De Novo Screening and Mirror Image Isomerization of Linear Peptides Targeting α7 Nicotinic Acetylcholine Receptor.

As ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) are widely distributed in the central and peripheral nervous systems and are associated with the pathogenesis of various degenerative neurological diseases. Here, we report the results of phage display-based de novo screening of an 11-residue linear peptide (named LKP1794) that targets the α7 nAChR, which is among the most abundant nAChR subtypes in the brain. Moreover, two d-peptides were generated through mirror image and/or primary sequence inverso isomerization (termed DRKP1794 and DKP1794) and displayed improved inhibitory effects (IC50 = 0.86 and 0.35 µM, respectively) on α7 nAChR compared with the parent l-peptide LKP1794 (IC50 = 2.48 µM), which markedly enhanced serum stability. A peptide-based fluorescence probe was developed using proteolytically resistant DKP1794 to specifically image the α7 nAChR in living cells. This work provides a new peptide tool to achieve inhibitory modulation and specifically image the α7 nAChR.

First report of Black Spot on Eucalyptus grandis × Eucalyptus urophylla caused by Pseudoplagiostoma eucalypti in Guangxi, China.

Eucalyptus grandis × Eucalyptus urophylla hybrid clone is an economically and ecologically important forest variety and is widely planted in Guangxi, China. Black spot, a newly found disease, occurred nearly 5333.3 hectares in an E. grandis × E. urophylla plantation of Qinlian forest farm (N: 21.866°, E: 108.921°) in Guangxi in October, 2019. Infected plants had lesions of black spots with watery margins on petioles and veins of E. grandis × E. urophylla. The size of spots ranged between 3 to 5 mm in diameter. When lesions expanded to girdle the petioles, wilt and death of leaves was observed, which subsequently affected growth of the trees. To isolate the causal agent, symptomatic plant tissues (leaves and petioles) were collected from two different sites, sampled from five plants each site. In the lab, infected tissues were surface sterilized with 75% ethanol for 10 seconds, then 2% sodium hypochlorite for 120 seconds, and rinsed with sterile distilled water three times. Small segments (5×5 mm) were cut from the margins of the lesions, then placed on potato dextrose agar (PDA) plates. The plates were incubated at 26°C in dark for 7 to 10 days. Fungal isolates YJ1 and YM6 with a similar morphology, which were obtained from 14 of 60 petioles and 19 of 60 veins respectively. These two colonies were initially light orange, then turned to olive brown as time progressed. Conidia were hyaline, smooth, aseptate, ellipsoidal, apex obtuse, and base tapering to flat protruding scar, 16.8 to 26.5µm long, and 6.6 to 10.4 µm wide (n=50). Some conidia had one or two guttules. The morphological characteristics were consistent with the description of Pseudoplagiostoma eucalypti Cheew., M. J. Wingf. & Crous (Cheewangkoon et al. 2010). For molecular identification, the internal transcribed spacer (ITS), ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 and T1/Bt2b, respectively (White et al. 1990; O'Donnell et al.1998; Glass and Donaldson 1995). Sequences of the two strains were deposited in GenBank (ITS: MT801070 and MT801071; BT2: MT829072 and MT829073). Phylogenetic tree was constructed with a maximum likelihood method, revealing that YJ1 and YM6 were on the same branch with P. eucalypti. Pathogenicity tests of the two strains were performed on three-month-old E. grandis × E. urophylla seedlings, by inoculating 6 wounded (by stabbing on petioles or veins) leaves of seedlings with mycelial PDA plugs (5 ×5 mm) from the edge of a 10-day old colony of strain YJ1 or YM6. Another 6 leaves were treated in the same manner but with PDA plugs as controls. All treatments were incubated in humidity chambers at 27°C and 80% relative humidity, under ambient light. All experiments were conducted three times. Lesions were observed at the points of inoculation, the petioles or veins turned black on inoculated leaves after 7 days, wilting of the leaves were also observed after 30 days, however the controls remained asymptomatic. Re-isolation was made and the fungus had same morphological measurements as the inoculated fungus, thus completing Koch's postulates. P. eucalypti had been reported as a pathogen of leaf spot on E. robusta in Taiwan island (Wang et al. 2016), leaf and shoot blight on E. pulverulenta in Japan (Inuma et al. 2015). To our knowledge, this is the first report of P. eucalypti affecting E. grandis × E. urophylla in mainland China. This report provides basis for the rational prevention and control of this new disease in the cultivation process of E. grandis × E. urophylla.

Total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates using side-chain Boc/tBu protecting groups.

A practical strategy for the total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates was developed. In this strategy, the Boc/tBu protecting groups are utilized for the side chains of Trp, His, Arg, Asp, and Glu, and is deprotected in borate buffer at 90 °C to avoid depurination of the oligonucleotide caused by strong acid treatment. The advantage of this strategy is that the abovementioned amino acids are readily available in the market and the side reaction of deguanidination of the Arg residue can be avoided. This side-chain Boc/tBu protection strategy will expand the applicability of total stepwise synthesis in the preparation of peptide-oligonucleotide conjugates.

The New Salicylaldehyde S,S-Propanedithioacetal Ester Enables N-to-C Sequential Native Chemical Ligation and Ser/Thr Ligation for Chemical Protein Synthesis.

