Light distribution and thermal effects in the rat brain under optogenetic stimulation

Optical brain stimulation gained a lot of attention in neuroscience due to its superior cell‐type specificity. In the design of illumination strategies, predicting the light propagation in a specific tissue is essential and requires knowledge of the optical properties of that tissue. We present the estimated absorption and reduced scattering in rodent brain tissue using non‐destructive contact spatially resolved spectroscopy (cSRS). The obtained absorption and scattering in the cortex, hippocampus and striatum are similar, but lower than in the thalamus, leading to a less deep but broader light penetration profile in the thalamus. Next, the light distribution was investigated for different stimulation protocols relevant for fiber‐optic based optogenetic experiments, using Monte Carlo simulation. A protocol specific analysis is proposed to evaluate the potential of thermally induced side effects.

     

Early mechanistic events in biotin dissociation from streptavidin

The streptavidin-biotin system has provided a unique opportunity to investigate the molecular details of ligand dissociation pathways. An underlying mechanistic question is whether ligand dissociation proceeds with a relatively ordered process of bond breaking and ligand escape. Here we report a joint computational and crystallographic study of the earliest events in biotin dissociation. In molecular dynamics potential of mean force simulations, a water molecule from a defined access channel intercalated into the hydrogen bond between Asp 128 and biotin, bridging them and stabilizing an intermediate state. In forced biotin dissociation simulations, this event led to subsequent bond breaking steps and ligand escape. In equilibrium simulations, the water molecule was sometimes observed to move back to the access channel with re-formation of the biotin hydrogen bond. Analysis of streptavidin crystal structures revealed a close overlap of crystallographically defined and simulated waters in the water access channel. These results suggest that biotin dissociation is initiated by stochastic coupling of water entry with lengthening of a specific biotin hydrogen-bonding interaction.     

Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms

Cholesterol 7α-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRα by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1α plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA.     

New prenylated isoflavonoids as protein tyrosine phosphatase 1B (PTP1B) inhibitors from Erythrina addisoniae

Bioassay-guided fractionation of the EtOAc extract of the root of Erythrina addisoniae (Leguminosae) resulted in the isolation of four new (1–4), along with 2 known prenylated isoflavonoids (5–6). The structures of the isolates were assigned on the basis of spectroscopic data analysis, focusing on interpretation of 1D and 2D NMR, and MS data. All the isolates were evaluated for their inhibitory effects on protein tyrosine phosphatase 1B (PTP1B), as well as their growth inhibition on MCF7, adriamycin-resistant MCF7 (MCF7/ADR), and MDA-MB-231 breast cancer cell lines. Compounds which exhibited PTP1B inhibitory activity (IC₅₀ values ranging from 4.6 ± 0.3 to 24.2 ± 2.1 μM) showed potential cytotoxic activity (IC₅₀ values ranging from 3.97 ± 0.17 to 11.4 ± 1.9 μM). Taken together, our data suggest that prenylated isoflavonoids, especially the isoflavone-type skeleton could be considered as new lead compounds against breast cancer via PTP1B inhibition.     

Palbinone and triterpenes from Moutan Cortex (Paeonia suffruticosa,Paeoniaceae) stimulate glucose uptake and glycogen synthesis via activation of AMPK in insulin-resistant human HepG2 Cells

Moutan Cortex is a well-known herb in traditional Korean, Chinese, and Japanese anti-diabetic formulae. In the current study, we investigated the metabolic effects of isolated triterpenes (1–7) in HepG2 cells under high glucose conditions. These compounds remakably stimulated AMP-activated protein kinase (AMPK), GSK-3β, and ACC phosphorylation. The compounds also increased glucose uptake and enhanced glycogen synthesis. Among these, compound 1 displayed the greatest potential anti-diabetic activity though the AMPK activation pathway. Compound 1 significantly increased the levels of phospho-AMPK, phospho-ACC, and phospho-GSK-3β and stimulated glucose uptake and glycogen synthesis in a dose-dependent manner. In conclusion, our results suggest that these compounds, especially compound 1, may have beneficial roles in glucose metabolism via the AMPK pathway.     

