Reduced genetic diversity and the success of the invasive peacock bass (Cichliformes: Cichlidae) / Diversidade genética reduzida e o sucesso da espécie invasora tucunaré (Cichliformes: Cichlidae)

Abstract Several species of Cichla successfully colonized lakes and reservoirs of Brazil, since the 1960's, causing serious damage to local wildlife. In this study, 135 peacock bass were collected in a reservoir complex in order to identify if they represented a single dominant species or multiple ones, as several Cichla species have been reported in the basin. Specimens were identified by color pattern, morphometric and meristic data, and using mitochondrial markers COI, 16S rDNA and Control Region (CR). Overlapping morphological data and similar coloration patterns prevented their identification using the taxonomic keys to species identification available in the literature. However, Bayesian and maximum likelihood from sequencing data demonstrated the occurrence of a single species, Cichla kelberi. A single haplotype was observed for the 16S and CR, while three were detected for COI, with a dominant haplotype present in 98.5% of the samples. The extreme low diversity of the transplanted C. kelberi evidenced a limited number of founding maternal lineages. The success of this colonization seems to rely mainly on abiotic factors, such as increased water transparency of lentic environments that favor visual predators that along with the absence of predators, have made C. kelberi a successful invader of these reservoirs.

Resumo Muitas espécies de Cichla colonizaram com sucesso lagos e reservatórios do Brasil desde os anos 1960, causando graves prejuízos à vida selvagem nesses locais. Neste estudo, 135 tucunarés foram coletados em um complexo de reservatórios a fim de identificar se representavam uma espécie dominante ou múltiplas espécies, uma vez que diversas espécies de Cichla foram registradas na bacia. Os espécimes foram identificados com base na coloração, dados morfométricos e merísticos, e por marcadores mitocondriais COI, 16S rDNA e Região Controle (RC). A sobreposição dos dados morfométricos e o padrão similar de coloração impediram a identificação utilizando as chaves de identificação disponíveis na literatura. Entretanto, as análises bayesiana e de máxima verossimilhança de dados moleculares demonstraram a ocorrência de uma única espécie, Cichla kelberi. Um único haplótipo foi observado para o 16S e RC, enquanto três foram detectados para o COI, com um haplótipo dominante presente em 98,5% das amostras. A baixa diversidade nos exemplares introduzidos de C. kelberi evidenciou um número limitado de linhagens maternas fundadoras. O sucesso da invasão parece depender de fatores abióticos, como a maior transparência da água de ambientes lênticos que favorece predadores visuais que, atrelado à ausência de predadores, fez do C. kelberi um invasor bem-sucedido nesses reservatórios.

Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding / Isolamento e caracterização de bactérias associadas ao trato intestinal do bicho-da-seda sob alimentação larval tratada com antibióticos

Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.

Genetic variations and phylogenetic relationship of genus Uromastyx from Punjab Pakistan / Variações genéticas e relação filgenética do gênero Uromastyx de Punjab, Paquistão

Abstract During the present study, specimens were collected from selected sites of Cholistan desert and Kalabagh Game Reserve, Punjab province, Pakistan. Each captured specimen was tagged with voucher number and morphometric measurements were taken. The average snout to vent length was 172.559±1.40 mm and average weight was 92.1±1.30 g. The DNA of Uromastyx hardwickii was amplified and sequenced using 16S rRNA primer set. The obtained DNA sequence has shown reliable and clear species identification. After trimming ambiguous bases, the obtained 16S rRNA fragment was 520 bp while 16S rRNA fragments aligned with closely matched sequence from NCBI comprised of 510 bp. Closely matched sequences of genus Uromastyx were retrieved from NCBI in blast searches. Neighbour-joining tree of genus Uromastyx was constructed based on p-distance using MEGA X. The mean intraspecific variation was 0.095±0.01 while intraspecific variation was ranging from 0-1%. Similarly, interspecific variation of Uromastyx hardwikii with Saara asmussi, Uromastyx alfredschmidti, Uromastyx geyri, Uromastyx thomasi, Uromastyx alfredschmidti was 0-12%, 0-19%, 0-19%, 0-20%, 12-19% respectively. The newly produced DNA was submitted to NCBI and accession number was obtained (MW052563.1). Results of current study provided information about the molecular and morphological identification of Genus Uromastyx. In our recommendation, comprehensive molecular based identification of Pakistan's reptiles is required to report any new or subspecies from country.

