Current Pubertal Development in Chinese Children and the Impact of Overnutrition, Lifestyle, and Perinatal Factors.

CONTEXT: Age of pubertal onset has been decreasing in many countries but there have been no data on pubertal development in Chinese children over the last decade. OBJECTIVE: The primary objective of the study was to evaluate the current status of sexual maturation in Chinese children and adolescents. Secondary objectives were to examine socioeconomic, lifestyle, and auxological associations with pubertal onset. METHODS: In this national, cross-sectional, community-based health survey, a multistage, stratified cluster random sampling method was used to select a nationally representative sample, consisting of 231 575 children and adolescents (123 232 boys and 108 343 girls) between 2017 and 2019. Growth parameters and pubertal staging were assessed by physical examination. RESULTS: Compared to 10 years previously, the median age of Tanner 2 breast development and menarche were similar at 9.65 years and 12.39 years respectively. However, male puberty occurred earlier with a median age of testicular volume ≥4 mL of 10.65 years. Pubertal onset did occur earlier at the extremes, with 3.3% of the girls with breast development at 6.5-6.99 years old, increasing to 5.8% by 7.5-7.99 years old. Early pubertal onset was also noted in boys, with a testicular volume ≥ 4 mL noted in 1.5% at 7.5-7.99 years, increasing to 3.5% at 8.5-8.99 years old. Obesity and overweight increased risk of developing earlier puberty relative to normal weight in both boys and girls. CONCLUSION: Over the past decade, pubertal development is occurring earlier in Chinese children. While the cause is multifactorial, overweight and obesity are associated with earlier puberty onset. The currently used normative pubertal data of precocious puberty may not be applicable to diagnose precocious puberty.

Structure-Activity Relationship Studies of Venglustat on NTMT1 Inhibition.

The protein N-terminal methyltransferase 1 (NTMT1) is implicated in neurogenesis, retinoblastoma, and cervical cancer. However, its pharmacological potentials have not been elucidated due to the lack of drug-like inhibitors. Here, we report the discovery of the first NTMT1 in vivo chemical probe GD433 by structure-guided optimization of our previously reported lead compound venglustat. GD433 (IC50 = 27 ± 1.1 nM) displays improved potency and selectivity than venglustat across biochemical, biophysical, and cellular assays. GD433 also displays good oral bioavailability and can serve as an in vivo chemical probe to dissect the pharmacological roles of Nα methylation. In addition, we also identified a close analogue (YD2160) that is inactive against NTMT1. The active inhibitor and negative control will serve as valuable tools to examine the physiological and pharmacological functions of NTMT1 catalytic activity.

Venglustat Inhibits Protein N-Terminal Methyltransferase 1 in a Substrate-Competitive Manner.

Venglustat is a known allosteric inhibitor for ceramide glycosyltransferase, investigated in diseases caused by lysosomal dysfunction. Here, we identified venglustat as a potent inhibitor (IC50 = 0.42 µM) of protein N-terminal methyltransferase 1 (NTMT1) by screening 58,130 compounds. Furthermore, venglustat exhibited selectivity for NTMT1 over 36 other methyltransferases. The crystal structure of NTMT1-venglustat and inhibition mechanism revealed that venglustat competitively binds at the peptide substrate site. Meanwhile, venglustat potently inhibited protein N-terminal methylation levels in cells (IC50 = 0.5 µM). Preliminary structure-activity relationships indicated that the quinuclidine and fluorophenyl parts of venglustat are important for NTMT1 inhibition. In summary, we confirmed that venglustat is a bona fide NTMT1 inhibitor, which would advance the study on the biological roles of NTMT1. Additionally, this is the first disclosure of NTMT1 as a new molecular target of venglustat, which would cast light on its mechanism of action to guide the clinical investigations.

Development of Cordyceps javanica BE01 with enhanced virulence against Hyphantria cunea using polyethylene glycol-mediated protoplast transformation.

