Heterozygous LRP1 deficiency causes developmental dysplasia of the hip by impairing triradiate chondrocytes differentiation due to inhibition of autophagy.

Developmental dysplasia of the hip (DDH) is one of the most common congenital skeletal malformations; however, its etiology remains unclear. Here, we conducted whole-exome sequencing in eight DDH families followed by targeted sequencing of 68 sporadic DDH patients. We identified likely pathogenic variants in the LRP1 (low-density lipoprotein receptor-related protein 1) gene in two families and seven unrelated patients. All patients harboring the LRP1 variants presented a typical DDH phenotype. The heterozygous Lrp1 knockout (KO) mouse (Lrp1+/-) showed phenotypes recapitulating the human DDH phenotypes, indicating Lrp1 loss of function causes DDH. Lrp1 knockin mice with a missense variant corresponding to a human variant identified in DDH (Lrp1R1783W) also presented DDH phenotypes, which were milder in heterozygotes and severer in homozygotes than those of the Lrp1 KO mouse. The timing of triradiate cartilage development was brought forward 1 or 2 wk earlier in the LRP-deficient mice, which leads to malformation of the acetabulum and femoral head. Furthermore, Lrp1 deficiency caused a significant decrease of chondrogenic ability in vitro. During the chondrogenic induction of mice bone marrow stem cells and ATDC5 (an inducible chondrogenic cell line), Lrp1 deficiency caused decreased autophagy levels with significant ß-catenin up-regulation and suppression of chondrocyte marker genes. The expression of chondrocyte markers was rescued by PNU-74654 (a ß-catenin antagonist) in an shRNA-Lrp1-expressed ATDC5 cell. Our study reveals a critical role of LRP1 in the etiology and pathogenesis of DDH, opening an avenue for its treatment.

iASMP: An interpretable in-silico predictive tool focusing on species-specific antimicrobial peptides.

Antimicrobial peptides (AMPs), a crucial part of the innate immune system, have been exploited as promising candidates for antibacterial agents. Many researchers have been devoting their efforts to develop novel AMPs in recent decades. In this term, many computational approaches have been developed to identify potential AMPs accurately. However, finding peptides specific to a particular bacterial species is challenging. Streptococcus mutans is a pathogen with an apparent cariogenic effect, and it is of great significance to study AMP that inhibit S. mutans for the prevention and treatment of caries. In this study, we proposed a sequence-based machine learning model, namely iASMP, to exactly identify potential anti-S. mutans peptides (ASMPs). After collecting ASMPs, the performances of models were compared by utilizing multiple feature descriptors and different classification algorithms. Among the baseline predictors, the model integrating the extra trees (ET) algorithm and the hybrid features exhibited optimal results. The feature selection method was utilized to remove redundant feature information to improve the model performance further. Finally, the proposed model achieved the maximum accuracy (ACC) of 0.962 on the training dataset and performed on the testing dataset with an ACC of 0.750. The results demonstrated that iASMP had an excellent predictive performance and was suitable for identifying potential ASMP. Furthermore, we also visualized the selected features and rationally explained the impact of individual features on the model output.

The effects of incorporation of the counterparts and mimics of L-lysine on the antimicrobial activity, hemolytic activity, cytotoxicity and tryptic stability of antimicrobial peptide polybia-MPII.

Due to the limited effects of conventional antibiotics on the increasing emergence of drug-resistant bacteria and fungi, novel antimicrobial agents were urgently needed to alleviate this phenomenon. Nowadays, antimicrobial peptides are believed to be a promising candidate for a new generation of antimicrobial drugs. Antimicrobial peptide polybia-MPII (MPII) was first isolated from the venom of the social wasp Polybia paulista with a broad spectrum of antimicrobial activity. In the present study, the counterparts and mimics of cationic amino acids of Lys, such as Arg, His, Orn, Dab and Dap were employed to substitute Lys in the sequence of MPII. The effects of the incorporation of these amino acids on its antimicrobial activity, hemolytic activity, cytotoxicity, enzyme stability and therapeutic potential were explored. Our results showed that although the incorporation of Arg could improve its antimicrobial activity, there is no improvement in enzyme stability. The incorporation of His makes MPII exert its antimicrobial activity in a pH-dependent manner. Notably, incorporating Dap could effectively decrease its hemolytic activity and cytotoxicity and enhance its enzyme stability against trypsin. In conclusion, this study would provide an effective strategy to improve the bioavailability and metabolic stability of AMPs while decrease their hemolytic activity and cytotoxicity.

GM-Pep: A High Efficiency Strategy to De Novo Design Functional Peptide Sequences.

