Exploring the potential of omics technologies in medicinal plant research: a review in Colombia / Explorando el potencial de las tecnologías ómicas en la investigación de plantas medicinales: una revisión en Colombia

This review presents advances in the implementation of high - throughput se quencing and its application to the knowledge of medicinal plants. We conducted a bibliographic search of papers published in PubMed, Science Direct, Google Scholar, Scopus, and Web of Science databases and analyzed the obtained data using VOSviewer (versi on 1.6.19). Given that medicinal plants are a source of specialized metabolites with immense therapeutic values and important pharmacological properties, plant researchers around the world have turned their attention toward them and have begun to examine t hem widely. Recent advances in sequencing technologies have reduced cost and time demands and accelerated medicinal plant research. Such research leverages full genome sequencing, as well as RNA (ribonucleic acid) sequencing and the analysis of the transcr iptome, to identify molecular markers of species and functional genes that control key biological traits, as well as to understand the biosynthetic pathways of bioactive metabolites and regulatory mechanisms of environmental responses. As such, the omics ( e.g., transcriptomics, metabolomics, proteomics, and genomics, among others) have been widely applied within the study of medicinal plants, although their usage in Colombia is still few and, in some areas, scarce. (185)

El extracto de cloroformo (CE) y las fracciones obtenidas de las raíces de Aldama arenaria se evaluaron para determinar su actividad antiproliferativa in vitro contra 10 líneas ce lulares tumorales humanas [leucemia (K - 562), mama (MCF - 7), ovario que expresa un fenotipo resistente a múltiples fármacos (NCI/ADR - RES), melanoma (UACC - 62), pulmón (NCI - H460), próstata (PC - 3), colon (HT29), ovario (OVCAR - 3), glioma (U251) y riñón (786 - 0)]. CE presentó actividad antiproliferativa débil a moderada (log GI 50 medio 1.07), mientras que las fracciones 3 y 4, enriquecidas con diterpenos de tipo pimarane [ent - pimara - 8 (14), ácido 15 - dien - 19 - oico y ent - 8(14),15 - pimaradien - 3 ß - ol], presentaron activid ad moderada a potente para la mayoría de las líneas celulares, con un log GI 50 medio de 0.62 y 0.59, respectivamente. Los resultados mostraron una acción antiproliferativa in vitro prometedora de las muestras obtenidas de A. arenaria , con los mejores resul tados para NCI/ADR - RES, HT29 y OVCAR - 3, y valores de TGI que van desde 5.95 a 28.71 µg.mL - 1, demostrando que los compuestos de esta clase pueden ser prototipos potenciales para el descubrimiento de nuevos agentes terapéuticos

Macular dystrophies associated with Stargardt-like phenotypes / Distrofias maculares associadas a fenótipos Stargardt-like

ABSTRACT Purpose: Stargardt-like phenotype has been described as associated with pathogenic variants besides the ABCA4 gene. This study aimed to describe four cases with retinal appearance of Stargardt disease phenotypes and unexpected molecular findings. Methods: This report reviewed medical records of four patients with macular dystrophy and clinical features of Stargardt disease. Ophthalmic examination, fundus imaging, and next-generation sequencing were performed to evaluate pathogenic variants related to the phenotypes. Results: Patients presented macular atrophy and pigmentary changes suggesting Stargardt disease. The phenotypes of the two patients were associated with autosomal dominant inheritance pattern genes (RIMS1 and CRX) and in the other two patients were associated with recessive dominant inheritance pattern genes (CRB1 and RDH12) with variants predicted to be pathogenic. Conclusion: Macular dystrophies may have phenotypic similarities to Stargardt-like phenotype associated with other genes besides the classic ones.

RESUMO Objetivo: Fenótipos Stargardt-like já foram asso-ciados a variantes patogênicas no gene ABCA4. O propósito desse estudo é descrever quatro pacientes com achados retinianos semelhantes a doença de Stargardt com resultados moleculares diferentes do esperado. Métodos: Esse relato fez a revisão de prontuários médicos de quatro pacientes com distrofia macular e achados clínicos sugestivos de doença de Stargardt. Foram realizados avaliação oftalmológica, exames de imagens e testes usando next generation sequencing para avaliar variantes patogênicas associadas aos fenótipos dos pacientes. Resultados: Os pacientes apresentavam atrofia macular e alterações pigmentares sugerindo achados clínicos de doença de Stargardt. Dois pacientes foram associados a genes com herança autossômica dominante (RIMS1 e CRX) e dois pacientes foram associados a genes com herança autossômica recessiva (CRB1 e RDH12) com variantes preditoras de serem patogênicas. Conclusão: Distrofias maculares podem ter similaridades fenotípicas com fenótipo de Stargardt-like associados a outros genes além dos classicamente já descritos.

