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.

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.

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.

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.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Application and Prospect of Nanopore Sequencing Technology in Etiological Diagnosis of Blood Stream Infection / 中国医学科学院学报

Blood stream infection (BSI),a blood-borne disease caused by microorganisms such as bacteria,fungi,and viruses,can lead to bacteremia,sepsis,and infectious shock,posing a serious threat to human life and health.Identifying the pathogen is central to the precise treatment of BSI.Traditional blood culture is the gold standard for pathogen identification,while it has limitations in clinical practice due to the long time consumption,production of false negative results,etc.Nanopore sequencing,as a new generation of sequencing technology,can rapidly detect pathogens,drug resistance genes,and virulence genes for the optimization of clinical treatment.This paper reviews the current status of nanopore sequencing technology in the diagnosis of BSI.

Advances of development and application amino acid biosensors / 生物工程学报

Amino acids are the basic building blocks of protein that are very important to the nutrition and health of humans and animals, and widely used in feed, food, medicine and daily chemicals. At present, amino acids are mainly produced from renewable raw materials by microbial fermentation, forming one of the important pillar industries of biomanufacturing in China. Amino acid-producing strains are mostly developed through random mutagenesis- and metabolic engineering-enabled strain breeding combined with strain screening. One of the key limitations to further improvement of production level is the lack of efficient, rapid, and accurate strain screening methods. Therefore, the development of high-throughput screening methods for amino acid strains is very important for the mining of key functional elements and the creation and screening of hyper-producing strains. This paper reviews the design of amino acid biosensors and their applications in the high-throughput evolution and screening of functional elements and hyper-producing strains, and the dynamic regulation of metabolic pathways. The challenges of existing amino acid biosensors and strategies for biosensor optimization are discussed. Finally, the importance of developing biosensors for amino acid derivatives is prospected.

Construction of a caries diagnosis model based on microbiome novelty score / 华西口腔医学杂志

OBJECTIVES@#This study aimed to analyze the bacteria in dental caries and establish an optimized dental-ca-ries diagnosis model based on 16S ribosomal RNA (rRNA) data of oral flora.@*METHODS@#We searched the public databa-ses of microbiomes including NCBI, MG-RAST, EMBL-EBI, and QIITA and collected data involved in the relevant research on human oral microbiomes worldwide. The samples in the caries dataset (1 703) were compared with healthy ones (20 540) by using the microbial search engine (MSE) to obtain the microbiome novelty score (MNS) and construct a caries diagnosis model based on this index. Nonparametric multivariate ANOVA was used to analyze and compare the impact of different host factors on the oral flora MNS, and the model was optimized by controlling related factors. Finally, the effect of the model was evaluated by receiver operating characteristic (ROC) curve analysis.@*RESULTS@#1) The oral microbiota distribution obviously differed among people with various oral-health statuses, and the species richness and species diversity index decreased. 2) ROC curve was used to evaluate the caries data set, and the area under ROC curve was AUC=0.67. 3) Among the five hosts' factors including caries status, country, age, decayed missing filled tooth (DMFT) indices, and sampling site displayed the strongest effect on MNS of samples (P=0.001). 4) The AUC of the model was 0.87, 0.74, 0.74, and 0.75 in high caries, medium caries, low caries samples in Chinese children, and mixed dental plaque samples after controlling host factors, respectively.@*CONCLUSIONS@#The model based on the analysis of 16S rRNA data of oral flora had good diagnostic efficiency.

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.

Different strategies of reducing non-viral nucleic acid of meta-virome in plasma: a comparative study / 中国输血杂志

