Complete Organelle Genome of the Desiccation-Tolerant (DT) Moss Tortula atrovirens and Comparative Analysis of the Pottiaceae Family.

Tortula atrovirens (Sm.) Lindb. is an important component of biological soil crusts and possesses an extraordinary tolerance against desiccation in dryland habitats. However, knowledge of the organelle genome of this desiccation-tolerant (DT) moss is still lacking. Here, we assembled the first reported Tortula organelle genome and conducted a comprehensive analysis within the Pottiaceae family. T. atrovirens exhibited the second largest chloroplast genome (129,646 bp) within the Pottiaceae, whereas its mitogenome (105,877 bp) and those of other mosses were smaller in size compared to other land plants. The chloroplast and mitochondrial genomes of T. atrovirens were characterized by the expansion of IR boundaries and the absence of homologous recombination-mediated by large repeats. A total of 57 RNA editing sites were detected through mapping RNA-seq data. Moreover, the gene content and order were highly conserved among the Pottiaceae organelle genomes. Phylogenetic analysis showed that bryophytes are paraphyletic, with their three lineages (hornworts, mosses, and liverworts) and vascular plants forming successive sister clades. Timmiella anomala is clearly separated from the monophyletic Pottiaceae, and T. atrovirens is closely related to Syntrichia filaris within the Pottioideae. In addition, we detected four hypervariable regions for candidate-molecular markers. Our findings provide valuable insights into the organelle genomes of T. atrovirens and the evolutionary relationships within the Pottiaceae family, facilitating future discovery of DT genetic resources from bryophytes.

Fabrication of lignocellulose/liquid metal-based conductive eutectic hydrogel composite for strain sensors.

High conductive and freeze-resistant hydrogels with adhesion function are ideal candidates for soft electronic devices. However, it remains a challenge to design appropriate conductive nanofillers to endow hydrogels with all these characteristics. Liquid metal (LM) exhibits exceptional electrical conductivity and convenient processability, rendering it a highly promising contender. Cellulose nanofibrils (CNFs) were employed as the interfacial stabilizer in synthesizing stable CNFs encapsulated LM solutions. Then the lignin was further coated on the surface of CNFs-LM (LCL) to prepare lignin-coated hybrid hydrogels. The obtained LCL displayed outstanding water-dispersible stability and were promising conductive nanofillers for hydrogels. During the fabrication of poly N-(hydroxymethyl) acrylamide (PHA) hydrogels, the LM was dispersed into LM particles with smaller sizes, leading to highly conductive LCL-PHA hydrogels (0.38 S·m-1). The prepared LCL-PHA hydrogels exhibited exceptional mechanical properties, including a strain at a break of 134.6 %, stress at a break of 22.7 Kpa, and a toughness of 16.3 KJ·m-3. Additionally, the LCL-PHA hydrogels demonstrated favorable electrical conductivity and adhesion. Notably, even after being subjected to freezing at -20 °C for 24 h, they remained suitable for effective real-time monitoring of all types of human activities, demonstrating superior environmental stability.

A potent synthetic nanobody with broad-spectrum activity neutralizes SARS-CoV-2 virus and the Omicron variant BA.1 through a unique binding mode.

The major challenge to controlling the COVID pandemic is the rapid mutation rate of the SARS-CoV-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is essential to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-CoV-2 variants. Here, we report a synthetic nanobody (named C5G2) obtained by phage display and subsequent antibody engineering. C5G2 has a single-digit nanomolar binding affinity to the RBD domain and inhibits its binding to ACE2 with an IC50 of 3.7 nM. Pseudovirus assays indicated that monovalent C5G2 could protect the cells from infection with SARS-CoV-2 wild-type virus and most of the viruses of concern, i.e., Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against Omicron BA.1 among all the variants, with an IC50 of 4.9 ng/mL. The cryo-EM structure of C5G2 in complex with the spike trimer showed that C5G2 binds to RBD mainly through its CDR3 at a conserved region that does not overlap with the ACE2 binding surface. Additionally, C5G2 binds simultaneously to the neighboring NTD domain of the spike trimer through the same CDR3 loop, which may further increase its potency against viral infection. Third, the steric hindrance caused by FR2 of C5G2 could inhibit the binding of ACE2 to RBD as well. Thus, this triple-function nanobody may serve as an effective drug for prophylaxis and therapy against Omicron as well as future variants.