Although peptides are regarded as ideal therapeutic agents, only a small proportion of the marketed drugs are peptides. In the past decade, pharmacists have paid great attention to the development of peptide therapeutics. Except a few approved chemically/rationally designed peptides, most attempts failed due to unsatisfactory efficacy or safety. Luckily, computation methods, such as artificial intelligence, have been utilized to accelerate the discovery of therapeutic peptides by predicting the activity, toxicity, and absorption, distribution, metabolism, and excretion of polypeptides. Usually, a specific biological activity of a peptide could be accurately determined by an interest-oriented binary classification constructed of a positive set and another un-experimentally validated negative set regardless of other characteristics, which suggests that it could be challenging to realize the comprehensive evaluation of the research object in the early stage of drug research and development. Herein, we proposed an integrated method (GM-Pep) that contained a conditional variational autoencoder model (CVAE) and a positive sample training multiclassifier (Deep-Multiclassifier) to effectively generate a single bioactive peptide sequence without toxicity and referential side effects. The results showed that our Deep-Multiclassifier model gave a sequence accuracy of up to 96.41% [toxicity (94.48%), antifungal (96.58%), antihypertensive (97.18%), and antibacterial (96.91%), respectively]. The properties of Deep-Multiclassifier and CVAE were validated through 12 first synthesized antibacterial peptides or compared to random peptides. The source code and data sets are available at https://github.com/TimothyChen225/GM-Pep.

Trehalose reduces bone loss in experimental biliary cirrhosis rats via ERK phosphorylation regulation by enhancing autophagosome formation.

Bone loss is a severe complication of primary biliary cirrhosis (PBC). Trehalose was intermittently administered in bile duct-ligated (BDL) male rats, a PBC-related osteoporosis model, for 4 weeks to reduce osteoporosis. Femoral bones were assessed ex vivo by micro computed tomography (CT) and histomorphometry. The potential mechanisms related to the reduction of osteoporosis were explored by evaluating the effect of trehalose on osteoblast autophagy, osteogenesis, osteoclastogenesis, and ERK phosphorylation. The results demonstrated that trehalose reduced osteoporosis of BDL rats and decreased osteoblast-mediated osteoclast differentiation by enhancing osteoblast autophagy to regulate osteoprotegerin (OPG) secretion. Hydroxychloroquine (HCQ) increased the expression of OPG and OPG/receptor activator genes for nuclear factor-κB ligand (RANKL) ratio, and reduced osteoblast-mediated osteoclastogenesis by inhibiting autophagy flux and inducing autophagosome formation. Furthermore, trehalose increased the phosphorylation of ERK1/2 in MC3T3-E1 cells, and the ERK inhibitor PD98059 reversed the upregulation of OPG gene and reduction of trehalose-induced osteoclastogeneis. The treatment with HCQ markedly increased the ERK phosphorylation. The correlation between autophagosome formation and ERK phosphorylation was confirmed in autophagy proteins (ATG) 4B or ATG5-deficient cells. Thus, trehalose could decrease osteoblast-mediated osteoclastogenesis and reduce PBC-related bone loss by regulating ERK phosphorylation via autophagosome formation.

The introduction of L-phenylalanine into antimicrobial peptide protonectin enhances the selective antibacterial activity of its derivative phe-Prt against Gram-positive bacteria.

Protonectin was a typical amphiphilic antimicrobial peptide with potent antimicrobial activity against Gram-positive and Gram-negative bacteria. In the present study, when its eleventh amino acid in the sequence was substituted by phenylalanine, the analog named phe-Prt showed potent antimicrobial activity against Gram-positive bacteria, but no antimicrobial activity against Gram-negative bacteria, indicating a significant selectivity between Gram-positive bacteria and Gram-negative bacteria. However, when Gram-negative bacteria were incubated with EDTA, the bacteria were susceptible to phe-Prt. Next, the binding effect of phe-Prt with LPS was determined. Our result showed that LPS could hamper the bactericidal activity of phe-Prt against Gram-positive bacteria. The result of zeta potential assay further confirmed the binding effect of phe-Prt with LPS for it could neutralize the surface charge of E. coli and LPS. Then, the effect of phe-Prt on the integrity of outer membrane of Gram-negative bacteria was determined. Our results showed that phe-Prt had a much weaker disturbance to the outer membrane of Gram-negative bacteria than the parent peptide protonectin. In summary, the introduction of L-phenylalanine into the sequence of antimicrobial peptide protonectin made phe-Prt show significant selectivity against Gram-positive bacteria, which could partly be attributed to the delay effect of LPS for phe-Prt to access to cell membrane. Although further study is still needed to clarify the exact mechanism of selectivity, the present study provided a strategy to develop antimicrobial peptides with selectivity toward Gram-positive and Gram-negative bacteria.