Resumo Durante o presente estudo, os espécimes foram coletados em locais selecionados do deserto do Cholistan e da Reserva de Caça de Kalabagh, província de Punjab, Paquistão. Cada espécime capturado foi etiquetado com o número do comprovante e medidas morfométricas foram realizadas. O comprimento médio do focinho à cloaca foi de 172,559 ± 1,40 mm, e o peso médio foi de 92,1 ± 1,30 g. O DNA de Uromastyx hardwickii foi amplificado e sequenciado usando o conjunto de primer 16S rRNA. A sequência de DNA obtida mostrou identificação de espécies confiável e clara. Após o corte de bases ambíguas, o fragmento de rRNA 16S obtido tinha 520 pb, enquanto os fragmentos de rRNA 16S alinhados com a sequência próxima do NCBI composta por 510 pb. Sequências semelhantes do gênero Uromastyx foram recuperadas do NCBI em pesquisas de explosão. A árvore de união de vizinhos do gênero Uromastyx foi construída com base na distância-p usando MEGA X. A variação intraespecífica média foi de 0,095 ± 0,01, enquanto a variação intraespecífica foi de 0-1%. Da mesma forma, a variação interespecífica de Uromastyx hardwikii com Saara asmussi, Uromastyx alfredschmidti, Uromastyx geyri, Uromastyx thomasi, Uromastyx alfredschmidti foi de 0-12%, 0-19%, 0-19%, 0-20%, 12-19%, respectivamente. O DNA recém-produzido foi submetido ao NCBI e o número de acesso foi obtido (MW052563.1). Os resultados do estudo atual forneceram informações sobre a identificação molecular e morfológica do Gênero Uromastyx. Em nossa recomendação, a identificação de base molecular abrangente de répteis do Paquistão é necessária para relatar qualquer nova ou subespécie do país.

Diversity of culturable endophytic fungi vary through time in Manihot esculenta Crantz / A diversidade de fungos endofíticos cultiváveis ​​varia ao longo do tempo em Manihot esculenta Crantz

Abstract Endophytic fungi are a ubiquituos group that colonize all plant species on earth. Studies comparing the location of endophytic fungi within the leaves and the sampling time in Manihot esculenta Crantz (cassava) are limited. In this study, mature leaves of M. esculenta from Panama were collected in order to compare the cultivable diversity of endophytic fungi and to determine their distribution within the leaves. A total of one hundred sixty endophytes belonging to 97 species representing 13 genera and 8 morphospecies determined as mycelia sterilia that containing 63 isolates were isolated. Cladosporium, Nigrospora, Periconia, and mycelia sterilia 1 and 3 were the most predominant isolated endophytes. We detected that endophytes varied across the sampling time, but not amongst locations within leaves. The endophytes composition across sampling and the location of endophytes within leaf was similar, except for Periconia and mycelia sterilia 3 and 7. The data generated in this study contribute to the knowledge on the biodiversity of endophytic fungi in Panama, and establish the bases for future research focused on understanding the function of endophytes in M. esculenta crops.

Resumo Os fungos endofíticos são um grupo ubiquituo que colonizam todas as espécies de plantas na terra. Os estudos que comparam a localização dos fungos endofíticos dentro das folhas de Manihot esculenta Crantz (mandioca) e o tempo de amostragem são muito escassos. Neste estudo, folhas maduras de M. esculenta foram coletadas do Panamá com a finalidade de comparar a diversidade cultivável de endófitos e determinar sua distribuição dentro das folhas. Um total de 170 endófitos foram isolados de 97 espécies que representam 13 gêneros e 8 morfoespécies determinadas como micélios esterilizados contendo 63 isolados. Os fungos Cladosporium, Nigrospora, Periconia e mycelia sterilia 1 e 3 foram os isolados mais predominantes. Também detectamos que os endófitos variaram ao longo do tempo de amostragem, mas não entre os locais dentro das folhas. A composição de endófitos na amostragem e localização de endófitos dentro da folha foi semelhante, exceto para Periconia e mycelia sterilia 3 e 7. Os dados gerados neste estudo contribuem para o conhecimento da biodiversidade de fungos endofíticos no Panamá e estabelecem as bases para pesquisas sobre o entendimento da função de endófitos em culturas de M. esculenta.