Engineering of Src Homology 2 Domain Leading to Sulfotyrosine Recognition With a High Affinity by Integrating a Distinctive Selection Theme and Next-Generation Sequencing.

Tyrosine sulfation plays a vital role in various biochemical reactions. Although sulfated tyrosine (sTyr) has a similar structure to phosphotyrosine (pTyr), the number of available sTyr sites is significantly less than that of pTyr sites, mainly because of the lack of effective sTyr probes. A few sTyr binders were identified on the basis of structural similarity by engineering the pTyr-binding pocket of an Src Homology 2 (SH2) domain through phage selections against sTyr peptides. Nevertheless, they still interact with pTyr peptides with comparable affinity. This study aims to identify sTyr superbinders using the SH2 domain as a template. We created a distinctive phage selection scheme that separately covered selections against sTyr and pTyr peptides, followed by next-generation sequencing (NGS). After selections, phage pools showed strong enzyme-linked immunosorbent assay (ELISA) signal intensities for both modified peptides, indicating that the variants evolved with a high affinity for these peptides, which causes difficulty in identifying sTyr-specific binders. In contrast, NGS data from selected pools showed significant differences, suggesting the enrichment of sTyr-specific variants during selections. Accordingly, we obtained the sTyr features based on NGS data analysis and prioritized a few potential sTyr binders. The variant SH2-4 showed a stronger affinity for sTyr than pTyr and was superior to previous sTyr binders as measured by the Biolayer Interferometry assay. In summary, we described the strategy of integrating NGS data mining with a novel selection scheme to identify sTyr superbinders.

Development in Detection Methods for the Expression of Surface-Displayed Proteins.

Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. A protein of interest is selected as the template and expressed on a molecular display platform such as a bacteriophage for engineering. Initially, the surface-displayed protein template needs to be checked against the desired target via ELISA to examine whether the functions of the displayed template remain intact. The ELISA signal is subject to the protein-target binding affinity. A low-affinity results in a weak ELISA signal which makes it difficult to determine whether the weak signal is because of low affinity or because of poor expression of the protein. Using a methyllysine-binding chromodomain protein Cbx1 that weakly binds to the histone H3K9me3 peptide, we developed and compared three different approaches to increase the signal-to-background ratio of ELISA measurements. We observed that the specific peptide-binding signal was enhanced by increasing the Cbx1 phage concentration on the ELISA plate. The introduction of previously known gain-of-function mutations to the Cbx1 protein significantly increased the ELISA signals. Moreover, we demonstrated that the H3K9me3-specific binding signal was enhanced by fusing Cbx1 with a high-affinity phosphotyrosine-binding protein and by coating the ELISA plate with a mixture of H3K9me3 and phosphotyrosine peptides. This approach also worked with binding to a lower affinity momomethyllysine peptide H3K9me1. These approaches may help improve ELISA experiments when dealing with low-affinity ligand-protein interactions.

The relative humidity-dependent viscosity of single quasi aerosol particles and possible implications for atmospheric aerosol chemistry.