Cordyceps javanica has promising application prospects as an entomopathogenic fungus with a wide range of hosts. To enhance the virulence of C. javanica, a polyethylene glycol (PEG)-mediated protoplast genetic transformation system was constructed. Strains overexpressing the subtilisin-like protease genes CJPRB and CJPRB1 and the tripeptidyl peptidase gene CJCLN2-1 were constructed with this system, and the effects of these strains on Hyphantria cunea were tested. The aminoglycoside G418 was used at 800 µg ml-1 to screen the transformants. C. javanica hyphae were degraded with an enzyme mixture to obtain protoplasts at 1.31 × 107 protoplasts ml-1. The transformation of 2 µg of DNA into 1,000 protoplasts was achieved with 20% PEG2000, and after 6 h of recovery, the transformation efficiency was 12.33 ± 1.42 transformants µg-1 plasmid. The LT50 values of CJPRB, CJPRB1, and CJCLN2-1-overexpressing C. javanica strains were 1.32-fold, 2.21-fold, and 2.14-fold higher than that of the wild-type (WT) strain, respectively. The three overexpression strains showed no significant differences from the WT strain in terms of colony growth, conidial yield, and conidial germination rate. However, the infection rate of the CJPRB1 strain was faster than that of the WT strain, with infection occurring within 4-5 days. The CJCLN2-1 strain had a significantly higher mortality rate than the WT strain within 4-10 days after infection. A C. javanica genetic transformation system was successfully constructed for the first time, and an overexpression strain exhibited enhanced virulence to H. cunea compared with the WT strain.

Improved Cell-Potent and Selective Peptidomimetic Inhibitors of Protein N-Terminal Methyltransferase 1.

Protein N-terminal methyltransferase 1 (NTMT1) recognizes a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) and transfers 1-3 methyl groups to the N-terminal region of its substrates. Guided by the co-crystal structures of NTMT1 in complex with the previously reported peptidomimetic inhibitor DC113, we designed and synthesized a series of new peptidomimetic inhibitors. Through a focused optimization of DC113, we discovered a new cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM). GD562 exhibited improved inhibition of the cellular N-terminal methylation levels of both the regulator of chromosome condensation 1 and the oncoprotein SET with an IC50 value of ~50 µM in human colorectal cancer HCT116 cells. Notably, the inhibitory activity of GD562 for the SET protein increased over 6-fold compared with the previously reported cell-potent inhibitor DC541. Furthermore, GD562 also exhibited over 100-fold selectivity for NTMT1 against several other methyltransferases. Thus, this study provides a valuable probe to investigate the biological functions of NTMT1.

Discovery of a potent and dual-selective bisubstrate inhibitor for protein arginine methyltransferase 4/5.

Protein arginine methyltransferases (PRMTs) have been implicated in the progression of many diseases. Understanding substrate recognition and specificity of individual PRMT would facilitate the discovery of selective inhibitors towards future drug discovery. Herein, we reported the design and synthesis of bisubstrate analogues for PRMTs that incorporate a S-adenosylmethionine (SAM) analogue moiety and a tripeptide through an alkyl substituted guanidino group. Compound AH237 is a potent and selective inhibitor for PRMT4 and PRMT5 with a half-maximal inhibition concentration (IC50) of 2.8 and 0.42 nmol/L, respectively. Computational studies provided a plausible explanation for the high potency and selectivity of AH237 for PRMT4/5 over other 40 methyltransferases. This proof-of-principle study outlines an applicable strategy to develop potent and selective bisubstrate inhibitors for PRMTs, providing valuable probes for future structural studies.

Structure-based Discovery of Cell-Potent Peptidomimetic Inhibitors for Protein N-Terminal Methyltransferase 1.

Protein N-terminal methyltransferases (NTMTs) catalyze the methylation of the α-N-terminal amines of proteins starting with an X-P-K/R motif. NTMT1 has been implicated in various cancers and in aging, implying its role as a potential therapeutic target. Through structural modifications of a lead NTMT1 inhibitor, BM30, we designed and synthesized a diverse set of inhibitors to probe the NTMT1 active site. The incorporation of a naphthyl group at the N-terminal region and an ortho-aminobenzoic amide at the C-terminal region of BM30 generates the top cell-potent inhibitor DC541, demonstrating increased activity on both purified NTMT1 (IC50 of 0.34 ± 0.02 µM) and the cellular α-N-terminal methylation level of regulator of chromosome condensation 1 (RCC1, IC50 value of 30 µM) in human colorectal cancer HT29 cells. Furthermore, DC541 exhibits over 300-fold selectivity to several methyltransferases. This study points out the direction for the development of more cell-potent inhibitors for NTMT1.

Selective Peptidomimetic Inhibitors of NTMT1/2: Rational Design, Synthesis, Characterization, and Crystallographic Studies.