The Regiocontrollable Enantioselective Synthesis of Chiral Trifluoromethyl-Containing Spiro-Pyrrolidine-Pyrazolone Compounds via Amino-Regulated 1,3-Proton Migration Reaction.

An amino-controlled regiodivergent asymmetric synthesis of CF3-containing spiro-pyrrolidine-pyrazolone compounds is described. With alkaloid-derived squaramide as catalyst, the 1,3-dipolar cycloaddition of α,ß-unsaturated pyrazolone with diethyl 2-((2,2,2-trifluoroethyl)imino) malonate offered adducts in excellent yields, dr, and ee. While the cyclohexanediamine-derived squaramide was employed, the reaction afforded a series of structure isomers through a switched umpolung reaction.

Multiple action mechanism and in vivo antimicrobial efficacy of antimicrobial peptide Jelleine-I.

With the extensive use of antibiotics in medicine, agriculture and food chemistry, the emergence of multi-drug resistant bacteria become more and more frequent and posed great threats to human health and life. So novel antimicrobial agents were urgently needed to defend the resistant bacteria. Jelleine-I was a small antimicrobial peptide (AMP) with eight amino acids in its sequence. It was believed to be an ideal template for developing antimicrobial agents. In the present study, the possible action mode against both gram-negative bacteria and gram-positive bacteria and in vivo antimicrobial activity was explored. Our results showed that Jelleine-I exhibits its antimicrobial activity mainly by disrupting the integrity of the cell membrane, which would not be affected by the conventional resistant mechanism. It also aims at some intracellular targets such as genomic DNA to inhibit the growth of microbes. In addition, the result of in vivo antimicrobial activity experiment showed that Jelleine-I performed a good therapeutic effect toward the mice with Escherichia coli infected peritonitis. Notably, Jelleine-I has negligible cytotoxicity toward the tested mammalian cells, indicating excellent cell selectivity between prokaryotic cells and eurkayotic cells. In summary, our results showed that Jelleine-I would be a potential candidate to be developed as a novel antimicrobial agent.

Life cycle assessment of single-use surgical and embedded filtration layer (EFL) reusable face mask.

BACKGROUND: The outbreak of the COVID-19 pandemic has led to an unprecedented amount of face mask consumption around the world. The increase in face mask consumption has brought focus to their environmental impact. To keep up with the increased demand for face masks, different variations of reusable face masks such as the embedded filtration layer (EFL) reusable face mask have emerged in the market. This study quantifies the environmental impact of the EFL reusable face mask and the single-use surgical face mask. METHODS: The life cycle assessment (LCA) study of the entire value chain from cradle-to-grave is applied to each face mask. Both face masks are evaluated over 1 functional unit (FU) of 31 12-h days for a single person. The ReCiPe method with the Hierachist perspective was applied. A total of nine impact categories as well as the generated waste of each face mask are evaluated. RESULTS: The results show that for 1 functional unit, the use of single-use surgical face mask and EFL reusable face mask will contribute 0.580 kg CO2-eq and 0.338 kg CO2-eq to climate change and generate 0.004 kg and 0.0004 kg of waste respectively. CONCLUSION: Comparing both face masks, the EFL reusable face mask will have a lower emission of at least 30% in terms of the generated waste and the impact categories considered, except for water depletion, freshwater eutrophication, marine eutrophication, and human toxicity.

Cu2+ reduces hemolytic activity of the antimicrobial peptide HMPI and enhances its trypsin resistance.

Nowadays, drug-resistant microbes are becoming a serious clinical problem threatening people's health and life. Antimicrobial peptides (AMPs) are believed to be potential alternatives of conventional antibiotics to combat the threat of drug-resistant microbes. However, the susceptibility of AMPs toward proteases is one of the major problems limiting their clinical use. In the present study, we reported the effect of Cu2+ on the bioactivity of AMP HMPI. We found that the addition of Cu2+ could improve the protease resistance of AMP HMPI without affecting its bioactivity. Notably, after the binding of Cu2+ with HMPI, the hemolytic activity of HMPI was greatly decreased. In addition, our results also demonstrated that the addition of Cu2+ increased the production of reactive oxygen species in the fungal cells, which may be a supplement for the antifungal activity of HMPI. In conclusion, the introduction of Cu2+ may provide an inorganic strategy to improve the stability and decrease the hemolytic activity of AMP HMPI, while maintaining its antifungal activity.