Viscosity is a fundamental physicochemical property of aerosol particles that influences chemical evolution, mass transfer rates, particle formation, etc. and also changes with ambient relative humidity (RH). However, the viscosity of real individual aerosol particles still remains less understood. Here, we developed a novel optical system based on dual optical tweezers to measure the viscosity of single suspending aerosol droplets under different RH conditions. In our experiment, a pair of quasi atmospheric aerosol droplets composed of organic and inorganic chemical substances were trapped and levitated by dual laser beams, respectively, and then collided and coalesced. The backscattering light signals and bright-field images of the dynamic coalescence process were recorded to infer the morphological relaxation time and the diameter of the composited droplet. Then, the viscosity of the droplet was calculated based on these measured values. The ambient RH of the aerosol droplets was controlled by varying the relative flow rates of dry and humidified nitrogen gas in a self-developed aerosol chamber. The viscosities of single aqueous droplets nebulized with solutes of sucrose, various sulfates and nitrates, and organic/inorganic mixtures were measured over the atmospheric RH range. Besides, the viscosities of the proxies of actual ambient aerosols in Beijing were investigated, which reasonably interpreted the aerosol chemistry transforming from sulfate dominating to nitrate dominating at the PM10 (particulate matter with an aerodynamic diameter of less than 10 µm) level in the last decade in Beijing. Furthermore, the hygroscopicity of droplets with a solute of organic/inorganic mixtures was researched to obtain a deep insight into the relationship between the viscosity and mass transfer process. Hence, we provide a robust approach for investigating the viscosity and hygroscopicity of the actual individual liquid PM10 aerosols.

Synergistic interaction of renewable nipagin and eugenol for aromatic copoly(ether ester) materials with desired performance.

Naturally occurring nipagin and eugenol were used as the collaborative starting materials for poly(ether ester) polymers. In this study, two series of nipagin and eugenol-derived copoly(ether ester)s, PHN11-xE1x and PHN11-xE2x (x = 0%, 5%, 10%, 15%, 20%), were prepared with renewable 1,6-hexanediol as a comonomer. The nipagin-derived component acts as the renewable surrogate of petroleum-based dimethyl terephthalate (DMT), while the eugenol-derived component acts as the cooperative property modifier of parent homopoly(ether ester) PHN1. 1,6-Hexanediol was chosen as the spacer because of its renewability, high boiling point, and short chain to enhance the glass transition temperatures (Tgs) of materials. The molecular weights and chemical structures were confirmed by gel permeation chromatograph (GPC), NMR and FTIR spectroscopies. Thermal and crystalline properties were studied by thermal gravimetric analysis (TGA), differential scanning calorimetric (DSC) and wide-angle X-ray diffraction (WXRD). The tensile assays were conducted to evaluate the mechanical properties. The results suggested that properties of this kind of poly(ether ester)s could be finely tuned by the relative content of two components for the desired applications (elastomer, rubbery) suitable for different scenarios from polyethylene glycol terephthalate (PET) and polybutylene terephthalate (PBT).

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple-mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two-orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross-reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr-containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.

Nondestructive Identification and Accurate Isolation of Single Cells through a Chip with Raman Optical Tweezers.

Raman optical tweezers (ROT) as a label-free technique plays an important role in single-cell study such as heterogeneity of tumor and microbial cells. Herein we designed a chip utilizing ROT to isolate a specific single cell. The chip was made from a polydimethylsiloxane (PDMS) slab and formed into a gourd-shaped reservoir with a connected channel on a cover glass. On the chip an individual cell could be isolated from a cell crowd and then extracted with ∼0.5 µL of phosphate-buffered saline (PBS) via pipet immediately after Raman spectral measurements of the same cell. As verification, we separated four different type of cells including BGC823 gastric cancer cells, erythrocytes, lymphocytes, and E. coli cells and quantifiably characterized the heterogeneity of the cancer cells, leukocyte subtype, and erythrocyte status, respectively. The average time of identifying and isolating a specific cell was 3 min. Cell morphology comparison and viability tests showed that the successful rate of single-cell isolation was about 90%. Thus, we believe our platform could further couple other single-cell techniques such as single-cell sequencing and become a multiperspective analytical approach at the level of a single cell.

Preparation of a multifunctional verapamil-loaded nano-carrier based on a self-assembling PEGylated prodrug.

