Identification of a psychiatric risk gene NISCH at 3p21.1 GWAS locus mediating dendritic spine morphogenesis and cognitive function.

BACKGROUND: Schizophrenia and bipolar disorder (BD) are believed to share clinical symptoms, genetic risk, etiological factors, and pathogenic mechanisms. We previously reported that single nucleotide polymorphisms spanning chromosome 3p21.1 showed significant associations with both schizophrenia and BD, and a risk SNP rs2251219 was in linkage disequilibrium with a human specific Alu polymorphism rs71052682, which showed enhancer effects on transcriptional activities using luciferase reporter assays in U251 and U87MG cells. METHODS: CRISPR/Cas9-directed genome editing, real-time quantitative PCR, and public Hi-C data were utilized to investigate the correlation between the Alu polymorphism rs71052682 and NISCH. Primary neuronal culture, immunofluorescence staining, co-immunoprecipitation, lentiviral vector production, intracranial stereotaxic injection, behavioral assessment, and drug treatment were used to examine the physiological impacts of Nischarin (encoded by NISCH). RESULTS: Deleting the Alu sequence in U251 and U87MG cells reduced mRNA expression of NISCH, the gene locates 180 kb from rs71052682, and Hi-C data in brain tissues confirmed the extensive chromatin contacts. These data suggested that the genetic risk of schizophrenia and BD predicted elevated NISCH expression, which was also consistent with the observed higher NISCH mRNA levels in the brain tissues from psychiatric patients compared with controls. We then found that overexpression of NISCH resulted in a significantly decreased density of mushroom dendritic spines with a simultaneously increased density of thin dendritic spines in primary cultured neurons. Intriguingly, elevated expression of this gene in mice also led to impaired spatial working memory in the Y-maze. Given that Nischarin is the target of anti-hypertensive agents clonidine and tizanidine, which have shown therapeutic effects in patients with schizophrenia and patients with BD in preliminary clinical trials, we demonstrated that treatment with those antihypertensive drugs could reduce NISCH mRNA expression and rescue the impaired working memory in mice. CONCLUSIONS: We identify a psychiatric risk gene NISCH at 3p21.1 GWAS locus influencing dendritic spine morphogenesis and cognitive function, and Nischarin may have potentials for future therapeutic development.

Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery.

Antidiabetic treatments aiming to reduce body weight are currently gaining increased interest. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist administered twice daily via s.c. injection, improves glycemic control, often with associated weight reduction. To further improve the therapeutic efficacy of exendin-4, we have developed a novel peptide engineering strategy that incorporates a serum protein binding motif onto a covalent side-chain staple and applied to the peptide to enhance its helicity and, as a consequence, its potency and serum half-life. We demonstrated that one of the resulting peptides, E6, has significantly improved half-life and glucose tolerance in an oral glucose tolerance test in rodents. Chronic treatment of E6 significantly decreased body weight and fasting blood glucose, improved lipid metabolism, and also reduced hepatic steatosis in diet-induced obese mice. Moreover, the high potency of E6 allowed us to administer this peptide using a dissolvable microstructure-based transdermal delivery system. Pharmacokinetic and pharmacodynamic studies in guinea pigs showed that a single 5-min application of a microstructure system containing E6 significantly improved glucose tolerance for 96 h. This delivery strategy may offer an effective and patient-friendly alternative to currently marketed GLP-1 injectables and can likely be extended to other peptide hormones.

Warming-induced northwestward migration of the East Asian monsoon rain belt from the Last Glacial Maximum to the mid-Holocene.

Glacial-interglacial changes in the distribution of C3/C4 vegetation on the Chinese Loess Plateau have been related to East Asian summer monsoon intensity and position, and could provide insights into future changes caused by global warming. Here, we present δ(13)C records of bulk organic matter since the Last Glacial Maximum (LGM) from 21 loess sections across the Loess Plateau. The δ(13)C values (range: -25‰ to -16‰) increased gradually both from the LGM to the mid-Holocene in each section and from northwest to southeast in each time interval. During the LGM, C4 biomass increased from <5% in the northwest to 10-20% in the southeast, while during the mid-Holocene C4 vegetation increased throughout the Plateau, with estimated biomass increasing from 10% to 20% in the northwest to >40% in the southeast. The spatial pattern of C4 biomass in both the LGM and the mid-Holocene closely resembles that of modern warm-season precipitation, and thus can serve as a robust analog for the contemporary East Asian summer monsoon rain belt. Using the 10-20% isolines for C4 biomass in the cold LGM as a reference, we derived a minimum 300-km northwestward migration of the monsoon rain belt for the warm Holocene. Our results strongly support the prediction that Earth's thermal equator will move northward in a warmer world. The southward displacement of the monsoon rain belt and the drying trend observed during the last few decades in northern China will soon reverse as global warming continues.