A genome-wide association study identifies new genes associated with developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is one of the most common congenital malformations and covers a spectrum of hip disorders from mild dysplasia to irreducible dislocation. The pathological mechanisms of DDH are poorly understood, which hampers the development of diagnostic tools and treatments. To gain insight into its disease mechanism, we explored the potential biological processes that underlie DDH by integrating pathway analysis tools and performing a genome-wide association study (GWAS). A total of 406 DDH-associated genes (P < 0.001) were identified by our GWAS using a Chinese Han cohort consisting of 386 DDH cases and 500 healthy controls (Set A). We verified the significant loci (P < 10-5 ) in another Chinese Han cohort consisting of 574 DDH patients and 569 healthy controls (Set B). An intronic Single Nucleotide Polymorphism (SNP) (rs61930502) showed significant association in Set A and Set B (P = 2.65 × 10-7 and 2.0 × 10-4 , respectively). The minor allele, rs61930502-A, which tended to prevent DDH showed a dominant effect. Heat shock 70 kDa protein 8 (HSPA8) showed the most direct interactions with other proteins which were coded by DDH-associated genes in the protein-protein interaction analysis. Interestingly, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested a relation between DDH and the genes involved in type II diabetes mellitus pathway (P = 0.0067). Our genetic and protein interaction evidence could open avenues for future studies of DDH.

The catalytic asymmetric synthesis of CF3-containing spiro-oxindole-pyrrolidine-pyrazolone compounds through squaramide-catalyzed 1,3-dipolar cycloaddition.

Pharmaceutically important compounds were synthesized through the organocatalytic 1,3-dipolar cycloaddition reaction. In the presence of a cinchonine-derived squaramide catalyst, the cycloaddition of N-2,2,2-trifluoroethylisatin ketimines with α,ß-unsaturated pyrazolones gave a spiro-pyrrolidine-linked oxindole and pyrazolone compound bearing four consecutive stereocenters and two vicinal spiroquaternary chiral centers, in excellent yields and stereoselectivities.

The asymmetric construction of CF3-containing spiro-thiazolone-pyrrolidine compounds via [3 + 2] cycloaddition.

The first organocatalytic method for the asymmetric construction of CF3-containing spiro-thiazolone-pyrrolidine compounds has been developed. This is one of the very few methods to reach spiro-thiazolones, and afforded the products in excellent yields and stereoselectivities catalysed by only 1 mol% cinchona alkaloid-derived catalyst.

The in vitro, in vivo antifungal activity and the action mode of Jelleine-I against Candida species.

Recently, the mortality of life-threatening fungal infections increased dramatically. However, there are few antifungals existed. Antimicrobial peptides (AMPs) as promising antifungal candidates have attracted much attention. Here, we present a small antimicrobial peptide Jelleine-I that had potent in vitro and in vivo antifungal activity. Negligible hemolytic activity and in vivo toxicity were observed. Selectivity index (SI) of Jelleine-I is at least 4.6 times higher than amphotericin B. Jelleine-I could increase the production of cellular ROS and bind with genome DNA. This may contribute to its antifungal activity. Furthermore, drug resistance is not induced when the fungal cells were repeatedly treated by Jelleine-I. In conclusion, our results suggest that Jelleine-I may have the potential to be developed as a novel antifungal agent.

Prevalence of patellofemoral pain and knee pain in the general population of Chinese young adults: a community-based questionnaire survey.

BACKGROUND: Previous studies that have described the prevalence of patellofemoral pain (PFP) have been limited to samples of military personnel or sporting populations, and convincing data in the general Chinese population are lacking. The present study defined the prevalence of PFP and knee pain in the general population of Chinese young adults and evaluated whether gender, age, or body mass index (BMI) were associated with PFP. METHODS: An anonymous online questionnaire survey was open to the general public in China. A self-report questionnaire was used to specifically identify PFP. The population aged 18-40 years was enrolled in the study and completed the questionnaire. The prevalence of PFP and knee pain in the overall sample and in subgroups stratified by sex, age, and BMI was estimated. Logistic regression analysis was conducted to determine if there was a significant association between PFP and sex, age, or BMI. RESULTS: A total of 1153 participants were enrolled in the study. The prevalence of PFP in the overall sample and among the male and female participants was 20.7, 20.3, and 21.2%, respectively. The prevalence of the knee pain in the overall sample and among the male and female participants was 35.6, 38.2, and 33.7%, respectively. The prevalence of PFP in the subgroups stratified by age and BMI did not differ significantly between the groups. Gender, age, and BMI did not have significant associations with the prevalence of PFP. CONCLUSION: PFP is common in the general Chinese population. Clinicians should direct more attention toward the early diagnosis of and interventions for PFP.

