Development of Phenyl-substituted Isoindolinone- and Benzimidazole-type Cereblon Ligands for Targeted Protein Degradation.

Thalidomide, pomalidomide and lenalidomide, collectively referred to as immunomodulatory imide drugs (IMiDs), are frequently employed in proteolysis-targeting chimeras (PROTACs) as cereblon (CRBN) E3 ligase-recruiting ligands. However, their molecular glue properties that co-opt the CRL4CRBN to degrade its non-natural substrates may lead to undesired off-target effects for the IMiD-based PROTAC degraders. Herein, we reported a small library of potent and cell-permeable CRBN ligands, which exert high selectivity over the well-known CRBN neo-substrates of IMiDs by structure-based design. They were further utilized to construct bromodomain-containing protein 4 (BRD4) degraders, which successfully depleted BRD4 in the tested cells. Overall, we reported a series of functionalized CRBN recruiters that circumvent the promiscuity from traditional IMiDs, and this study is informative to the development of selective CRBN-recruiting PROTACs for many other therapeutic targets.

Microstructure and Mechanical Properties of Weaving Wire and Arc Additive Manufactured AZ91 Magnesium Alloy Based on Cold Metal Transfer Technique.

Based on the cold metal transfer (CMT) technique, a deposited wall of AZ91 magnesium alloy was fabricated by weaving wire and arc additive manufacturing (WAAM), the shaping, microstructure, and mechanical properties of the sample with the weaving arc were characterized and discussed by compared with the sample without the weaving arc, and the effects of the weaving arc on grain refinement and property enhancement of the AZ91 component by CMT-WAAM process were investigated. After introducing the weaving arc, the effective rate of the deposited wall could be increased from 84.2% to 91.0%, and the temperature gradient of the molten pool could be reduced with an increase in constitutional undercooling. The equiaxed α-Mg grains became more equiaxial due to the dendrite remelting, and the ß-Mg17Al12 phases distributed uniformly induced by the forced convection after introducing the weaving arc. Compared to the deposited component fabricated by the CMT-WAAM process without the weaving arc, the average ultimate tensile strength and elongation of the component by weaving the CMT-WAAM process both increased. The weaving CMT-WAAM component showed isotropy and has better performance than the traditional cast AZ91 alloy.

A platform for the rapid synthesis of molecular glues (Rapid-Glue) under miniaturized conditions for direct biological screening.

Molecular glues, functioning via inducing degradation of the target protein while having similar molecular weight as traditional small molecule drugs, are emerging as a promising modality for the development of therapeutic agents. However, the development of molecular glues is limited by the lack of general principles and systematic methods. Not surprisingly, most molecular glues have been identified serendipitously or through phenotypic screening of large libraries. However, the preparation of large and diverse molecular glue libraries is not an easy task and requires extensive resources. We previously developed platforms for rapid synthesis of proteolysis targeting chimeras (PROTACs) that can be used directly for biological screening with minimal resources. Herein, we report a platform of rapid synthesis of molecular glues (Rapid-Glue) via a micromolar scale coupling reaction between hydrazide motif on the E3 ligase ligands and commercially available aldehydes with diverse structures. A pilot library of 1520 compounds is generated under miniaturized conditions in a high throughput manner without any further manipulation including purification after the synthesis. Through this platform, we identified two highly selective GSPT1 molecular glues through direct screening in cell-based assays. Three additional analogues were prepared from readily available starting materials by replacing the hydrolytic labile acylhydrazone linker with a more stable amide linker based on the two hits. All three analogues showed significant GSPT1 degradation activity and two of them possess comparable activity to the corresponding hit. The feasibility of our strategy is thus verified. Further studies by increasing the diversity and size of the library followed by appropriate assays will likely yield distinct molecular glues targeting novel neo-substrates.

Soil C, N and P stoichiometry in different forest stand types in the middle and lower reaches of the Beijiang River, China.