Anti-SARS-CoV-2 activity of small molecule inhibitors of cathepsin L / 药学学报

The coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which has led to serious worldwide economic burden. Due to the continuous emergence of variants, vaccines and monoclonal antibodies are only partial effective against infections caused by distinct strains of SARS-CoV-2. Therefore, it is still of great importance to call for the development of broad-spectrum and effective small molecule drugs to combat both current and future outbreaks triggered by SARS-CoV-2. Cathepsin L (CatL) cleaves the spike glycoprotein (S) of SARS-CoV-2, playing an indispensable role in enhancing virus entry into host cells. Therefore CatL is one of the ideal targets for the development of pan-coronavirus inhibitor-based drugs. In this study, a CatL enzyme inhibitor screening model was established based on fluorescein labeled substrate. Two CatL inhibitors IMB 6290 and IMB 8014 with low cytotoxicity were obtained through high-throughput screening, the half inhibition concentrations (IC50) of which were 11.53 ± 0.68 and 1.56 ± 1.10 μmol·L-1, respectively. SDS-PAGE and cell-cell fusion experiments confirmed that the compounds inhibited the hydrolysis of S protein by CatL in a concentration-dependent manner. Surface plasmon resonance (SPR) detection showed that both compounds exhibited moderate binding affinity with CatL. Molecular docking revealed the binding mode between the compound and the CatL active pocket. The pseudovirus experiment further confirmed the inhibitory effects of IMB 8014 on the S protein mediated entry process. In vitro pharmacokinetic evaluation indicated that the compounds had relatively good drug-likeness properties. Our research suggested that these two compounds have the potential to be further developed as antiviral drugs for COVID-19 treatment.

Characterization and phylogenetic analysis of chloroplast genome of Cynanchum wallichii and Cynanchum otophyllum / 药学学报

italic>Cynanchum wallichii and Cynanchum otophyllum belong to the genus Cynanchum in the family Apocynaceae, and are important medicinal plants. In this study, we sequenced and assembled the chloroplast genomes of C. wallichii and C. otophyllum, and performed a phylogenetic analysis of the structural characteristics of their chloroplast genomes and their phylogenetic positions. The results showed that the chloroplast genomes of both C. wallichii and C. otophyllum had a typical tetrad structure, with 133 genes annotated, and the total GC contents of both were similar. Codon preference analysis showed that the relative synonymous codon usage in the chloroplast genomes of C. wallichii and C. otophyllum differed slightly, but the differences were not significant, and there was a strong A or U preference at the third codon position. In both chloroplast genomes, 91 and 103 simple sequence repeats were detected respectively, and the largest proportion of A/T type repeats. Nucleotide polymorphism analysis showed that the nucleotide diversity of the intergenic sequences in the chloroplast genome of genus Cynanchum were generally higher than those of the common gene sequences. A pair of primers was designed based on the high variation region of the chloroplast genome to identify C. wallichii and C. otophyllum. The phylogenetic analysis showed that the C. wallichii and Cynanchum thesioides were the closest relatives, while the C. otophyllum, Cynanchum bungei and Cynanchum wilfordii formed a stable monophyletic clade within the genus Cynanchum, and the three species were closely related. The comparative analysis of the chloroplast genomic characteristics and phylogeny of C. wallichii and C. otophyllum will provide a theoretical basis for the species identification of the two plants and for the study of genetic diversity and phylogeny of the genus Cynanchum.

Establishment of a high-throughput screening platform based on drug repurposing targeting alpha-1-acid glycoprotein and discovery of potential weight loss drugs / 药学实践杂志

Objective Alpha-1-acid glycoprotein （ORM） was a new target for the development of weight loss drugs. To search for potential weight loss drugs that could target ORM from the compound library of already marketed drugs based on drug repurposing. Methods The pGL4.20-ORM1 promoter recombinant plasmid was contructed and validated, and then a lentiviral vector was utilized to establish stable AML12 cell lines expressing ORM1 promoter-LUC-PURO. This cell line was employed for high-throughput screening of compounds from the marketed drug library, and the luminescence value of the cells was characterized by enzyme marker. Results Primary screening and secondary screening of 1 470 compounds identified 42 compounds that increased ORM1 promoter expression and could be used for further weight loss effect assessment. Conclusion This study successfully constructed LV-AML12-ORM1 promoter-LUC-PURO stable expression cell lines using lentiviral vectors, laying a foundation for efficient and stable screening of weight loss drugs targeting ORM.