Protein N-terminal methyltransferases (NTMTs) methylate the α-N-terminal amines of proteins starting with the canonical X-P-K/R motif. Genetic studies imply that NTMT1 regulates cell mitosis and DNA damage repair. Herein, we report the rational design and development of the first potent peptidomimetic inhibitor for NTMT1/2. Biochemical and cocrystallization studies manifest that BM30 (with a half-maximal inhibitory concentration of 0.89 ± 0.10 µM) is a competitive inhibitor to the peptide substrate and noncompetitive to the cofactor S-adenosylmethionine. BM30 exhibits over 100-fold selectivity to NTMT1/2 among a panel of 41 MTs, indicating its potential to achieve high selectivity when targeting the peptide substrate binding site of NTMT1/2. Its cell-permeable analogue DC432 (IC50 of 54 ± 4 nM) decreases the N-terminal methylation level of the regulator of chromosome condensation 1 and SET proteins in HCT116 cells. This proof-of principle study provides valuable probes for NTMT1/2 and highlights the opportunity to develop more cell-potent inhibitors to elucidate the function of NTMTs in the future.

Probing the Plasticity in the Active Site of Protein N-terminal Methyltransferase 1 Using Bisubstrate Analogues.

The bisubstrate analogue strategy is a promising approach to develop potent and selective inhibitors for protein methyltransferases. Herein, the interactions of a series of bisubstrate analogues with protein N-terminal methyltransferase 1 (NTMT1) were examined to probe the molecular properties of the active site of NTMT1. Our results indicate that a 2-C to 4-C atom linker enables its respective bisubstrate analogue to occupy both substrate- and cofactor-binding sites of NTMT1, but the bisubstrate analogue with a 5-C atom linker only interacts with the substrate-binding site and functions as a substrate. Furthermore, the 4-C atom linker is the optimal and produces the most potent inhibitor (Ki,app = 130 ± 40 pM) for NTMT1 to date, displaying more than 3000-fold selectivity for other methyltransferases and even for its homologue NTMT2. This study reveals the molecular basis for the plasticity of the active site of NTMT1. Additionally, our study outlines general guidance on the development of bisubstrate inhibitors for any methyltransferases.

Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors.

Methyltransferases (MTases) play diverse roles in cellular processes. Aberrant methylation levels have been implicated in many diseases, indicating the need for the identification and development of small molecule inhibitors for each MTase. Specific inhibitors can serve as probes to investigate the function and validate therapeutic potential for the respective MTase. High-throughput screening (HTS) is a powerful method to identify initial hits for further optimization. Here, we report the development of a fluorescence-based MTase assay and compare this format with the recently developed MTase-Glo luminescence assay for application in HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model system, we miniaturized to 1536-well quantitative HTS format. Through a pilot screen of 1428 pharmacologically active compounds and subsequent validation, we discovered that MTase-Glo produced lower false positive rates than the fluorescence-based MTase assay. Nevertheless, both assays displayed robust performance along with low reagent requirements and can potentially be employed as general HTS formats for the discovery of inhibitors for any MTase.

[Usage of ethnomedicine on COVID-19 in China].

In December 2019, an outbreak of viral pneumonia began in Wuhan, Hubei Province, which caused the spread of infectious pneumonia to a certain extent in China and neighboring countries and regions, and triggered the epidemic crisis. The coronavirus disease 2019(COVID-19) is an acute respiratory infectious disease listed as a B infectious disease, which is managed according to standards for A infectious disease. Traditional Chinese medicine and integrated traditional Chinese and Western medicine have played an active role in the prevention and control of this epidemic. China's ethnomedicine has recognized infectious diseases since ancient times, and formed a medical system including theory, therapies, formula and herbal medicines for such diseases. Since the outbreak of the COVID-19 epidemic, Tibet Autonomous Region, Qinghai Province, Inner Mongolia Autonomous Region, Xinjiang Uygur Autonomous Region and Chuxiong Autonomous Prefecture of Yunnan, Qiandongnan Autonomous Prefecture of Guizhou have issued the prevention and control programs for COVID-19 using Tibetan, Mongolian, Uygur, Yi and Miao medicines. These programs reflect the wisdom of ethnomedicine in preventing and treating diseases, which have successfully extracted prescriptions and preventive measures for the outbreak of the epidemic from their own medical theories and traditional experiences. In this paper, we summarized and explained the prescriptions and medicinal materials of ethnomedicine in these programs, and the origin of Tibetan medicine prescriptions and Mongolian medicine prescriptions in ancient books were studied. These become the common characteristics of medical prevention and treatment programs for ethnomedicine to formulate therapeutic programs under the guidance of traditional medicine theories, recommend prescriptions and prevention and treatment methods with characteristics of ethnomedicine, and focus on the conve-nience and standardization. However, strengthening the support of science and technology and the popularization to the public, and improving the participation of ethnomedicine in national public health services and the capacity-building to deal with sudden and critical diseases are key contents in the development of ethnomedicine in the future.