In an effort to prove the inherent side effects of doxorubicin (DOX) and potentially revoke the effects of drug resistance exhibited by cancer cells, we have designed a multifunctional DOX-delivery nano-carrier system able to encapsulate the drug resistance reversal agent Verapamil HCl (VRP·HCl). Hydrophilic short-chain polyethylene glycol (i.e., mPEG) was covalently linked to hydrophobic DOX and a benzoic imine linkage was used to form a linear amphiphilic PEGylated prodrug, namely mPEG-b-DOX. In aqueous solution, the amphiphilic PEG-b-DOX is able to self-assemble to form stable nanoparticles with a DOX loading content of approximately 40 wt% and a diameter of ∼ 143 nm. The resulting nanoparticles can simultaneously serve as an anticancer drug conjugate and as a drug carrier system. Here, the hydrophilic VRP could be encapsulated into the nano-carriers via a conventional dialysis method. The loading efficiency in mPEG-b-DOX nano-carrier was determined to be 53.97% and the loading content was found to be 7.71 wt%. The VRP-loaded nano-carriers grew slightly in size, to a diameter of ∼ 177 nm. We found that the release of DOX and VRP was much faster at a lower pH value. The biological activity of the nano-carriers were evaluated in vitro and compared with the DOX-loaded system. In doing so we found that the VRP-loaded nano-carrier features a much higher antitumor activity. Furthermore, the combined-system exhibits a significantly enhanced cytotoxicity with an elevated apoptosis rate observed for MCF-7/ADR used as a cell line in this in vitro study. This combinatory system and promising candidate for applications involving DOX chemotherapy proved to be easy to prepare and could be characterized in terms of biocompatibility, biodegradability, loading capacity, pH responsiveness and reversal of drug resistance.

PH responsive polypeptide based polymeric micelles for anticancer drug delivery.

A pH-responsive polymeric micelle based on poly(aspartamide) derivative was explored as an efficient acid-triggered anticancer drug delivery system. Poly(α,ß-l-asparthydrazide) (PAHy) was prepared by aminolysis reaction of polysuccinimide with hydrazine hydrate. Poly(ethylene glycol) and aliphatic chain (C18) were conjugated onto PAHy to afford an amphiphilic copolymer with acid-liable hydrazone bonds. The structure of the resulting copolymer and its self-assembled micelles were confirmed by (1) H NMR, FTIR, DLS, and TEM. Furthermore, doxorubicin (DOX) was loaded into the polymeric micelles via the hydrophobic interaction between the C18 group and DOX molecules, and the π-π staking between the hydrazone conjugated DOX and free DOX molecules. Results showed that the DOX loaded nanoparticle (NP) was relatively stable under physiological conditions, while the DOX was quickly released in response to acidity due to the shedding of mPEG shells and dissociating of C18 segments because of the pH-cleavage of intermediate hydrazone bonds. In addition, the DOX loaded micelles presented a high cytotoxic activity against tumor cells in vitro. This pH responsive NP has appeared highly promising for the targeted intracellular delivery of hydrophobic chemotherapeutics in cancer therapy.

[Detection of inorganic elements in parasitic plants and their hosts].

Parasitic plants are destructive agricultural pests. Today, parasitic plants have been recognized as serious pests causing considerable economic damage on crop and woods in China. Parasites are among the most destructive weeds known, and more and more people begin to pay more attention to the relationship between parasitic plant and host. Two cistanches and their hosts were analyzed and characterized by ICP-AES. The contents of K, P, Na, Ca, Mg, Fe, B, Cu, Zn, Al and Mn in Tamarix chinensis, Haloxylon ammodendron, Cistanche deserticola and Cistanche tubulosa were determined. The results showed that the spectra of main elements of the two cistanches and their hosts are similar, but the content of each element is different. The content of K, P, N and Ca is higher than other element, The content of K and P in Cistanche deserticola and Cistanche tubulosa is higher than the content of theirs hosts. The present study provides a new scientific foundation for further study and general application of parasitic plant.

[Analysis of the content of mineral elements in Scutellaria baicalensis, skullcap tea and its solution].