Evolution of chemistry and selection technology for DNA-encoded library

DNA-encoded chemical library (DEL) links the power of amplifiable genetics and the non-self-replicating chemical phenotypes, generating a diverse chemical world. In analogy with the biological world, the DEL world can evolve by using a chemical central dogma, wherein DNA replicates using the PCR reactions to amplify the genetic codes, DNA sequencing transcripts the genetic information, and DNA-compatible synthesis translates into chemical phenotypes. Importantly, DNA-compatible synthesis is the key to expanding the DEL chemical space. Besides, the evolution-driven selection system pushes the chemicals to evolve under the selective pressure, i.e., desired selection strategies. In this perspective, we summarized recent advances in expanding DEL synthetic toolbox and panning strategies, which will shed light on the drug discovery harnessing in vitro evolution of chemicals via DEL.

A multidimensional platform of patient-derived tumors identifies drug susceptibilities for clinical lenvatinib resistance

Lenvatinib, a second-generation multi-receptor tyrosine kinase inhibitor approved by the FDA for first-line treatment of advanced liver cancer, facing limitations due to drug resistance. Here, we applied a multidimensional, high-throughput screening platform comprising patient-derived resistant liver tumor cells (PDCs), organoids (PDOs), and xenografts (PDXs) to identify drug susceptibilities for conquering lenvatinib resistance in clinically relevant settings. Expansion and passaging of PDCs and PDOs from resistant patient liver tumors retained functional fidelity to lenvatinib treatment, expediting drug repurposing screens. Pharmacological screening identified romidepsin, YM155, apitolisib, NVP-TAE684 and dasatinib as potential antitumor agents in lenvatinib-resistant PDC and PDO models. Notably, romidepsin treatment enhanced antitumor response in syngeneic mouse models by triggering immunogenic tumor cell death and blocking the EGFR signaling pathway. A combination of romidepsin and immunotherapy achieved robust and synergistic antitumor effects against lenvatinib resistance in humanized immunocompetent PDX models. Collectively, our findings suggest that patient-derived liver cancer models effectively recapitulate lenvatinib resistance observed in clinical settings and expedite drug discovery for advanced liver cancer, providing a feasible multidimensional platform for personalized medicine.

High diagnostic yield with algorithmic molecular approach on hereditary neuropathies

SUMMARY OBJECTIVE: Charcot-Marie-Tooth disease covers a group of inherited peripheral neuropathies. The aim of this study was to investigate the effect of targeted next-generation sequencing panels on the molecular diagnosis of Charcot-Marie-Tooth disease and its subtypes in routine clinical practice, and also to show the limitations and importance of next-generation sequencing in the diagnosis of Charcot-Marie-Tooth diseases. METHODS: This is a retrospective study. Three different molecular methods (multiplex ligation probe amplification, next-generation sequencing, and whole-exome sequencing) were used to detect the mutations related to Charcot-Marie-Tooth disease. RESULTS: In total, 64 patients (33 males and 31 females) with suspected Charcot-Marie-Tooth disease were analyzed for molecular etiology. In all, 25 (39%) patients were diagnosed by multiplex ligation probe amplification. With an extra 11 patients with normal PMP22 multiplex ligation probe amplification results that were consulted to our laboratory for further genetic analysis, a total of 50 patients underwent next-generation sequencing for targeted gene panels associated with Charcot-Marie-Tooth disease. Notably, 18 (36%) patients had pathogenic/likely pathogenic variants. Whole-exome sequencing was performed on five patients with normal next-generation sequencing results; the diagnostic yield by whole-exome sequencing was 80% and it was higher in the childhood group. CONCLUSION: The molecular etiology in Charcot-Marie-Tooth disease patients can be determined according to pre-test evaluation, deciding the inheritance type with pedigree analysis, the clinical phenotype, and an algorithm for the genetic analysis. The presence of patients without a molecular diagnosis in all the literature suggests that there are new genes or mechanisms waiting to be discovered in the etiology of Charcot-Marie-Tooth disease.

Intervention of next-generation sequencing in diagnosis of Alzheimer's disease: challenges and future prospects / Intervenção do sequenciamento de nova geração no diagnóstico da doença de Alzheimer: desafios e perspectivas futuras

ABSTRACT Clinical diagnosis of several neurodegenerative disorders based on clinical phenotype is challenging due to its heterogeneous nature and overlapping disease manifestations. Therefore, the identification of underlying genetic mechanisms is of paramount importance for better diagnosis and therapeutic regimens. With the emergence of next-generation sequencing, it becomes easier to identify all gene variants in the genome simultaneously, with a system-wide and unbiased approach. Presently various bioinformatics databases are maintained on discovered gene variants and phenotypic indications are available online. Since individuals are unique in their genome, evaluation based on their genetic makeup helps evolve the diagnosis, counselling, and treatment process at the personal level. This article aims to briefly summarize the utilization of next-generation sequencing in deciphering the genetic causes of Alzheimer's disease and address the limitations of whole genome and exome sequencing.