Genomic constellations of RVA detected in Brazil from 1986 to 2016: a temporal and geographical distribution and occurrence of reassortments / Constelações genômicas de RVA detectadas no Brasil de 1986 a 2016: uma distribuição temporal e geográfica e ocorrência de rearranjos

INTRODUCTION: Species A rotavirus (RVA) infections are a major cause of severe gastroenteritis in children of <5 years worldwide. In Brazil, before vaccination, RVA was associated with 3.5 million episodes of acute diarrheal disease per year. Due to the segmented nature of their genomes, rotaviruses can exchange genes during co-infections, and generate new virus strains and new reinfections. OBJECTIVE: To evaluate the genomic diversity of RVA isolated in Brazil in 30 years, between 1986 to 2016, to investigate possible changes in the frequency of genotype constellations before and after the implementation of the vaccine. METHODS: In total, 4,474 nucleotide sequences were obtained from the Virus Variation Database. Genomic constellation was compared, and the proportion of rotavirus genotypes was analyzed by time and geographic region. RESULTS: Our results showed that major known genotypes were circulating in the country during the period under analysis, with a prevalence of the G1P[8] Wa-like genotype, decreasing only in the period immediately after the introduction of the vaccine. Regarding the geographical distribution, most of our constellations, 62 (39.2%), and 50 (31.6%) were concentrated in the North and Northeast regions. Our analysis also showed the circulation of multiple strains during the periods when the DS-1-like and AU-1-like genotypes were co-circulating with the Wa-like genotype. CONCLUSION: Therefore, it is likely that inter-genogroup reassortments are still occurring in Brazil and so it is important to establish an efficient surveillance system to follow the emergence of novel reassorted strains that might not be targeted by the vaccine.

INTRODUÇÃO: As infecções por rotavírus A (RVA) são uma das principais causas de gastroenterite grave em crianças <5 anos em todo o mundo. No Brasil, antes da vacinação, o RVA estava associado a 3,5 milhões de episódios de diarreia aguda por ano. Devido à natureza segmentada de seus genomas, os rotavírus podem trocar genes durante as coinfecções, gerar novas cepas de vírus e novas reinfecções. OBJETIVO: Avaliar a diversidade genômica de RVA isolados no Brasil entre 1986 a 2016 para investigar possíveis alterações na frequência das constelações de genótipos antes e após a implantação da vacina. MÉTODOS: No total, 4.474 sequências de nucleotídeos foram obtidas do Banco de Dados de Variação de Vírus. A constelação genômica foi comparada e a proporção dos genótipos de rotavírus foi analisada por tempo e região geográfica. RESULTADOS: Nossos resultados mostraram que os principais genótipos conhecidos circulavam no país no período em análise, com prevalência do genótipo G1P[8] Wa-like, diminuindo apenas no período imediatamente após a introdução da vacina. Em relação à distribuição geográfica, a maioria das nossas constelações, 62 (39,2%) e 50 (31,6%), concentrava-se nas regiões Norte e Nordeste. Nossa análise também mostrou a circulação de cepas múltiplas durante os períodos em que os genótipos DS-1-like e AU-1-like estavam co-circulando com o genótipo Wa-like. CONCLUSÃO: Portanto, é provável que rearranjos inter-genogrupos ainda estejam ocorrendo no Brasil e por isso é importante estabelecer um sistema de vigilância eficiente para acompanhar o surgimento de novas cepas rearranjadas que podem não ser protegidas pela vacina.

Drug resistance and genomic characteristics of Salmonella enterica serovar London from clinical and food sources in Hangzhou City from 2017 to 2021 / 中华预防医学杂志

Objective: To analyze the drug resistance and genomic characteristics of Salmonella enterica serovar London isolated from clinical and food sources in Hangzhou City from 2017 to 2021. Methods: A total of 91 Salmonella enterica serovar London strains isolated from Hangzhou City from 2017 to 2021 were analyzed for drug susceptibility, pulsed field gel electrophoresis (PFGE) typing and whole genome sequencing. Multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST) and detection of drug resistance genes were performed by using the sequencing data. Phylogenetic analysis was conducted to compare the 91 genomes from Hangzhou City with 347 genomes from public databases. Results: No significant difference in the drug resistance rate was observed between clinical strains and food strains to 18 drugs in Hangzhou City(all P>0.05), and the multidrug resistance (MDR) rate was 75.8% (69/91). Most strains were resistant to 7 drug classes simultaneously. One strain was resistant to Polymyxin E as well as positive for mcr-1.1, and 50.5% (46/91) of the strains were resistant to Azithromycin and were positive for mph(A). All 91 Salmonella enterica serovar London strains were ST155, which were subdivided into 44 molecular types by PFGE and 82 types by cgMLST. Phylogenetic analysis showed that most strains from Hangzhou City (83/91) were clustered together, and a small number of human isolates from Europe, North America and pork isolates from Hubei and Shenzhen were mixed in the cluster. Other strains from Hangzhou City (8/91) were closely related to strains from Europe, America and Southeast Asia. Strains isolated from pork were the most closely related to clinical strains. Conclusion: The epidemic of Salmonella enterica serovar London in Hangzhou City is mainly caused by the spread of ST155 strains, which is mainly transmitted locally. At the same time, cross-region transmission to Europe, North America, Southeast Asia, and other provinces and cities in China may also occur. There is no significant difference in the drug resistance rate between clinical strains and food strains, and a high level of MDR is found in the strains. Clinical infection of Salmonella enterica serovar London may be closely related to pork consumption in Hangzhou City.