We examined the vertical distribution characteristics of soil organic carbon (C), total nitrogen (N), total phosphorus (P) and their ecological stoichiometric ratios in 0-80 cm soil profile under three forest stand types in the middle and lower reaches of the Beijiang River, including broad-leaved forest, coniferous forest, and mixed coniferous and broad-leaved forest. The results showed that soil C, N and P contents of the three forest stand types were 12.17-14.25, 1.14-1.31, and 0.27-0.30 g·kg-1, respectively. The contents of C and N decreased with the increases of soil depth. The content of C and N in each soil layer showed that coniferous and broad-leaved mixed forest > coniferous forest > broad-leaved forest. There was no significant difference in P content among the three stand types, and there was no obvious variation in the vertical profile. The soil C/N, C/P, and N/P of the three forest types were 11.2-11.3, 49.0-60.3, and 4.5-5.7, respectively. There was no significant difference in soil C/N among the three stand types. The highest soil C/P and N/P were found in the mixed forest. There was no interaction between soil depth and stand type in affecting soil C, N, P contents and their stoichiometric ratios. There was significant positive correlation between C and N, and between N and C/P in each stand type and soil layer. Soil C/P and N/P had stronger ecological indicating effects on stand types. The coniferous and broad-leaved mixed forest was strongly limited by P availability.

Optimization of CMT Characteristic Parameters for Swing Arc Additive Manufacturing of AZ91 Magnesium Alloy Based on Process Stability Analysis.

The droplet transfer behavior and stability of the swing arc additive manufacturing process of AZ91 magnesium alloy based on the cold metal transfer (CMT) technique were studied by analyzing the electrical waveforms and high-speed droplet images as well as the forces on the droplet, and the Vilarinho regularity index for short-circuit transfer (IVSC) based on variation coefficients was used to characterize the stability of the swing arc deposition process. The effect of the CMT characteristic parameters on the process stability was investigated; then, the optimization of the CMT characteristic parameters was realized based on the process stability analysis. The results show that the arc shape changed during the swing arc deposition process; thus, a horizontal component of the arc force was generated, which significantly affected the stability of the droplet transition. The burn phase current I_sc_wait presented a linear function relation with IVSC, while the other three characteristic parameters, i.e., boost phase current I_boost, boost phase duration t_I_boost and short-circuiting current I_sc2, all had a quadratic correlation with IVSC. A relation model of the CMT characteristic parameters and IVSC was established based on the rotatable 3D central composite design; then, the optimization of the CMT characteristic parameters was realized using a multiple-response desirability function approach.

GraphDPA: Predicting drug-pathway associations by graph convolutional networks.

Pathway-based drug discovery is a promising strategy for the discovery of drugs with low toxicity and side effects. However, identifying the associations between drug and targeting pathways is challenging for this method. The formation of various biomolecular interaction databases and the development of neural network technology provide new ways for the large-scale prediction of drug-pathway associations. This article proposes a new model called GraphDPA, which represents the drug and pathway-gene association as a graph. We use graph convolutional networks (GCN) to learn the features of the drug and pathway and predict the drug-pathway association. The results show that GraphDPA can predict drug-pathway associations with high accuracy, which verify the potential of the GCN in drug discovery.

A platform for the rapid synthesis of proteolysis targeting chimeras (Rapid-TAC) under miniaturized conditions.

Proteolysis targeting chimera (PROTAC) is one of the most frequently used technologies for targeted protein degradation. PROTACs are composed of target protein ligand, E3 ligase ligand and a linker between them. Traditional methods for the development of PROTACs involve step-by-step synthesis and are time consuming. Herein, we report a platform for the rapid synthesis of PROTACs (Rapid-TAC) via a traceless coupling reaction between ortho-phthalaldehyde (OPA) motif on the ligand of targeting protein and an amine fucntional group on the commercially available partial PROTAC library that is composed of different E3 ligase ligands and various types and lengths of linkers. Under our optimized miniaturized conditions, the full PROTACs can be synthesized in a high throughput manner and the products can be directly used for screening without any further manipulations including purification. We demonstrated the utility of this platform by quickly identifying active degraders for androgen receptor (AR) and BRD4 with DC50 of 41.9 nM and 8.9 nM, respectively. It is expected that this Rapid-TAC platform can be easily extended to many other targets, thus lowering the barrier to access this novel modelity for small molecule drug discovery and faciliate structure activity relationship studies.

Effective use of sequence information to predict CRISPR-Cas9 off-target.

The CRISPR/Cas9 gene-editing system is the third-generation gene-editing technology that has been widely used in biomedical applications. However, off-target effects occurring CRISPR/Cas9 system has been a challenging problem it faces in practical applications. Although many predictive models have been developed to predict off-target activities, current models do not effectively use sequence pair information. There is still room for improved accuracy. This study aims to effectively use sequence pair information to improve the model's performance for predicting off-target activities. We propose a new coding scheme for coding sequence pairs and design a new model called CRISPR-IP for predicting off-target activity. Our coding scheme distinguishes regions with different functions in the sequence pairs through the function channel. Moreover, it distinguishes between bases and base pairs using type channels, effectively representing the sequence pair information. The CRISPR-IP model is based on CNN, BiLSTM, and the attention layer to learn features of sequence pairs. We performed performance verification on two data sets and found that our coding scheme can represent sequence pair information effectively, and the CRISPR-IP model performance is better than others. Data and source codes are available at https://github.com/BioinfoVirgo/CRISPR-IP.

