyne compounds affecting the selectivity of the resulting product. Utilizing the hydroxyethyl oxazole derivatives obtained in this research could aid in the development of various peroxisome proliferator-activated receptor agonist derivatives.

Post-kidney transplant soluble Klotho levels are determined by pretransplant soluble Klotho levels in both living donors and recipients.

BACKGROUND: Soluble Klotho (sKl), the free form of membrane-bound Klotho predominantly expressed in the kidney, is detectable in serum and may have multiple pleiotropic effects. Patients with end-stage kidney disease are possibly sKl deficient, and kidney transplantation is the treatment of choice in these patients; however, little is known about changes in posttransplant sKl level and the factors influencing these changes. METHODS: We conducted a prospective longitudinal study to examine changes in posttransplant sKl level in recipients for 12 months after living-donor kidney transplantation and analyzed correlations between posttransplant changes in sKl levels and various influencing factors in both recipients and donors. RESULTS: 29 kidney transplant recipients and their living donors were included for analysis. The results showed that sKl levels transiently decreased at 1 week posttransplant but progressively increased thereafter for 12 months. Multivariable linear regression analysis showed that body surface area-adjusted donor sKl levels were associated with posttransplant increases in recipient sKl levels at 12 months. In addition, pretransplant recipient sKl levels and body surface area-adjusted donor sKl levels were identified as an independent predictor of 12-month posttransplant sKl levels. CONCLUSION: Pretransplant sKl levels in both kidney recipients and living donors are a strong determinant of sKl levels after kidney transplantation.

Construction of 7-Diethylaminocoumarins Promoted by an Electron-Withdrawing Group.

The coumarin skeleton has been a focus of attention for many years, and its fluorescence properties vary depending on the substituents. Fluorescent coumarin derivatives are useful tools for many strategies have been developed for their synthesis. Although 7-diethylaminocoumarin has excellent fluorescence properties, it is unstable. We have developed a facile strategy for the synthesis of 7-diethylaminocoumarin derivatives by increasing the electrophilicity of the ynone moiety to promote nucleophilic addition reactions and cyclization. The reaction tolerates a variety of substitutions at the 4-position.

Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay.

Peroxisome proliferator-activated receptor γ (PPARγ) is a molecular target of metabolic syndrome and inflammatory disease. PPARγ is an important nuclear receptor and numerous PPARγ ligands were developed to date; thus, efficient assay methods are important. Here, we investigated the incorporation of 7-diethylamino coumarin into the PPARγ agonist rosiglitazone and used the compound in a binding assay for PPARγ. PPARγ-ligand-incorporated 7-methoxycoumarin, 1, showed weak fluorescence intensity in a previous report. We synthesized PPARγ-ligand-incorporating coumarin, 2, in this report, and it enhanced the fluorescence intensity. The PPARγ ligand 2 maintained the rosiglitazone activity. The obtained partial agonist 6 appeared to act through a novel mechanism. The fluorescence intensity of 2 and 6 increased by binding to the ligand binding domain (LBD) of PPARγ and the affinity of reported PPARγ ligands were evaluated using the probe.

Skeletal and mineral metabolic effects of risedronate in a rat model of high-turnover renal osteodystrophy.

INTRODUCTION: High-turnover bone disease is a major consequence of SHPT and may explain the high risk for fracture in patients with advanced chronic kidney disease (CKD). Bisphosphonates suppress bone turnover and improve bone strength, but their effects have not been fully characterized in advanced CKD with severe SHPT. Bisphosphonates also increase 1,25-dihydroxyvitamin D levels in normal and uremic rats, but the underlying mechanism remains to be determined. MATERIALS AND METHODS: We investigated the skeletal and mineral metabolic effects of RIS, a pyridinyl bisphosphonate, in rats with severe SHPT induced by 5/6 nephrectomy plus a high phosphate diet. RESULTS: Nephrectomized rats developed severe SHPT, along with hyperphosphatemia, low 1,25-dihydroxyvitamin D, and markedly increased FGF23. Moreover, these rats exhibited characteristic features of high-turnover renal osteodystrophy, including increased indices of trabecular bone turnover, decreased cortical bone thickness, inferior cortical biomechanical properties, and a prominent increase in peritrabecular fibrosis. RIS treatment increased bone volume and partially attenuated trabecular bone remodeling, cortical bone loss, and mechanical properties, whereas it produced a marked improvement in peritrabecular fibrosis along with a corresponding decrease in osteogenic gene markers. RIS treatment also suppressed the elevation of FGF23, which was associated with increased 1,25-dihydroxyvitamin D. CONCLUSIONS: In a rat model of severe SHPT, treatment with RIS partially attenuated histological manifestations of high-turnover bone disease. RIS treatment also suppressed the elevation of FGF23, which may explain the increased 1,25-dihydroxyvitamin D production during the treatment.

Preventive effect of early introduction of everolimus and reduced-exposure tacrolimus on renal interstitial fibrosis in de novo living-donor renal transplant recipients.