The content of nine mineral elements in the root, stem and leaf of 1-3 years old Scutellaria baicalensis, skullcap tea, and its solution was determined by the inductively coupled plasma atomic emission spectrometry (ICP-AES). The results show that the main mineral elements in the root, stem and leaf are similar. The main mineral elements include K, Ca, Mg, P, Al and Fe. The content ratio of Zn, Mn, Cu, Fe, Al, P, Mg, Ca and K in the leaf of Scutellaria haicalensis is 1 : 3 : 6 : 15 : 18 19 : 41 : 333 : 423. The contents of Fe, Mn, P, Mg and Ca in the stems and leaves of Scutellaria baicalensis increase with the increase of cultivation period. There were K, Ca, Mg and other mineral elements in the skullcap tea, and Fe, Zn and Mg were easy to be dissolved, the dissolution rates were 61.8%, 55.4% and 61.4% respectively. This study can provide scientific basis for rational utilization of the above ground resources of Scutellaria baicalensis.

[Mineral elements content of C. deserticola at different growth stages].

ICP - AES technology was used to determine the mineral elements content of Cistanche deserticola Ma (C. deserticola). The results showed that: (1) At succulent stem stages, the content of K was the highest in 5 macroelements, it can reach to 9.45 mg x g(-1), and the proportion for K : Na : P : Ca : Mg was 12 : 3.4 : 1.6 : 1.4 : 1. Among 5 microelements, the content of Fe was the highest and can reach to 97.31 microg x g(-1), and the proportion for Fe : Cu : Mn: Zn : Cu : B was 25 : 3.7 : 3.5 : 1.2 : 1. (2) At reproductive growth stages, the contents of Ca, Mg, Fe, Zn, Cu, Mn and B at unearthed stage were significantly higher than that at the underearth stage, especially for the content of Fe and Mn, they can respectively reach to 697. 55 and 38.75 microg x g(-1) at capsule formative stage. The contents of Fe and Mn at capsule formative stage were almost 7.2 and 8.3 times than that at succulent stem stage. (3) At reproductive growth stages, the aerial part will exclude Na and accumulate P, K, Ca, Mg, Fe, Mn, Zn, Cu and B. This result of the study will be a scientific basis for evaluating the quality of C. deserticola.

[Determination of trace elements in holoparasitism Cistanche tubulosa].

The trace elements in Chinese herbal medicines are important nutritional elements for human health. Application of ICP-AES was applied to detect Fe, Mn, Cu, Zn and Mg of the different parts and size grading on growth of Cistanche tubulosa. The results showed that there were comparatively rich trace elements in Cistanche tubulosa, and the trace elements of the different parts and size grading on growth were significantly different. Traditionally, the succulent stem and 200-500 grams of Cistanche tubulosa are common in the use of medicinal herbs, and the authors found that the iron, manganese, copper, zinc and magnesium were 242.18, 17.85, 7.96, 6.64 and 1 357.50 microg x g(-1) in the succulent stem of Cistanche tubulosa. There were 174.21, 4.48, 8.11, 4.31 and 1 126.93 microg x g(-1) in 200-500 grams of Cistanche tubulosa. It provided useful data for discussing the relationship between the content of the trace elements and the efficacy of Cistanche tubulosa.

[Effects of S-3307 on photosynthesis of Ligusticum chuanxiong].

OBJECTIVE: To study the effects of S-3307 spraying time and density on photosynthetic characteristic of Ligusticum chuanxiong. METHOD: The photosynthetic characteristic of L. chuanxiong under different S-3307 spraying time and density was studied by plot cultivation experiment. RESULT: The content of chlorophyll a and chlorophyll b in leaf increased when the spraying density was 20, 40, 80 mg x L(-1), while the net photosynthetic rate was the maximum. When the spraying density was 160 mg x L(-1), the content of chlorophyll a and chlorophyll as well as net photosynthetic rate were not increased. CONCLUSION: S-3307 spraying can raise the photosynthetic capacity of L. chuanxiong and promote the form of assimilation products.