Poly-G for tumor matched samples chronicles the evolution of human colorectal cancer / 中华肿瘤杂志

Objective: To analyze poly-guanine (poly-G) genotypes and construct the phylogenetic tree of colorectal cancer (CRC) and provide an efficient and convenient method for the study of intra-tumor heterogeneity and tumor metastasis pathway. Methods: The clinicopathological information of patients with primary colorectal cancer resection with regional lymph node metastases were retrospectively collected in the Department of General Surgery, General Hospital of Tianjin Medical University from January 2017 to December 2017. The paraffin sections of the paired tumor samples were performed consecutively, and multi-region microdissection was performed after histogene staining. The phenol-chloroform extraction and ethanol precipitation scheme was used to obtain DNA, and Poly-G multiplex PCR amplification and capillary electrophoresis detection were performed. The correlation between Poly-G mutation frequency and clinicopathological parameters was analyzed. Based on the difference of Poly-G genotypes between paired samples, the distance matrix was calculated, and the phylogenetic tree was constructed to clarify the tumor metastasis pathway. Results: A total of 237 paired samples were collected from 20 patients including 134 primary lesions, 66 lymph node metastases, 37 normal tissues, and Poly-G mutation was detected in 20 patients (100%). The mutation frequency of Poly-G in low and undifferentiated patients was (74.10±23.11)%, higher than that in high and medium differentiated patients [(31.36±12.04)%, P<0.001]. In microsatellite instability patients, the mutation frequency of Poly-G was (68.19±24.80)%, which was higher than that in microsatellite stable patients [(32.40±14.90)%, P=0.003]. The Poly-G mutation frequency was not correlated with age, gender, and pathological staging (all P>0.05). Based on Poly-G genotype difference of the paired samples, the phylogenetic trees of 20 patients were constructed, showing the evolution process of the tumor, especially the subclonal origins of lymph node metastasis. Conclusion: Poly-G mutations accumulate in the occurrence and development of CRC, and can be used as genetic markers to generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica / 中西医结合学报

OBJECTIVE@#AP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes, such as the regulation of biosynthesis of active lignan. However, few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica. This study establishes a complete picture of the AP2/ERF superfamily in I. indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.@*METHODS@#To identify the IiAP2/ERF superfamily genes, the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool. Bioinformatic analyses were conducted to investigate the protein structure, motif composition, chromosome location, phylogenetic relationship, and interaction network of the IiAP2/ERF superfamily genes. The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.@*RESULTS@#One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I. indigotica genome database in this study. By sequence alignment and phylogenetic analysis, the IiAP2/ERF genes were classified into 5 groups including AP2, ERF, DREB (dehydration-responsive element-binding factor), Soloist and RAV (related to abscisic acid insensitive 3/viviparous 1) subfamilies. Among which, 122 members were unevenly distributed across seven chromosomes. Sequence alignment showed that I. indigotica and A. thaliana had 30 pairs of orthologous genes, and we constructed their interaction network. The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I. indigotica. Members that may regulate lignan biosynthesis in roots were also preliminarily identified. Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up- or down-regulated under salt or drought treatment, among which, 33 IiAP2/ERF genes were regulated by both stresses.@*CONCLUSION@#This study undertook a genome-wide characterization of the AP2/ERF superfamily in I. indigotica, providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Population structure of food-borne Staphylococcus aureus in China / 中华流行病学杂志