Discovery of Bisubstrate Inhibitors for Protein N-Terminal Methyltransferase 1.

Protein N-terminal methyltransferase 1 (NTMT1) plays an important role in regulating mitosis and DNA repair. Here, we describe the discovery of a potent NTMT1 bisubstrate inhibitor 4 (IC50 = 35 ± 2 nM) that exhibits greater than 100-fold selectivity against a panel of methyltransferases. We also report the first crystal structure of NTMT1 in complex with an inhibitor, which revealed that 4 occupies substrate and cofactor binding sites of NTMT1.

An asparagine/glycine switch governs product specificity of human N-terminal methyltransferase NTMT2.

α-N-terminal methylation of proteins is an important post-translational modification that is catalyzed by two different N-terminal methyltransferases, namely NTMT1 and NTMT2. Previous studies have suggested that NTMT1 is a tri-methyltransferase, whereas NTMT2 is a mono-methyltransferase. Here, we report the first crystal structures, to our knowledge, of NTMT2 in binary complex with S-adenosyl-L-methionine as well as in ternary complex with S-adenosyl-L-homocysteine and a substrate peptide. Our structural observations combined with biochemical studies reveal that NTMT2 is also able to di-/tri-methylate the GPKRIA peptide and di-methylate the PPKRIA peptide, otherwise it is predominantly a mono-methyltransferase. The residue N89 of NTMT2 serves as a gatekeeper residue that regulates the binding of unmethylated versus monomethylated substrate peptide. Structural comparison of NTMT1 and NTMT2 prompts us to design a N89G mutant of NTMT2 that can profoundly alter its catalytic activities and product specificities.

IMPACT OF IMMUNE RESPONSE OF A PARASITIC BEETLE Dastarcus helophoroides ON ITS HOST BEETLE Monochamus alternatus.

Dastarcus helophoroides is an ectoparasitoid beetle of Monochamus alternatus, and the parasitism by D. helophoroides larvae remarkably influenced on the immune responses of M. alternatus larvae in many aspects. The hemolymph melanization reactions in the hosts were inhibited 1 h and 24 h postparasitization. The phenoloxidase activities of hemolymph were significantly stimulated 4 h postparasitization and inhibited 12 h postparasitization, and back to control level. The antibacterial activities of hemolymph in the parasitized hosts were significantly lower than that in the unparasitized ones 1 h postparasitization. By 72 h postparasitism, the total hemocyte numbers of the parasitized larvae declined to not more than one-seconds of the number collected from the unparasitized larvae. All sampled hemolymph held the capability of nodulation, and there were fluctuations in the number of nodules the hemocytes made. However, there were no significant differences between unparasitized and parasitized larvae at each time point in the hemagglutination activity and the ratios of spreading hemocytes. In conclusion, D. helophoroides larvae could regulate M. alternatus immune system and resulted in the changes in host immune responses.

Design, synthesis and biological evaluation of novel imidazo[4,5-c]pyridinecarboxamide derivatives as PARP-1 inhibitors.

A series of novel cyclic amine-substituted imidazo[4,5-c]pyridinecarboxamide analogs were designed and synthesized. All the target compounds were evaluated for their PARP inhibition activity, and the result indicated that most of the compounds possessed inhibitory effect on PARP at the concentration of 1µM, among which compound 8d (IC50=0.528 µM) was selected for evaluating the antitumor effect in vivo. The result showed the antitumor efficacy of the compound 8d and cisplatin combination group in a mouse A549 model is similar with that of the ABT-888 and cisplatin combination group.

[Study on chemical constituents from essential oil of Hypericum patulum].

OBJECTIVE: To investigate the chemical constituents from the essential oil of Hypericum patulum, and provide the scientific basis for exploitation. METHODS: The essential oil was extracted by steam distillation. The chemical constituents of the essential oil were analyzed by GC-MS. The relative contents of these constituents were calculated using square peaks to normalization. RESULTS: 72 peaks were separated and 24 constituents were identified, which constituted about 79.50% of the total essential oil. CONCLUSION: The main constituents are alpha-pinene (18.14%), 2,4a,5,6,7,8-hexahydro-3,5,5,9-tetramethyl-,(R)-1H-benzocycloheptene (13.64%), beta-caryophyllene (9.41%), longifolene (6.23%), etc.