Oxygen isotopes of East Asian dinosaurs reveal exceptionally cold Early Cretaceous climates.

Early Cretaceous vertebrate assemblages from East Asia and particularly the Jehol Biota of northeastern China flourished during a period of highly debated climatic history. While the unique characters of these continental faunas have been the subject of various speculations about their biogeographic history, little attention has been paid to their possible climatic causes. Here we address this question using the oxygen isotope composition of apatite phosphate (δ ) from various reptile remains recovered from China, Thailand, and Japan. δ values indicate that cold terrestrial climates prevailed at least in this part of Asia during the Barremian-early Albian interval. Estimated mean air temperatures of about 10 ± 4 °C at midlatitudes (∼ 42 °N) correspond to present day cool temperate climatic conditions. Such low temperatures are in agreement with previous reports of cold marine temperatures during this part of the Early Cretaceous, as well as with the widespread occurrence of the temperate fossil wood genus Xenoxylon and the absence of thermophilic reptiles such as crocodilians in northeastern China. The unique character of the Jehol Biota is thus not only the result of its evolutionary and biogeographical history but is also due to rather cold local climatic conditions linked to the paleolatitudinal position of northeastern China and global icehouse climates that prevailed during this part of the Early Cretaceous.

PEGylated iminodiacetic acid zinc complex stabilizes cationic RNA-bearing nanoparticles.

Self-assembling nanoparticles comprising cationic polymers are of interest for the delivery of oligonucleotide-based therapeutics. Unfortunately, exposure of the nanoparticle cationic surface to plasma and plasma proteins compromises particle stability and circulating half-life. Herein, we report that improved nanoparticle stability can be achieved through temporary grafting of PEG to the nanoparticle surface. Grafting is induced through zinc complexation between PEG-IDA and the exposed polyhistidylated polylysine (H-K) cationic polymer of pre-formed nanoparticles.

Stabilization and Transdermal Delivery of an Investigational Peptide Using MicroCor® Solid-State Dissolving Microstructure Arrays.

The formulation of biotherapeutics presents unique challenges especially with regard to physical and chemical stability and often requires refrigerated storage conditions of final drug products. Peptide A is an example of a developmental compound which showed significant stability challenges when prepared as a liquid formulation for a subcutaneous injection. The aim of the present study was to evaluate whether Peptide A can be successfully formulated in MicroCor® microstructure arrays (MSAs) as an alternative delivery option. MSAs contain a high density of dissolving microstructures allowing for transdermal delivery. In the present work, a 5600-needle MSA (~200 µm long microstructures, 2 cm2 array) was prepared with a therapeutically-relevant dose of Peptide A. The array was shown to be stable under room-temperature storage conditions for 3 months. On in vivo application to Yucatan minipigs, Peptide-A-loaded MSAs demonstrated only mild and transient skin irritation and a very high efficiency of peptide transfer from dissolving microstructures into the skin resulting in absolute bioavailability of 74%. This transdermal bioavailability was very similar to the 73% bioavailability obtained from a subcutaneous injection. This technical feasibility study demonstrated that MicroCor® technology represents a viable option for delivery of Peptide A with significant improvements in peptide stability.

Smart polymer-streptavidin conjugates.

The conjugation of stimuli-responsive, or "smart," polymers to streptavidin is described. The polymer is synthesized with a thiol-reactive end-group, which is used to end-graft the polymer to cysteine or lysine side-chains that are genetically engineered into controlled positions on the streptavidin surface. The conjugation positions are chosen on the basis of their location relative to the binding site, together with the criteria that they be solvent accessible and thus reactive. The polymer composition can be controlled to impart responsiveness to temperature, pH, and/or specific wavelengths of light. These signals are sent to the polymer, which serves as an antennae and actuator to gate biotin or biotinylated protein association with the streptavidin binding sites. The molecular switching and gating activity is directed by the reversible polymer transition between a hydrophilic, expanded coil and a more hydrophobic, collapsed state that is smaller in volume. The differences in the polymer steric properties serve to block or allow ligand access to the binding site. The control of polymer molecular weight is a particularly important design parameter for these molecular gates.