Development of Selective Histone Deacetylase 6 (HDAC6) Degraders Recruiting Von Hippel-Lindau (VHL) E3 Ubiquitin Ligase.

Histone deacetylase 6 (HDAC6) is involved in multiple cellular processes such as aggresome formation, protein stability, and cell motility. Numerous HDAC6-selective inhibitors have been developed as cellular chemical tools to elucidate the function of HDAC6. Since HDAC6 has multiple domains that cannot be studied by HDAC6-selective inhibitors, CRISPR-CAS9 and siRNA/shRNA have been employed to elucidate the nonenzymatic functions of HDAC6. However, these genetic methods have many limitations. Proteolysis targeting chimera (PROTAC) is an emerging technology for the development of small molecules that can quickly remove the entire protein in cells. We previously developed multifunctional HDAC6 degraders that can recruit cereblon (CRBN) E3 ubiquitin ligase. These HDAC6 degraders can degrade not only HDAC6 but also neo-substrates of CRBN. They are excellent candidates for the development of anticancer therapeutics, but the multifunctional nature of the CRBN-based HDAC6 degraders has limited their utility as specific chemical probes for the study of HDAC6-related cellular pathways. Herein we report the development of the first cell-permeable HDAC6-selective degraders employing Von Hippel-Lindau (VHL) E3 ubiquitin ligase, which does not have any known neo-substrates. The DC50's of the most potent compound 3j are 7.1 nM and 4.3 nM in human MM1S and mouse 4935 cell lines, respectively. The D max's of 3j in these two cell lines are 90% and 57%, respectively.

Carbon dynamics in three subtropical forest ecosystems in China.

The carbon sequestration capacity of the forest ecosystem normally increases overage due to the carbon dynamic in below canopy and soil. The carbon dynamic is reflective of the forest characteristics and their interactions with climate, topographic, and soil conditions. In this study, we measured the carbon content and carbon density of canopy, shrub, understory vegetation, litter, and soil, and assessed carbon dynamics in three forest ecosystems (Cunninghamia lanceolate, Pinus massoniana, and Evergreen broad-leaved forests) with a combination of data from Fujian Provincial forest resource inventory. This study showed that the carbon content of the canopy layers increased over time, and the carbon content of the topsoil (0-30 cm) in the young forests was significantly higher than that in other age groups in Cunninghamia lanceolata forest and Pinus massoniana forest. Due to the carbon differences in the soil layer, the carbon stocks of the C. lanceolata forest and the P. massoniana forest declined from 1996 to 2007, but the carbon stocks of Evergreen broad-leaved forest increased. Besides, using the traditional carbon content coefficient (0.5) might underestimate the carbon sequestration potential of these forest ecosystems, especially for the mature forests. The coniferous forests displayed a short-term reduction in the carbon stocks of ecosystems between 10 and 20 years after afforestation, and the decline cannot be ignored in the carbon budget.

The complete chloroplast genome sequence of Butea monosperma (Fabaceae).

Butea monosperma, an importantmedicinal plantin Fabaceae, is mainly distributed in southern Asia. In this study, we reported the complete chloroplast (cp) genome of B. monosperma assembled with Illumina sequencing data. The whole cp genome of this species is 151,925 bp in length, consisting of two inverted repeat regions (IR, 25,083 bp), one large single-copy region (LSC, 83,541 bp), and one small single-copy region (SSC, 18,218 bp).A total of 128 genes were annotated for the chloroplast genome, including 83 protein-coding genes, 37 tRNAs and 8 rRNAs. Phylogenetic analysis indicated that B. monosperma was closely related to the genus Lespedeza.

Development of Multifunctional Histone Deacetylase 6 Degraders with Potent Antimyeloma Activity.