RESUMO O diagnóstico clínico de vários distúrbios neurodegenerativos com base no fenótipo clínico é difícil devido à sua natureza heterogênea e às manifestações da doença que se sobrepõem. Portanto, a identificação dos mecanismos genéticos subjacentes é de suma importância para um melhor diagnóstico e regimes terapêuticos. Com o surgimento do sequenciamento de próxima geração, o diagnóstico se tornou mais acessível com uma abordagem imparcial em todo o sistema para identificar simultaneamente todas as variantes de genes no genoma. Atualmente, vários bancos de dados de bioinformática sobre variantes genéticas descobertas e indicações fenotípicas estão disponíveis online. Uma vez que os indivíduos são únicos em seu genoma, a avaliação com base em sua composição genética ajudou na evolução do processo de diagnóstico, aconselhamento e tratamento em nível pessoal. Este artigo teve como objetivo resumir brevemente a utilização do sequenciamento de próxima geração para decifrar as causas genéticas da doença de Alzheimer (DA) e abordar as limitações do sequenciamento completo do genoma e do exoma.

Genome sequencing reveals molecular subgroups in oral epithelial dysplasia

Abstract This study aimed to analyze the molecular characteristics of oral epithelial dysplasia (OED), highlighting the pathways and variants of genes that are frequently mutated in oral squamous cell carcinoma (OSCC) and other cancers. Ten archival OED cases were retrieved for retrospective clinicopathological analysis and exome sequencing. Comparative genomic analysis was performed between high-grade dysplasia (HGD) and low-grade dysplasia (LGD), focusing on 57 well-known cancer genes, of which 10 were previously described as the most mutated in OSCC. HGD cases had significantly more variants; however, a similar mutational landscape to OSCC was observed in both groups. CASP8+FAT1/HRAS, TP53, and miscellaneous molecular signatures were also present. FAT1 is the gene that is most affected by pathogenic variants. Hierarchical divisive clustering showed division between the two groups: "HGD-like cluster" with 4HGD and 2LGD and "LGD-like cluster" with 4 LGD. MLL4 pathogenic variants were exclusively in the "LGD-like cluster". TP53 was affected in one case of HGD; however, its pathway was usually altered. We describe new insights into the genetic basis of epithelial malignant transformation by genomic analysis, highlighting those associated with FAT1 and TP53. Some LGDs presented a similar mutational landscape to HGD after cluster analysis. Perhaps molecular alterations have not yet been reflected in histomorphology. The relative risk of malignant transformation in this molecular subgroup should be addressed in future studies.

Community structure of endophytic fungi in roots and leaves of Fagopyrum mill and Avena sativa in a Chinese northern cold region

In order to explore the endophytic fungi of Fagopyrum Mill and Avena sativa, Illumina Miseq high-throughput sequencing was used to analyze the community structure and diversity of endophytic fungi in leaves and roots of buckwheat and oat at the mature stage. The results of community structure showed that there were 205 operational taxonomic units (OTUs) in buckwheat roots and 181 OTUs in buckwheat leaves based on 97% sequence similarity level. There were 152 OTUs and 127 OTUs in the root and the leaf of oat, respectively. At the phylum level, Ascomycota and Basidiomycota were the dominant endophytic fungi in buckwheat roots and leaves, while Ascomycota was the dominant endophytic fungus in oat roots and leaves. Alpha diversity analysis showed that the Ace index, Chao index and Shannon index of buckwheat roots were higher than that of buckwheat leaves, and the three indices of oat roots were also higher than that of oat leaves, indicating that the richness and diversity of endophytic fungi community in roots were higher than that in leaves. Biomarkers were found by significant difference analysis in buckwheat and oat. The endophytic functional groups of buckwheat and oat were mainly distributed in Pathotroph and Saprotroph. The results of this study laid a foundation for fully exploiting the dominant endophytic fungal resources of buckwheat and oat and further developing microbial fertilizers.