【Objective】 To explore the influence of common methods of reducing non-viral nucleic acid on the abundance of plasma virus group. 【Methods】 Three kinds of library construction, five kinds of centrifugation conditions, two kinds of filters, four kinds of enzymes and four concentrations of chloroform were used to treat plasma samples added quantitatively 2.16 mL of pseudorabies virus(PRV) and 2.16 mL of porcine parvovirus(PPV). A total of 21.6 mL of plasma samples were processed, including 54 samples. Subsequently, nucleic acid was extracted, mitochondrial DNA(mtDNA) and two viruses were quantitated, the library of the next generation sequencing was constructed, Illumina NovaSeq 6000 was used for the next generation sequencing. The sequencing data were compared with Kraken Py 2.0 software, and the species annotation analysis was conducted. The corresponding species classification information of each segment was obtained to analyze the impact of different reducing non-viral nucleic acid methods on the relative abundance of microorganisms and two indicator viruses. 【Results】 After sequencing by Illumina NovaSeq 6000, 306.27 GB raw data and 193.17 GB clean data were obtained, with Q20>90%, Q30>85%, Error Rate of 0.03%, and average GC Content of 45.02%. The DNA library construction process significantly increased the proportion of microbial sequences and the PRV abundance [(91.8±0.5)%](P<0.05); RNA library construction and combined library construction can increase the abundance of Pestivirus, an RNA virus, and the PRV abundance was(17.7±3.3)% and(8.1±1.5)% respectively. The Ct value of mtDNA was increased and the proportion of human sequence decreased to less than(89.5±1)%, while the proportion of microbial sequence increased to (2.4±0.03)% after treatment of five centrifugation conditions(P<0.05); After centrifugation at 4℃, 100 g, 30 min, the PRV abundance was increased to (40.6±6)%, and centrifugation at 4℃, 4 000 g, 45 min reduced the PRV abundance to (4.1±0.01)%(P<0.05). Both of 0.22-μm filter and 0.45-μm filter increased the Ct value of mtDNA to above 25.56±0.13, decreased the proportion of human sequence to less than (86.1±0.6)%, increased the proportion of microbial sequence to (3.1±0.1)% and (3.4±0.2)%, and decreased the PRV abundance to (1.6±0.3)% and (4.1±0.7)%(P<0.05), while there was no statistical difference in the effect on PPV concentration and abundance. DNase Ⅰ and Benzonase increased the Ct value of PPV to 25.65±0.06 and 25.36±0.45, decreased the proportion of human sequence to (81.7±5.6)% and (72.8±6.7)%, and increased the proportion of microbial sequence and PRV abundance to (11.0±4.1)% and (16.1±4.7)%, (55.8±2.3)% and (39.0±8.9)%, respectively(P<0 05); After treatment with RNase A, the Ct value of PRV increased to 25.20±0.11, and the human sequence proportion decreased to (85.4±5.6)%(P<0 05); Lysozyme had no effect on removing non-viral nucleic acid. The chloroform of 1%, 5%, 10% and 20% increased Ct value of PRV and mtDNA to no less than 27.17±0.21 and 25.68±0.04; Only 10% chloroform increased the proportion of microbial sequences to (3.1±1.2)%(P<0.05); The abundance of PRV with 1% and 5% chloroform treatment was increased to (48.7±13.3)% and (42.1±5.5)%(P<0.05), while 10% and 20% chloroform reduced PRV abundance to (1.0±0.5)% and (3.4±2.8)%(P<0.05). There was no statistical difference in the effect of chloroform with four contents on PPV abundance. 【Conclusion】 Centrifugation at 4℃, 5 000 g, 10 min is suitable for increasing the overall abundance of virus, and centrifugation at 4℃, 100 g, 30 min is suitable for increasing the content of virus similar to PRV. 0.45-μm filter, DNase Ⅰ, Benzonase and low concentration chloroform can effectively reduce the proportion of non-viral nucleic acid sequence in plasma to increase the abundance of the indicated virus group. Thus, the enrichment effect of plasma meta-virome is closely related to the nature of the virus, and the appropriate virus enrichment method should be selected according to the research purpose to establish the corresponding enrichment strategy.

The technologies of small molecule ligand-target protein interaction and high throughput ligand screening / 药学学报

The interaction of drug and target protein is a critical part of new drug discovery. It is the premise for drugs to exert therapeutic effects by targeting specific binding sites of target proteins and thereby affecting its pharmacological activity. Currently, a variety of techniques are exploited to detect the interaction between drug ligands and target proteins. For example, cellular thermal shift assay (CETSA) and differential scanning fluorimetry (DSF) based on thermodynamics, mass spectrometry and nuclear magnetic resonance technology, etc. In addition, high-throughput ligand screening technology provides technical convenience for the search of specific ligand, and is a powerful tool to efficiently identify the interaction between drug ligand and target protein. Here, we summarize the detection techniques of interaction between small molecules and target proteins, and discuss the application of high-throughput ligand screening technology in drug research.

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.