Histone deacetylase 6 (HDAC6) primarily catalyzes the removal of acetyl group from the side chain of acetylated lysine residues in cytoplasmic proteins such as α-tubulin and HSP90. HDAC6 is involved in multiple disease-relevant pathways. Based on the proteolysis targeting chimera strategy, we previously developed the first HDAC6 degrader by tethering a pan-HDAC inhibitor with cereblon (CRBN) E3 ubiquitin ligase ligand. We herein report our new generation of multifunctional HDAC6 degraders by tethering selective HDAC6 inhibitor Nexturastat A with CRBN ligand that can synergize with HDAC6 degradation for the antiproliferation of multiple myeloma (MM). This new class of degraders exhibited improved potency and selectivity for the degradation of HDAC6. After the optimization of the linker length and linking positions, we discovered potent HDAC6 degraders with nanomolar DC50 and promising antiproliferation activity in multiple myeloma (MM) cells.

A predictive nondestructive model for the covariation of tree height, diameter, and stem volume scaling relationships.

Metabolic scaling theory (MST) posits that the scaling exponents among plant height H, diameter D, and biomass M will covary across phyletically diverse species. However, the relationships between scaling exponents and normalization constants remain unclear. Therefore, we developed a predictive model for the covariation of H, D, and stem volume V scaling relationships and used data from Chinese fir (Cunninghamia lanceolata) in Jiangxi province, China to test it. As predicted by the model and supported by the data, normalization constants are positively correlated with their associated scaling exponents for D vs. V and H vs. V, whereas normalization constants are negatively correlated with the scaling exponents of H vs. D. The prediction model also yielded reliable estimations of V (mean absolute percentage error = 10.5 ± 0.32 SE across 12 model calibrated sites). These results (1) support a totally new covariation scaling model, (2) indicate that differences in stem volume scaling relationships at the intra-specific level are driven by anatomical or ecophysiological responses to site quality and/or management practices, and (3) provide an accurate non-destructive method for predicting Chinese fir stem volume.

Ratio of microRNA-122/155 in isoniazid-induced acute liver injury in mice.

Liver injury is a major hindrance to the treatment of tuberculosis (TB) patients due to the primary side effects associated with anti-TB drugs. Several investigations have identified sensitive biomarkers for the early diagnosis of anti-TB drug-induced liver injury (ADLI), including the use of microRNAs (miRNAs/miRs). However, the association between miR-122/155 and ADLI remains unknown. Thus, the present study used reverse transcription-quantitative polymerase chain reaction to observe changes in tissue miR-122/155 expression levels during the course of liver injury in mice. Liver injury was induced by the administration of isoniazid (INH), a first-line anti-TB drug. miR-122/155 expression levels were quantified at seven time points throughout 1 day (0.25, 0.75, 1.5, 6, 12, 18 and 24 h) based on the pharmacokinetics of INH in mice. Notably, over the timecourse of INH-induced liver injury, the tissue miR-122 expression level significantly decreased at 0.25 h, which is the peak concentration time of INH, compared with the control group (P<0.05). The change was more rapid than that of the serum aminotransferase and miR-155, which were significantly increased at 0.75 h. In addition, the pathological score correlated with the ratio of miR-122/miR-155 (r=-0.779; P<0.01). In conclusion, the miR-122/155 ratio may be utilized as a sensitive biomarker for ADLI, which could contribute to the early diagnosis of patients following anti-TB treatment.

[Characteristics of leaf carbon, nitrogen and phosphorus stoichiometry in relation to plant size of Machilus pauhoi].

To explore the effects of stand age on variation patterns of leaf C, N, P stoichiometric characteristics of Machilus pauhoi, two stands, i.e., 9 and 13 years old, were selected. The relationships between leaf nutrient contents (C, N and P) and diameters at breast height (DBH) of individual plants were analyzed. The data revealed that the individual variations of seedlings in M. pauhoi stands were strengthened with the stand development. The stand age had significant effects on leaf C, N, P contents and C:N ratio but not on C:P and N: P ratios. Specifically, the mean values of leaf C, N, P contents and N:P ratio in the 9-year-old stand were lower than those in the 13-year-old stand, whereas, inverse pattern of C:N and C:P ratios were found in the two stands. Furthermore, leaf N and P stoichiometry varied significantly within the stand. Specifically, leaf N and P contents, as well as their stoichiometric ratios, linearly correlated with DBH in the 9-year-old stand. On the contrary, leaf N and P stoichiometry showed quadratic correlation in 13-year-old stand (except leaf C:N which linearly correlated with DBH). Lastly, nutrient transfer rates of leaf N and P in the 9-year-old stand were higher than that in 13-year-old stand, and the discrepancies of leaf nutrient transfer strategy between growing and non-growing seasons were caused by the different growth phases and environmental conditions.