The combination of distinct peptide ligation techniques to facilitate chemical protein synthesis represents one of the long-standing goals in the field. A new combination ligation method of N-to-C sequential native chemical ligation and Ser/Thr ligation (NCL-STL) is described for the first time. This method relies on the peptide salicylaldehyde S,S-propanedithioacetal (SALPDT)-ester prepared by a new 1,3-propanedithiol-mediated reaction. The peptide SALPDT-ester, which is compatible with NCL, can be fully activated by N-chlorosuccinimide (NCS)/AgNO3 in aqueous solution to afford peptide SAL-ester for use in the subsequent STL. The practicality of the combined NCL-STL method is illustrated by the synthesis of S-palmitoylated matrix-2 (S-palm M2) ion channel from Influenza A virus and S-palmitoylated interferon-induced transmembrane protein 3 (S-palm IFITM3). This approach expands the multiple-segments peptide ligation toolkit for producing important and complex custom-made protein samples by chemical protein synthesis.

Chemical Synthesis of Native S-Palmitoylated Membrane Proteins through Removable-Backbone-Modification-Assisted Ser/Thr Ligation.

The preparation of native S-palmitoylated (S-palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S-palm membrane proteins by removable-backbone-modification-assisted Ser/Thr ligation (RBMGABA -assisted STL). This method involves two critical steps: 1) synthesis of S-palm peptides by a new γ-aminobutyric acid based RBM (RBMGABA ) strategy, and 2) ligation of the S-palm RBM-modified peptides to give the desired S-palm product by the STL method. The utility of the RBMGABA -assisted STL method was demonstrated by the synthesis of rabbit S-palm sarcolipin (SLN) and S-palm matrix-2 (M2) ion channel. The synthesis of S-palm membrane proteins highlights the importance of developing non-NCL methods for chemical protein synthesis.

Ginkgolides-loaded soybean phospholipid-stabilized nanosuspension with improved storage stability and in vivo bioavailability.

The purpose of this study was to investigate the effects of soybean phospholipid, as a steric stabilizer, on improving dissolution rate, storage stability and bioavailability of ginkgolides. The ginkgolides coarse powder, hydroxypropyl methylcellulose (HPMC), soybean phospholipid and sodium dodecyl sulfate (SDS) were mixed and wet-milled to prepare nanosuspension S1. Nanosuspension S2 was obtained by the same technique except adding the soybean phospholipid. Results of particle size showed that particle size (D50) of S1 significantly decreased from 44.25 µm to 0.373 µm. Results of differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and transmission electron microscope (TEM) showed that ginkgolides in nanosuspension still maintained its crystallinity, and the nanoparticles were all nearly circular and uniformly dispersed. Then, pellets F1 and F2 were prepared by layering S1 and S2 onto the microcrystalline cellulose (MCC) spheres, respectively. The dissolution rate of ginkgolide A (GA) and ginkgolide B (GB) in F1 was 98.3% and 97.7% in 30 min, respectively. It was much higher than F2 (89.0% and 86.5%) and coarse powder of ginkgolides (22.3% and 24.6%). According to the results of stability test, the storage stability of F1 was improved compared with F2. In addition, compared with coarse powder of ginkgolides, the relative bioavailability of GA and GB in F1 were up to (221.84 ±â€¯106.67) % and (437.45 ±â€¯336.43) %, respectively. The above results demonstrated that soybean phospholipid added to the nanosuspension played an important role in improving drug dissolution rate, storage stability and in vivo bioavailability: (1) The amphiphilic soybean phospholipid interacted with the drug, with the hydrophobic part adsorbed on the surface of the poorly soluble drug and the hydrophilic part exposed to the aqueous medium. This increases the wettability of the nanoparticles, which ensure a good redispersibility of the drug particles. (2) It could self-assemble to form an interfacial phospholipid film by surrounding the individual nanoparticles, which can produce enough steric hindrance to prevent nanoparticles from aggregation and ensure a rapid dissolution rate. (3) Soybean phospholipid and its hydrolysate formed strong micellar solubilizing vehicles with bile salts in vivo, stimulated the absorption process of ginkgolides. Thus, soybean phospholipid was a promising steric stabilizer in nanosuspension drug delivery system.

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase.

During the total chemical synthesis of the water-soluble globular Haemophilus Influenzae DNA ligase (Hin-Lig), we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 m guanidine hydrochloride. Conventional peptide performance-improving strategies, such as installation of a terminal/side-chain Arg tag or O-acyl isopeptide, failed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state, in addition to good peptide solubility, is a prerequisite for successful peptide ligation.

Interfacial molecular interactions based on the conformation recognition between the insoluble antitumor drug AD-1 and DSPC.

In this study, molecular interactions between the anti-cancer agent 20(R)-25-methoxyl-dammarane-3ß, 12ß, 20-triol (AD-1) and phospholipid 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) were investigated using the Langmuir film balance technique. The characteristics of binary Langmuir monolayers consisting of DSPC and AD-1 were conducted on the basis of the surface pressure-area per molecule (π-A) isotherms. It was found that the drug was able to become efficiently inserted into preformed DSPC monolayers, indicating a preferential interaction between AD-1 and DSPC. For the examined lateral pressure at 20mN/m, the largest negative values of ΔGex were found for the AD-1/DSPC monolayer, which should be the most stable. Based on the calculated values of ΔGex, we found that the AD-1/DSPC systems exhibited the best mixed characteristics when the molar fraction of the AD-1 was 0.8; at that relative concentration, the AD-1 molecules can mix better and interact with the phospholipid molecules. In addition, the drug-DSPC binary supramolecular structure was also deposited on the mica plates as shown by atomic force microscopy (AFM). Finally, molecular docking calculations explained satisfactorily that, based on the conformations interactions (conformation recognition), even at an AD-1/DSPC molar ratio as high as 8:2, the interfacial stabilization of the AD-1/DSPC system was fairly strong due to hydrophobic interactions. A higher loading capacity of DSPC might be possible, as it is associated with a more flexible geometrical environment, which allows these supramolecular structures to accept larger increases in drug loading upon steric binding.