High-Throughput Sequencing Technology for Detection of Gene Mutations in Myeloid Malignancies and Its Clinical Prognostic Significance / 中国实验血液学杂志

OBJECTIVE@#To detect the gene mutations in patients with myeloid malignancies by high-throughput sequencing and explore the correlation between gene mutations and prognosis.@*METHODS@#A retrospective analysis was performed on 56 patients with myeloid malignancies who were hospitalized in the department of hematology, Peking University International Hospital from January 2020 to May 2021. The genetic mutations of the patients were detected by next-generation sequencing technology, and the correlation between the genetic mutations and prognosis of myeloid malignancies was analyzed.@*RESULTS@#In 56 patients, the number of mutated genes detected in a single patient is 0-9, with a median of 3. Sequencing results showed that the most common mutated genes were RUNX1(21.4%), TET2(17.9%), DNMT3A(17.9%), TP53(14.3%) and ASXL1(14.3%), among which the most common mutations occurred in the signaling pathway-related genes (23.3%) and the transcription factor genes (18.3%). 84% of the patients carried multiple mutated genes (≥2), and correlation analysis showed there were obvious co-occurring mutations between WT1 and FLT3, NPM1 and FLT3-ITD, and MYC and FLT3. TP53 mutation was more common in MDS patients.The overall survival time of patients with NRAS mutation was significantly shortened (P =0.049). The prognosis of patients with TP53 mutation was poor compared with those without TP53 mutation, but the difference wasn't statistically significant (P =0.08).@*CONCLUSION@#The application of next-generation sequencing technology is of great significance in myeloid malignancies, which is helpful to better understand the pathogenesis of the disease, to judge the prognosis and to find possible therapeutic targets.

Preliminary study on high throughput screening small molecules targeting Gram-negative bacilli outer membrane protein BamA / 中华检验医学杂志

Objective:High-throughput screening to obtain small molecular compounds against Gram-negative bacilli by targeting BamA outer membrane protein.Methods:The sybyl-X2.1 software was used to perform high-throughput virtual screening of small molecular compounds in Chemdiv compound library based on the molecular docking. The top 150 hits by high-throughput screening were re-screened through in vitro biological experiments. The top 4 small molecules with obvious antibacterial activity were selected for in-depth molecular docking analysis, and the small molecule 8308-0401 with the highest docking score was selected for further experiments. The antibacterial effect of 8308-0401 combined with rifampicin was tested by checkerboard assay. Finally, the affinity between 8308-0401 and BamA was tested by plasma surface resonance assay. Results:The docking score of the top 150 hits calculated by high-throughput virtual screening had a mean value of 5.63. In vitro biological experiments showed that small molecules 8308-0401, 8365-1335, C066-2507 and L582-0346 exhibited strong antibacterial activity. Among those molecules, 8308-0401 showed the highest molecular docking score, and synergistic antibacterial activity against both types of strains and clinical isolates when combined with rifampicin. 8308-0401 has a strong affinity to BamA with binding a constant of 182 μmol/L. Conclusion:The small molecule 8308-0401 exerts antibacterial activity against Gram negative bacilli by targeting the outer membrane protein BamA.

Clinical laboratory genetic diagnosis of Marfan syndrome / 中华检验医学杂志

Objective:To establish the clinical laboratory genetic diagnosis procedures for Marfan syndrome (MFS) and carry out clinical laboratory genetic diagnosis for MFS families.Methods:The second generation high-throughput sequencing was used to sequence and analyze the FBN1 gene of two MFS families who visited to Fuwai Central China Cardiovascular Hospital (Heart Center of Henan People′s Hospital) from January to December 2020, and then Sanger sequencing was used to verify the second generation high-throughput sequencing results. At the same time, the sanger sequencing of mutation sites was performed on normal family members and 100 healthy people to identify the pathogenic mutations of FBN1 gene in the MFS families. The pregnant women of two families were guided for prenatal diagnosis in the second trimester of pregnancy.Results:The clinical laboratory diagnosis of MFS showed that two MFS patients had the pathogenic mutation of c.2560T>C heterozygous mutation and c.6772T>C heterozygous mutation in FBN1 gene, respectively. The mutation was not observed in 100 healthy people and normal members in two families. The prenatal diagnosis showed that there was a heterozygous mutation of FBN1 gene c.2560T>C in the first fetus of the MFS family, which was MFS. There was no mutation in the FBN1 gene in the second fetus of the MFS family, so it was recommended to continue the pregnancy. The results of postpartum follow-up were consistent with the results of clinical laboratory diagnosis.Conclusion:The clinical laboratory genetic diagnosis procedures for MFS have been established successfully, which provides an important reference for clarifying the clinical diagnosis of MFS.