BACKGROUND: To improve the long-term outcomes following renal transplantation, prevention of renal-allograft interstitial fibrosis (IF), mainly due to calcineurin inhibitors, is an important therapeutic target. Everolimus (EVR) was reported to have antifibrotic effects. We aimed to investigate the safety, efficacy, and IF of our modified immunosuppressive regimen, which includes early introduction of EVR and reduced-exposure tacrolimus (Tac) (EVR group), and compare it with the standard-exposure tacrolimus-based regimen (Tac group) in de novo living-donor renal recipients. METHODS: In this retrospective, single-center cohort study, we compared the 2-year clinical courses between the two groups according to intention to treat. Additionally, in patients in whom biopsies were obtained at 1 h, 3 months, and 12 months post-transplant, we compared IF between the groups using imaging analysis. RESULTS: Overall, 47 patients were included (EVR group, n = 22; Tac group, n = 25). There were no significant differences in renal function and incidences of rejection and viral infections between the groups at the 2-year post-transplant follow-up. However, pathologic imaging analysis (n = 34) revealed chronological progression of IF in the Tac group during the first year post-transplant and no changes in the EVR group (fibrosis rate at 3 months: 20.8 vs. 13.6%, p < 0.001; at 12 months: 24.7 vs. 14.7%, p < 0.001, respectively). CONCLUSION: Our modified immunosuppressive regimen may have an antifibrotic effect on transplanted kidneys without loss of safety and efficacy.

Correction to: Preventive effect of early introduction of everolimus and reduced­exposure tacrolimus on renal interstitial fibrosis in de novo living­donor renal transplant recipients.

In the Original publication of the article, the co-author name has been misspelled as "Yousuke Nakagawa".

Calmodulin as a protein linker and a regulator of adaptor/scaffold proteins.

Calmodulin (CaM) is a universal regulator for a huge number of proteins in all eukaryotic cells. Best known is its function as a calcium-dependent modulator of the activity of enzymes, such as protein kinases and phosphatases, as well as other signaling proteins including membrane receptors, channels and structural proteins. However, less well known is the fact that CaM can also function as a Ca2+-dependent adaptor protein, either by bridging between different domains of the same protein or by linking two identical or different target proteins together. These activities are possible due to the fact that CaM contains two independently-folded Ca2+ binding lobes that are able to interact differentially and to some degree separately with targets proteins. In addition, CaM can interact with and regulates several proteins that function exclusively as adaptors. This review provides an overview over our present knowledge concerning the structural and functional aspects of the role of CaM as an adaptor protein and as a regulator of known adaptor/scaffold proteins.

Metal Microcapsules Prepared via Electroless Plating at Liquid-Liquid Interface.

We report the facile preparation of metal microcapsules via the formation of a Pickering emulsion, stabilized by catalytic palladium nanoparticles (PdNPs), and subsequent electroless plating at an oil-water interface induced by the adsorbed PdNPs. Metal microcapsules with smooth metal shells are formed by simply agitating the mixture of the plating solution and 1,2-dichloroethane containing poly(vinylpyrrolidone)-coated PdNPs, without external heating or electrical sources as energy inputs. We found that the metal microcapsules had thin copper metal shells with a relatively smooth surface and metallic luster. The metal shells were composed of more than 99 atom % copper in the form of Cu and Cu2O. We believe that this simple metal microcapsule preparation method may be beneficial to produce novel functional microcapsules with metal shells.

Construction of a series of intermediates in the ß-oxidation pathway from THA to EPA via DHA in free acid form.

ß-Oxidation of most fatty acids occurs in the mitochondria. However, ß-oxidation for ω-3 polyunsaturated fatty acids (PUFAs) is distinct from abundant fatty acids and occurs in the peroxisomes. Since little is known about peroxisomal ß-oxidation, here we report the synthesis of proposed intermediates of ω-3 PUFA ß-oxidation steps in free fatty acid form having a conjugated double bond, a ß-hydroxyl group, a ß-olefin and a ß-carbonyl group. These fatty acids can serve as authentic samples for biological experiments.

Uterine rupture due to placenta percreta in the first trimester of a pregnancy subsequent to a transverse uterine fundal cesarean section: A case report.

Transverse uterine fundal cesarean section in cases of total placenta previa reduces blood loss, but its influence on subsequent pregnancies, including the uterine rupture risk, remains unclear. We report a case of uterine rupture due to placenta percreta in the first trimester in a 43-year-old woman who underwent transverse uterine fundal incision in a previous pregnancy (at 40 years old). The patient did not undergo assessment of the uterine scare after the previous operation. Oocyte donation and in vitro fertilization at another institution resulted in the current pregnancy. At 11 weeks 3 days, she was admitted to the emergency department because of sudden severe abdominal pain. Ultrasound showed massive accumulation of free fluid in the peritoneal cavity and the fetus was outside the uterine cavity; uterine rupture was diagnosed. During emergency laparotomy, the uterine rupture was detected at exactly the previous incision site; a total hysterectomy was performed. Pregnancy after a transverse uterine fundal cesarean section is at high risk. As uterine scar dehiscence might have caused the uterine rupture, wounds should be evaluated before allowing subsequent pregnancies.