A new species of the genus Leptolalax(Anura:Megophryidae) from southern Vietnam

We describe a new species of megophryid frog from Phu Yen Province in southern Vietnam. Leptolalax macrops sp. nov. is distinguished from its congeners by a combination of the following morphological attributes:(1) body size medium(SVL 28.0–29.3 mm in three adult males, 30.3 mm in single adult female);(2) supra-axillary glands present, creamy white; ventrolateral glands indistinct;(3) tympanum externally distinct;(4) dorsal skin roughly granular with larger tubercles, dermal ridges on dorsum absent;(5) rudimentary webbing present between fingers Ⅰ–Ⅱ and Ⅱ–Ⅲ; rudimentary webbing between all toes; fingers and toes without dermal fringes;(6) in life ventral surface greyish-violet with white speckling;(7) supratympanic fold distinct, dark brown in life;(8) iris bicolored, typically golden in upper half, fading to golden green in lower half;(9) tibia short(TbL/SVL 0.44–0.45 in males); and(10) eyes large and protuberant(ED/SVL 0.15–0.16 in males).From all congeners for which comparable sequences are available, the new species differs markedly in the 16 S rRNA mitochondrial gene sequence(P-distance5.7%). The new species is currently known only from montane evergreen tropical forests of Song Hinh District, Phu Yen Province, and M'Drak District of Dak Lak Province at elevations of 470–630 m a.s.l.. We suggest the new species should be considered as Data Deficient following the IUCN's Red List categories. We also report a previously unknown Leptolalax mt DNA lineage from an evergreen tropical forest in the Hoa Thinh District of Phu Yen Province,which may also represent an undescribed species.     

Topical application of ALK5 inhibitor A-83-01 reduces burn wound contraction in rats by suppressing myofibroblast population

Burn scar contracture that follows the healing of deep dermal burns causes severe deformation and functional impairment. However, its current therapeutic interventions are limited with unsatisfactory outcomes. When we treated deep second-degree burns in rat skin with activin-like kinase 5 (ALK5) inhibitor A-83-01, it reduced wound contraction and enhanced the area of re-epithelialization so that the overall time for wound closing was not altered. In addition, it reduced myofibroblast population in the dermis of burn scar with a diminished deposition of its biomarker proteins such as α-SMA and collagen. Treatment of rat dermal fibroblast with A-83-01 inhibited transforming growth factor-β1 (TGF-β1)-dependent induction of α-SMA and collagen type I. Taken together, these results suggest that topical application of ALK5 inhibitor A-83-01 could be effective in preventing the contraction of burn wound without delaying the wound closure by virtue of its inhibitory activity against the TGF-β-induced increase of myofibroblast population.     

A Simple,Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

The human tryptase locus on chromosome 16 contains one gene encoding only β-tryptase and another encoding either β-tryptase or the homologous α-tryptase, providing α:β gene ratios of 0∶4, 1∶3 or 2∶2 in the diploid genome, these genotypes being of potential clinical relevance in severe atopy. Using an EcoRV restriction site in α- but not β- tryptase, PCR products, spanning intron 1 to exon 5, were used to determine α/β-tryptase gene ratios using non-radioactive labels, including ethidium bromide labeling of all PCR products, and either digoxigenin-primer or DY682-primer labeling of only the final PCR cycle products. Sensitivity increased ∼60-fold with each final PCR cycle labeling technique. Ethidium bromide labeling underestimated amounts of α-tryptase, presumably because heteroduplexes of α/β-tryptase amplimers, formed during annealing, were EcoRV resistant. In contrast, both final PCR cycle labeling techniques precisely quantified these gene ratios, because only homoduplexes were labeled. Using the DY682-primer was most efficient, because PCR/EcoRV products could be analyzed directly in the gel; while digoxigenin-labeled products required transfer to a nitrocellulose membrane followed by immunoblotting. This technique for determining the α/β-tryptase genotype is sensitive, accurate, simple and safe, and should permit high-throughput screening to detect potential phenotype-genotype relations for α/β-tryptases, and for other closely related alleles.     

Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 degrees C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium deltaG degrees, deltaH degrees, deltaS degrees, deltaC degrees(p), and activation deltaG++ of S45A at 37 degrees C are 13.7+/-0.1 kcal/mol, -21.1+/-0.5 kcal/mol, -23.7+/-1.8 cal/mol K, -223+/-12 cal/mol K, and 20.0+/-2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the deltaH++ and deltaS++ of dissociation, 25.8+/-1.2 kcal/mol and 18.7+/-4.3 cal/mol K. The large increases of deltaH++ and deltaS++ in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.