Fetal RHD screening from circulating cell-free DNA in RhD-negative pregnant women / 中华围产医学杂志

Objective:To investigate the RHD genotypes of RhD-negative pregnant women and explore the optimum strategy for fetal RHD screening among this population in the region. Methods:This prospective study recruited 33 cases of RhD-negative singleton pregnancies at ≥12 weeks of gestation in Nanjing Drum Tower Hospital from March to November 2021. On the basis of RHD genotyping, quantitative real-time polymerase chain reaction (PCR) was used to amplify the exons 5 and 10 of RHD gene in the circulating cell-free DNA of RhD-negative pregnant women harboring whole RHD gene deletion and RHD-CE(2-9)- D. High-throughput sequencing was performed to detect chr1:25648453 locus from circulating cell-free DNA in plasma of RhD-negative pregnant women harboring RHD 1227A mutation to screen the fetal RhD blood group. Neonatal umbilical cord blood samples were collected for verifying fetal RHD genotyping. Descriptive statistical analysis was used. Results:Whole RHD gene deletion homozygous genotype ( n=20, 60.6%), RHD-CE(2-9) -D/whole RHD gene deletion heterozygous genotype ( n=5, 21.2%), RHD 1227A/whole RHD gene deletion heterozygous genotype ( n=7, 15.2%) and RHD 711delC/whole RHD gene deletion heterozygous genotype ( n=1) were identified in the 33 RhD-negative pregnant women. In the 25 cases with whole RHD gene deletion homozygous genotype or RHD-CE(2-9)- D/whole RHD gene deletion heterozygous genotype, 22 fetuses were RhD-positive and three were RhD-negative based on prenatal screening, which were confirmed by the neonatal serological test results after birth. In the seven cases carrying RHD 1227A/whole RHD gene deletion heterozygous genotype, all fetuses were RhD-positive, which were consistent with the results of serological detection after delivery. The case harboring RHD 711delC/whole RHD gene deletion heterozygous genotype did not receive fetal RHD screening. Conclusions:This study suggests that whole RHD gene deletion homozygous genotype is the most common allele in RhD-negative population in this area, followed by RHD 1227A/whole RHD gene deletion heterozygous genotype and RHD- CE(2-9)- D/whole RHD gene deletion heterozygous genotype. For women with whole RHD gene deletion homozygous genotype, RHD- CE(2-9)- D, or RHD 1227A mutation, fetal RHD screening with quantitative real-time PCR and high-throughput sequencing are important for the management of RhD-negative pregnant women.

Analysis of differential expression profiles of Piwi-interacting RNA in diabetic nephropathy patients / 中华肾脏病杂志

Objective:To investigate the correlation between Piwi-interacting RNA (piRNA) and diabetic nephropathy (DN).Methods:The differential expression profiles of piRNAs in renal tissues of patients with DN (experimental group) and renal tissues adjacent to tumors of patients with renal tumors (control group) were detected by high-throughput sequencing. The biological function of differentially expressed piRNAs was described by gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis. Real-time fluorescence quantitative PCR was used to detect the serum expression level of target piRNAs in patients with DN. Spearman correlation analysis was used to analyze the correlation between serum target piRNAs and clinical indexes of patients with DN.Results:The results of high throughput sequencing showed that there were 127 differentially expressed piRNAs between DN group and control group, with screening condition of |log 2(fold changes)|≥2 and P<0.05. Among them, there were 99 up-regulated piRNAs and 28 down-regulated piRNAs. The top 5 up-regulated piRNAs were piRNA-hsa-161686, piRNA-hsa-349255, piRNA-hsa-355720, piRNA-hsa-151229 and piRNA-hsa-154959, respectively. The top 5 down-regulated piRNAs were piRNA-hsa-1929960, piRNA-hsa-174194, piRNA-hsa- 148658, piRNA-hsa-172594 and piRNA-hsa-172421, respectively. The PCR verification results of 3 up-regulated genes and 3 down-regulated genes with low P values and high expression levels showed that serum expression level of piRNA-hsa-77976 was significantly down-regulated in patients with DN ( P=0.028), which was consistent with that of sequencing, while the expression levels of other genes were inconsistent with the sequencing results or had no statistical significance. Bioinformatics analysis results predicted that significantly differentially expressed piRNAs might participate in the regulation of DN through Rap1, Ras, PI3K-Akt and axon guiding pathways. The results of correlation analysis showed that the expression level of piRNA-hsa-77976 was negatively correlated with blood urea nitrogen ( r=-0.584, P=0.028), serum creatinine ( r=-0.637, P=0.014), cystatin C ( r=-0.738, P=0.003) and β2 microglobulin ( r=-0.822, P<0.001), and positively correlated with estimated glomerular filtration rate ( r=0.661, P=0.010). Conclusion:The differential expression of piRNA is closely related to DN, and may be used as a new biomarker for the diagnosis and prognosis of DN.