Molecular characterization of mutations associated with resistance among 72 multidrug-resistant strains of Mycobacterium tuberculosis by whole genome sequencing / 中国热带医学

@#Abstract: Objective　To understand the characteristics of mutations associated with resistance among 72 multidrug-resistant tuberculosis (MDR-TB) strains using whole genome sequencing (WGS) and to evaluate the performance of WGS for predicting MDR-TB drug resistance. Methods　The clinical strains isolated from patients who visited the outpatient department of Tianjin Center for Tuberculosis Control from January to September in 2020 were collected. Identification tests using p-nitrobenzoic acid (PNB) medium were performed. Drug susceptibility tests (proportion method) on L-J medium were performed. After excluding duplicate strains, 72 MDR-TB strains were selected for WGS. Data were analyzed by using online databases and the phenotypic drug susceptibility test results were compared with resistance profiles predicted by WGS. Results　All of 72 MDR-TB strains belonged to linage 2, and there was no significant difference in rate of pre-extensive drug-resistant tuberculosis (pre-XDR-TB) between modern type and ancestral type (χ2=0.287, P=0.592). A total of 81 mutation types were found from resistance-related genes for 12 anti-tuberculosis drugs, and the common mutation types in different drug-resistant strains were: streptomycin (SM): rpsL Lys43Arg; isoniazid (INH): katG Ser315Thr; rifampicin (RIF): rpoB Ser450Leu; ethambutol (EMB): embB Met306Val; ofloxacin (OFX), levofloxacin (LFX), moxifloxacin (MFX): gyrA Asp94Gly; kanamycin (KAM), capreomycin (CAP), amikacin (AMK): rrs 1401a>g; para-aminosalicylic acid (PAS): folC Ile43Thr. Nine mutation types were found in 9 prothionamide (PTO)-resistant strains, one type for each strain. The sensitivity and specificity of WGS for predicting resistance to different drugs were SM: 98.15% and 88.89%, INH: 90.28% and -, RIF: 98.62% and -, EMB: 79.49% and75.76%, OFX: 97.30% and 85.71%, KAM: 85.71% and 98.46%, PAS: 27.27% and 95.08%, PTO: 81.82% and 60.66%, CAP: 60.00% and 98.51%, LFX: 97.22% and 83.33%, MFX: 97.30% and 85.71%, AMK:100.00% and 100.00%, respectively. Conclusion　WGS is a rapid and promising method which has high consistency with the phenotypic drug sensitivity test. Therefore, it has good application prospects in predicting drug resistance in MDR-TB.

Analysis of microbial diversity in the midgut of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus / 中国热带医学

@#Abstract: Objective　To investigate the composition and diversity of midgut microbial community of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus (SFTSV)． Methods　The midgut DNA of three group Haemaphysalis longicornis infected with SFTSV was extracted, and the 16S rDNA gene of the sample was sequenced by HiSeq platform. The composition and diversity of endosymbiotic microbial community were clarified by OTU cluster analysis and alpha diversity analysis. Results　The midgut microbial clusters of the three groups infected with SFTSV were 143, 113, 163 OTUs respectively; the sparsity curve and abundance grade curve showed that the data had sufficient sequencing depth, and the midgut of Haemaphysalis longicornis infected with SFTSV was rich in microbial composition, but the species distribution was uneven. The analysis of microbial community composition showed that Proteobacteria, Firmicutes and Actinobacteria were the main dominant bacteria at the phyla level. At the class level, Gammaproteobacteria, Bacilli, Betaproteobacteria and Actinomycetia were the main dominant bacteria. At the order level, Legionellales, Bacillales, Burkholderiales and Actinomycetales were the main dominant orders. At the family level, Coxiellaceae, Bacillaceae, Moraxellaceae and Rhodococcaceae were the main dominant families. At the genus level, the relative abundance of Coxiella was the highest, followed by Aeribaillus and Azonexus. Alpha diversity analysis showed that the average Shannon index was 139.67, the average Simpson index was 0.48, the average Chao index was 145.06, and the average ACE index was 147.11. Conclusions　The species diversity of intestinal microorganisms in Haemaphysalis longicornis infected with SFTSV is rich. The results provide a basis for further exploring the interaction between intestinal microbes of Haemaphysalis longicornis and SFTSV and developing new ideas for the prevention and control of ticks and tick-borne diseases.