On-line measurements of delta(15)N in biological fluids by a modified continuous-flow elemental analyzer with an isotope-ratio mass spectrometer.

A modified continuous-flow elemental analyzer coupled to an isotope-ratio mass spectrometer (modified EA-IRMS) was tested for on-line delta(15)N measurement on urea solution and biological fluids (e.g. urine). The elemental analyzer configuration was adapted by adding a U-shaped cold trap and an X-pattern four-way valve for on-line trapping/venting of water from the liquid samples. Results indicate that the delta(15)N ratios show little variation (standard deviation (SD) = 0.05 per thousand) with a sample size above the equivalent N yield of 0.2 mg urea (0.092 mg N) when the mass spectrometer conditions were carefully optimized. By contrast, a significant logarithmic decrease in delta(15)N with sample size was observed but this can be offset by applying a linearity correction or blank correction when the sample size is between equivalent N yields of 0.05 and 0.2 mg urea. The blank corrected delta(15)N ratios give an overall precision of approximately 0.16 per thousand whereas the average precision for delta(15)N corrected using combined linearity and shift correction is 0.05 per thousand. The relatively large variation in blank corrected delta(15)N values may be attributed to the variability of the blank delta(15)N in the sequence. Therefore, the blank correction should be carefully performed in routine measurements. As a result, the linearity range of a modified EA-IRMS can be extended to a minimum sample size of 0.023 mg N. In addition, the reproducibility of the new system is good, as indicated by the precision (<0.2 per thousand) for a set of standards and unknowns. The data show that fluids containing nitrogen can be successfully analyzed in the modified EA-IRMS.

Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate.

A system has been developed for reversibly binding and thermoprecipitating biotinylated macromolecules. A high off-rate Ser45Ala (S45A) streptavidin mutant has been covalently conjugated to poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer. The resulting conjugate is shown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide in response to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be released from the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release step has been shown to be unique to the mutant streptavidin conjugate-a conjugate of wild type (WT) streptavidin and PNIPAAm does not release bound biotinylated molecules upon treatment with excess free biotin. The capture efficiency (fraction of target molecule precipitated from solution) of the S45A-PNIPAAm conjugate is similar to that of the WT-PNIPAAm conjugate for the biotinylated IgG target molecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAm conjugate). The release efficiency (fraction of originally precipitated target molecule released after treatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgG target and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the use of a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.

Photoswitching of ligand association with a photoresponsive polymer-protein conjugate.

Light-regulated molecular switches that reversibly control biomolecular function could provide new opportunities for controlling activity in diagnostics, affinity separations, bioprocessing, therapeutics, and bioelectronics applications. Here we show that site-specific conjugation of light-responsive polymers near the biotin-binding pocket of streptavidin provides control of ligand binding affinity in response to UV and visible light irradiation. Two different light-responsive polymers were utilized that display opposite photoresponsive solubility changes under UV or visible (vis) light irradiation in aqueous solutions. At 40 degrees C, the N,N-dimethylacrylamide (DMA)-co-4-phenylazophenyl acrylate (AZAA) copolymer (DMAA) was soluble under UV irradiation and precipitated under visible light, while the DMA-co-N-4-phenylazophenyl acrylamide (AZAAm) copolymer (DMAAm) was soluble under visible irradiation and precipitated under UV light. Both polymers were synthesized with a vinyl sulfone terminus and conjugated to the Glu116Cys (E116C) streptavidin mutant via thiol coupling. The DMAA-streptavidin conjugate bound biotin efficiently when the polymer was in the soluble state under UV irradiation, but under visible irradiation, the polymer collapsed and blocked free biotin association. Furthermore, if biotin was allowed to bind when the polymer was in the soluble state under UV irradiation, then when the polymer was collapsed by visible light, the streptavidin released the bound biotin. The DMAAm-streptavidin conjugate showed the opposite response, with association of biotin allowed under visible light irradiation and blocked under UV irradiation. The photoresponses of the streptavidin conjugates thus correspond to the original photoresponsive phase transition properties of the polymer switches triggered by the cis-trans isomerization of the diazo chromophores.

Affinity separation using an Fv antibody fragment-"smart" polymer conjugate.

Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.