Establishment and characterization of high-throughput culture platform for kidney organoids / 中华肾脏病杂志

Objective:To establish and identify a high-throughput culture platform for induced pluripotent stem cells to differentiated kidney organoids.Methods:Human urine-derived induced pluripotent stem cells were selected and plated at a suitable cell density, and differentiated using small molecule compounds such as CHIR99021/fibroblast growth factor 9/heparin during day 1-6. On day 7, cells with appropriate density were digested and resuspended, than added to a 96-well 3D culture plate for 24 hours. After the cells formed spheroids, fibroblast growth factor 9 and heparin were added to induce differentiation till day 24. The immunofluorescence and transmission electron microscopy were used to compare the differences of kidney organoids obtained by the reported differentiation protocol (transwell protocol) method and high-throughput culture platform.Results:Kidney organoids were successfully differentiated by two protocols. Immunofluorescence results showed that LTL, GATA-3, and synaptopodin, which were major kidney cell markers, were all expressed, and mature renal organoids were formed. The results of transmission electron microscopy showed that the kidney organoids successfully developed foot processes, the unique cellular feature of the glomerular podocytes, which were evenly distributed and neatly interspersed with each other. At the same intermediate mesodermal cell count of 1.0×10 7, approximately 7 renal organoids were obtained by the transwell protocol, while approximately 1 000 renal organoids were obtained by the high-throughput culture platform. Conclusion:A high-throughput culture platform for kidney organoids is successfully established, and a large amount of mature kidney organoids with complete structure and function can be obtained. The differentiation efficiency of kidney organoids is greatly improved.

Analysis of the nail microbiome in patients with onychomycosis by high-throughput sequencing of 16S rDNA and internal transcribed spacer regions / 中华皮肤科杂志

Objective:To investigate differences in bacterial and fungal microbiome between infected nails and healthy nails among patients with onychomycosis.Methods:Nail scraping samples were collected from infected nails and healthy nails of 31 patients with onychomycosis, who visited Dalian Dermatosis Hospital from August 2020 to July 2021. The total DNA of nail microbiota was extracted, and the V3-V4 regions of the bacterial 16S rDNA gene and the fungal internal transcribed spacer (ITS) region were amplified and sequenced using Illumina technology. The USEARCH and mothur softwares were used for data cluster analysis to obtain the operational taxonomic units (OTUs) , Wilcoxon rank sum test was used to analyze α diversity, analysis of similarities (ANOSIM) was performed to analyze β diversity, linear discriminant analysis of effect size (LEfSe) was performed to evaluate the species difference.Results:Among the 31 patients with onychomycosis, 16 were males and 15 were females. According to the age, they were divided into young group (18 - 35 years old, 10 cases) , middle-aged group (36 - 60 years old, 11 cases) , and elderly group (over 60 years old, 10 cases) . As the α-diversity analysis revealed, the infected nail group showed significantly decreased Shannon index ( W = 290, P = 0.007) , but significantly increased Simpson index ( W = 663, P = 0.010) compared with the healthy nail group, suggesting that the diversity and evenness of bacterial communities were lower in the infected nail group than in the healthy nail group; however, there was no significant difference in the diversity of fungal communities between the infected nail group and healthy nail group. The β-diversity analysis based on the unweighted-UniFrac distance matrix showed no significant difference in the fungal or bacterial community composition between the infected nail group and healthy nail group (bacterial communities: R = 0.0052, P = 0.331; fungal communities: R = 0.0036, P = 0.337) ; the β-diversity analysis based on the weighted-UniFrac distance matrix showed significant differences in the abundance of bacterial and fungal communities between the two groups (both P = 0.001) . In terms of the species composition, the bacterial flora with significantly decreased abundance in the infected nail group compared with the healthy nail group included Bacteroidetes, Proteobacteria, Betaproteobacteria, Burkholderiales, Ralstonia, Sphingomonas and Streptococcus, while the abundance of Bacilli, Bacillales and Staphylococcus was significantly higher in the infected nail group than in the healthy nail group. Compared with the healthy nail group, the fungal flora with significantly increased abundance in the infected nail group included Eurotiomycetes, Onygenales, Leotiomycetes-ord-incertae-sedis, Arthrodermataceae, Periconia, Erysiphe, Tilletiopsis, Trichophyton, Erysiphe cruciferarum, Trichophyton rubrum, Malassezia sympodialis, while the abundance of Saccharomycetes, Saccharomycetales, Saccharomycetaceae, Dothioraceae, Candida and Alternaria was significantly lower in the infected nail group than in the healthy nail group. Conclusion:The diversity and abundance of bacterial communities significantly differed between infected nails and healthy nails among patients with onychomycosis, while only the abundance of fungal communities differed between the two groups, and perhaps there was correlations between some bacterial and fungal communities.