Asymmetric Synthesis of α-Trifluoromethyl Pyrrolidines through Organocatalyzed 1,3-Dipolar Cycloaddition Reaction.

The optically active α-trifluoromethyl pyrrolidines have been achieved through organocatalyzed 1,3-dipolar cycloaddition reaction first. With diphenylprolinol trimethylsilyl ether as catalyst and in the presence of 3,5-dinitrobenzoic acid, the reaction of trifluoroethylamine-derived ketimine with 2-enals gave α-trifluoromethyl pyrrolidines bearing three contiguous stereogenic centers in excellent diastereoselectivies, stereoselectivities, and yields.

Asymmetric synthesis of CF3-containing tetrahydroquinoline via a thiourea-catalyzed cascade reaction.

An organocatalytic asymmetric method for the synthesis of 2-CF3 tetrahydroquinoline has been achieved. The cascade reaction of 2-aminochalcones with ß-CF3 nitroalkenes afforded the products bearing three contiguous stereogenic centers in good yields with excellent diastereoselectivities and enantioselectivities.

Antimicrobial activity and stability of protonectin with D-amino acid substitutions.

The misuse and overuse of antibiotics result in the emergence of resistant bacteria and fungi, which make an urgent need of the new antimicrobial agents. Nowadays, antimicrobial peptides have attracted great attention of researchers. However, the low physiological stability in biological system limits the application of naturally occurring antimicrobial peptides as novel therapeutics. In the present study, we synthesized derivatives of protonectin by substituting all the amino acid residues or the cationic lysine residue with the corresponding D-amino acids. Both the D-enantiomer of protonectin (D-prt) and D-Lys-protonectin (D-Lys-prt) exhibited strong antimicrobial activity against bacteria and fungi. Moreover, D-prt showed strong stability against trypsin, chymotrypsin and the human serum, while D-Lys-prt only showed strong stability against trypsin. Circular dichroism analysis revealed that D-Lys-prt still kept typical α-helical structure in the membrane mimicking environment, while D-prt showed left hand α-helical structure. In addition, propidium iodide uptake assay and bacteria and fungi killing experiments indicated that all D-amino acid substitution or partially D-amino acid substitution analogs could disrupt the integrity of membrane and lead the cell death. In summary, these findings suggested that D-prt and D-Lys-prt might be promising candidate antibiotic agents for therapeutic application against resistant bacteria and fungi infection. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

D-amino acid substitution enhances the stability of antimicrobial peptide polybia-CP.

With the increasing emergence of resistant microbes toward conventional antimicrobial agents, there is an urgent need for the development of antimicrobial agents with novel action mode. Antimicrobial peptides (AMPs) are believed to be one kind of ideal alternatives. However, AMPs can be easily degraded by protease, which limited their therapeutic use. In the present study, D-amino acid substitution strategy was employed to enhance the stability of polybia-CP. We investigated the stability of peptides against the degradation of trypsin and chymotrypsin by determining the antimicrobial activity or determining the HPLC profile of peptides after incubation with proteases. Our results showed that both the all D-amino acid derivative (D-CP) and partial D-lysine substitution derivative (D-lys-CP) have an improved stability against trypsin and chymotrypsin. Although D-CP takes left-hand α-helical conformation and D-lys-CP loses some α-helical content, both of the D-amino acid-substituted derivatives maintain their parental peptides' membrane active action mode. In addition, D-lys-CP showed a slight weaker antimicrobial activity than polybia-CP, but the hemolytic activity decreased greatly. These results suggest that D-CP and D-lys-CP can offer strategy to improve the property of AMPs and may be leading compounds for the development of novel antimicrobial agents.

Antimicrobial peptide protonectin disturbs the membrane integrity and induces ROS production in yeast cells.

Candidiasis is often observed in immunocompromised patients and is the 4th most common cause of bloodstream infections. However, antifungals are limited, so novel antifungal agents are urgently needed. Antimicrobial peptides (AMPs) are considered as potential alternatives of conventional antibiotics. In the present study, antimicrobial peptide protonectin was chemically synthesized and its antifungal activity and mode of action were studied. Our results showed that protonectin has potent antifungal activity and fungicidal activity against the tested fungi cells. Its action mode involved the disruption of the membrane integrity and the inducing of the production of cellular ROS. Furthermore, protonectin could inhibit the formation of biofilm and kill the adherent fungi cells. In conclusion, with the increase of fungal infection, protonectin may offer a new strategy and be considered as a potential therapeutic agent against fungal disease.