Recombinant expression, antimicrobial activity and mechanism of action of tritrpticin analogs containing fluoro-tryptophan residues.

The increase in antibiotic-resistant bacterial infections has prompted significant academic research into new therapeutic agents targeted against these pathogens. Antimicrobial peptides (AMPs) appear as promising candidates, due their potent antimicrobial activity and their ubiquitous presence in almost all organisms. Tritrpticin is a member of this family of peptides and has been shown to exert a strong antimicrobial activity against several bacterial strains. Tritrpticin's main structural characteristic is the presence of three consecutive Trp residues at the center of the peptide. These residues play an important role in the activity of tritrpticin against Escherichia coli. In this work, a recombinant version of tritrpticin was produced in E. coli using calmodulin as a fusion protein expression tag to overcome the toxicity of the peptide. When used in combination with glyphosate, an inhibitor of the endogenous synthesis of aromatic amino acids, this expression system allowed for the incorporation of fluorinated Trp analogs at very high levels (>90%). The antimicrobial activity of the 4-, 5- and 6-fluoro-Trp-containing tritrpticins against E. coli was as strong as the activity of the native peptide. Similarly, the tritrpticin analogs exhibited comparable abilities to perturb and permeabilize synthetic lipid bilayers as well as the outer and inner membrane of E. coli. Furthermore, the use of 19F NMR spectroscopy established that each individual fluoro-Trp residue interacts differently with SDS micelles, supporting the idea that each Trp in the original tritrpticin plays a different role in the perturbing/permeabilizing activity of the peptide. Moreover, our work demonstrates that the use of fluoro-Trp in solvent perturbation 19F NMR experiments provides detailed site-specific information on the insertion of the Trp residues in biological membrane mimetics. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation.

BACKGROUND: The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated. RESULTS: Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in K D value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium. CONCLUSIONS: Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.

Indium-Catalyzed Regioselective ß-Alkylation of Pyrroles with Carbonyl Compounds and Hydrosilanes and Its Application to Construction of a Quaternary Carbon Center with a ß-Pyrrolyl Group.

Treatment of N-substituted pyrroles with carbonyl compounds and nucleophiles under indium catalysis was found to be a promising method for preparing ß-alkylpyrroles without contamination by α-alkylpyrroles. With this methodology, a variety of alkyl groups, which are primary, secondary, and tertiary as well as cyclic and functionalized types, can be introduced in place onto the pyrrole ring. The simplicity performable as a catalytic one-step process is one of the important features of this reaction. The substituent on the nitrogen atom of the product ß-alkylpyrrole can be removed easily by literature procedures. Therefore, the indium-catalyzed ß-alkylation plus the N-deprotection is a powerful system for all six variations, which are N-substituted and N-unsubstituted ß-alkylpyrroles having primary, secondary, and tertiary alkyl groups. Our method is applicable to synthesizing, albeit in two steps, ß-pyrrolyl-group-connected unsymmetrical tetraarylmethanes that have not been addressed thus far. Mechanistic studies showed the following three aspects: (1) dipyrrolylalkanes produced in situ from the pyrrole and carbonyl compound are key intermediates, (2) the selective ß-alkylation is attributed to the selective elimination of an α-pyrrolyl group from the dipyrrolylalkane intermediates, and (3) the indium Lewis acid catalyst is indispensable for the progress of both stages.

Pyoderma gangrenosum in a pregnant patient: A case report and literature review.

Pyoderma gangrenosum (PG) is a rare ulcerative skin disease that usually starts as a pustular lesion and rapidly progresses to a painful ulcer with undermined violaceous borders. The occurrence of PG during pregnancy is uncommon. We describe a case of a pregnant patient with PG who was diagnosed as having ulcerative colitis after delivery. Obstetricians need to understand the pathogenesis of PG and its associated conditions because it is important to make a proper diagnosis and provide targeted therapy.

[Pulmonary Mycobacterium Avium-Complex (MAC) Disease Differentially Diagnosed from Metastasis of Testicular Cancer : A Case Report].

A 45 year-old-man was admitted to our hospital because of discomfort in his left scrotum. He had a left testicular tumor. We performed high orchiectomy and pathological findings revealed testicular cancer. He was treated with bleomycin, etoposide and cisplatin. Computed tomography showed a new mass in the left lung after 3 cycles of the chemotherapy. Because of its rapid growth, the tumor was thought to be a metastasis lesion of testicular cancer or pulmonary infection. Transbronchial lung biopsy showed an invasion of multinucleated giant cells and granuloma. The culture and polymerase chain reaction of the bronchial sputum were positive for myobacterium avium-complex (MAC). From these findings, the left lung tumor was diagnosed as pulmonary MAC disease. He received partial resection of the left lung and the lesion was diagnosed as granuloma. There was no recurrence of testicular cancer or pulmonary disease after the surgery.