Effects of modified Buzhong Yiqi Decoction on intestinal microflora in a rat model of chronic obstructive pulmonary disease / 中国基层医药

Objective:To investigate the effects of modified Buzhong Yiqi Decoction on intestinal microflora in a rat model of chronic obstructive pulmonary disease. Methods:From April to June 2021, 60 specific pathogen-free Wistar rats were selected for this study. They were randomly divided into blank control, model, traditional Chinese medicine, and western medicine groups with 15 rats per group. Rat models of chronic obstructive pulmonary disease with lung and spleen deficiency were established in all groups except the blank control group. Rat models in the traditional Chinese medicine and western medicine groups were administered modified Buzhong Yiqi Decoction and synbiotics. Rat models in the model and blank control groups were identically administered 0.9% sodium chloride injection. After 7 days, the feces of rats in each group were collected for 16S rRNA sequencing of intestinal flora. Effective sequences were clustered to obtain operational taxonomic units for principal coordinate analysis, species composition analysis, and alpha diversity analysis. The effects of modified Buzhong Yiqi Decoction on the structure, diversity, and abundance changes of intestinal flora were analyzed. Results:The dominant bacteria in the traditional Chinese medicine and western medicine groups were Firmicutes, while the dominant bacteria in the blank control and model groups were Bacteroides. The dominant bacterial groups in each group were mainly Lactobacillus and Bacteroides. Alpha diversity analysis showed that the Shannon index in the community diversity indices of traditional Chinese medicine, western medicine, and blank control groups was (3.65 ± 0.35), (3.65 ± 0.36), and (3.59 ± 0.20), respectively, which were significantly higher than (3.37 ± 0.26) in the model group ( t = 2.49, 2.44, 2.60, all P < 0.05). There was no significant difference in the Shannon index among traditional Chinese medicine, western medicine, and blank control groups (all P > 0.05). The Sobs index of the traditional Chinese medicine, western medicine, and blank control group was (458.67 ± 73.11), (454.80 ± 95.13), and (525.93 ± 101.88), respectively, which were significantly higher than (337.40 ± 37.49) in the model group ( t = 5.72, 4.45, 6.73, all P < 0.05). The Sobs index in the blank control group was higher than that in the western medicine group. There was no significant difference in the Sobs index between blank control and traditional Chinese medicine groups and between western medicine and traditional Chinese medicine groups (both P > 0.05). Principal coordinate analysis revealed that compared with the blank control group, Actinomycetes decreased and Proteobacteria and Desulfurization bacteria increased at the phylum level in the model group, while compared with the blank control group, Bacteroides, Rombutzia,Trichospirillus, and Parabacteroides increased, and Prevotella, Clostridium, Brucella, and Ruminococcus decreased at the genus level. Compared with the western medicine group, Bacillus, Prevotella, Brucella, and Prevotellidae in the traditional Chinese medicine group increased, while Clostridium, Pectinobacter, Christensen, and Trichospirillus decreased in the traditional Chinese medicine group. There was a statistically significant difference in the composition of the bacterial population between groups (all P < 0.05). Conclusion:There is an imbalance in intestinal microecology in a rat model of chronic obstructive pulmonary disease. Modified Buzhong Yiqi Decoction can regulate the intestinal microecology environment, improve the structure of intestinal flora, and increase the diversity and abundance of intestinal flora.

Genetic profiles of phenylalanine hydroxylase gene mutations in neonates with phenylketonuria in Xinjiang / 中国新生儿科杂志

Objective:To study the genetic profiles of phenylalanine hydroxylase (PAH) gene mutations in neonates with phenylketonuria (PKU) in Xinjiang.Methods:From January 2015 to December 2021,neonates born and genetically diagnosed with PKU in our region were retrospectively included. The genetic profiles of different ethnic groups were analyzed and compared with PKU patients from central, northwest and northern regions of China.Results:A total of 131 neonates with PKU were enrolled, including 82 Han, 25 Hui and 20 Uyghur patients, 4 cases of other ethnic groups. 46, 20 and 14 types of pathogenic variants were detected in each ethnic group with detection rates of 95.1% (156/164), 66.0% (33/50), and 60.0% (24/40), respectively. The variants were mainly missense mutations and located in exons 2, 3, 6,7 and 11. The most common loci in Hui patients were c.158G>A (18.2%), c.728G>A (18.2%) and c.898G>T (9.1%). The most common loci in Uyghur patients were c.158G>A (33.3%), c.355C>T (12.5%) and c.1068C>A (8.3%). c. 898G>T might be most unique in Hui patients and c.355C>T most unique in Uyghur patients in Xinjiang. A novel variant of PAH gene, c.828G>C (p.M276I) in exon 7 was identified. Compared with northern, central and northwestern regions of China, PKU patients in Xinjiang had significantly higher incidence of c.158G>A mutation and lower incidence of c.728G>A mutation ( P<0.05). Conclusions:Missense mutations of PAH gene are common in some regions of Xinjiang. The compositions of PAH gene variations are similar to northwest and northern China with significant differences in hotspots of mutations.