Objective: To understand the population structure of food-borne Staphylococcus (S.) aureus in China. Methods: Whole genome sequencing was used to analyze 763 food-borne S. aureus strains from 16 provinces in China from 2006 to 2020. Multilocus sequence typing (MLST), staphylococcal protein A gene (spa) typing, and staphylococcal chromosome cassettemec (SCCmec) typing were conducted, and minimum spanning tree based on ST types (STs) was constructed by BioNumerics 7.5 software. Thirty-one S. aureus strains isolated from imported food products were also included in constructing the genome phylogenetic tree. Results: A total of 90 STs (20 novel types) and 160 spa types were detected in the 763 S. aureus isolates. The 72 STs (72/90, 80.0%) were related to 22 clone complexes. The predominant clone complexes were CC7, CC1, CC5, CC398, CC188, CC59, CC6, CC88, CC15, and CC25, accounting for 82.44% (629/763) of the total. The STs and spa types in the predominant clone complexes changed over the years. The methicillin-resistant S. aureus (MRSA) detection rate was 7.60%, and 7 SCCmec types were identified. The ST59-t437-Ⅳa (17.24%, 10/58), ST239-t030-Ⅲ (12.07%, 7/58), ST59-t437-Ⅴb (8.62%, 5/58), ST338-t437-Ⅴb (6.90%, 4/58) and ST338-t441-Ⅴb (6.90%, 4/58) were the main types in MRSA strains. The genome phylogenetic tree had two clades, and the strains with the same CC, ST, and spa types clustered together. All CC7 methicillin sensitive S. aureus strains were included in Clade1, while 21 clone complexes and all MRSA strains were in Clade2. The MRSA strains clustered according to the SCCmec and STs. The strains from imported food products in CC398, CC7, CC30, CC12, and CC188 had far distances from Chinese strains in the tree. Conclusions: In this study, the predominant clone complexes of food-borne strains were CC7, CC1, CC5, CC398, CC188, CC59, CC6, CC88, CC15, and CC25, which overlapped with the previously reported clone complexes of hospital and community-associated strains in China, suggesting that close attention needs to be paid to food, a vehicle of pathogen transmission in community and food poisoning.

Establishment of PCR assays and genetic polymorphism analysis of genes encoding Clostridium perfringens β2 toxin from different sources / 中华流行病学杂志

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β2 toxin (cpb2) and atypical-cpb2 (aty-cpb2), analyze the epidemiological characteristics and genetic polymorphism of the cpb2 of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb2 of 188 Clostridium perfringens strains were examined by PCR; the cpb2 sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb2-library based on 110 strains carrying the cpb2 were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb2 (con-cpb2) and aty-cpb2. Results: The specificity of PCR assay for the cpb2 and aty-cpb2 was verified. The PCR results for cpb2 amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb2, 94 types A strains carried aty-cpb2, 6 types A strains carried con-cpb2, and 7 types F strains carried aty-cpb2. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb2 toxin was developed, and the previous PCR method for detecting aty-cpb2 was improved. aty-cpb2 is the primary gene encoding of β2 toxin. There is a significant nucleotide sequence variance between the various cpb2 genotypes.

Genetic characterization of varicella-zoster virus in people aged 20 years and under in Yichang City of Hubei Province, 2019-2020 / 中华流行病学杂志

Objective: To analyze the genetic characteristics of varicella-zoster virus (VZV) in people aged 20 years and under in Yichang City of Hubei Province from 2019 to 2020. Methods: Based on the Yichang Health Big Data Platform, we investigated cases 20 and under clinically diagnosed as herpes zoster in three hospitals from March 2019 to September 2020. Collecting vesicle fluid and throat swab samples of the cases and completing questionnaires to obtain basic information. Real-time fluorescent quantitative PCR was used for positive identification of the virus. PCR amplification of VZV's open reading frame (ORF) and sequencing of the products to determine the VZV genotype. Analyze mutations at some specific single nucleotide polymorphism (SNP) sites. Results: Among 46 cases of herpes zoster, the male to female ratio was 1.3∶1 (26∶20) and the age ranged from 7 to 20 years old. Fifteen cases had been vaccinated against varicella, including 13 and 2 cases of 1 and 2 doses, respectively. VZV strains were detected in 34 samples (73.91%), all belonging to Clade 2. Phylogenetic tree analysis of the nucleotide of ORF22 showed, compared with Clade 2 referenced strains, the sequence matching degree of nucleotide for all 34 samples was 99.0% to 100.0%. Conclusion: The main VZV strain causing herpes zoster in people aged 20 years and under in Yichang from 2019 to 2020 was Clade 2.

Analysis of the characteristics of viral infections in children with diarrhea in Beijing from 2018 to 2022 / 中华预防医学杂志

Objective: To explore the characteristics of viral infections in children with diarrhea in Beijing from 2018 to 2022. Methods: Real-time PCR and enzyme-linked immunosorbent assay were used to detect viral nucleic acid of Norovirus (NoV), Sappovirus (SaV), Astrovirus (AstV), Enteric Adenovirus (AdV) or antigen of Rotavirus (RV) in 748 stool samples collected from Beijing Capital Institute of Pediatrics from January 2018 to December 2021. Subsequently, the reverse transcription PCR or PCR method was used to amplify the target gene of the positive samples after the initial screening, followed by sequencing, genotyping and evolution analysis, so as to obtain the characteristics of these viruses. Phylogenetic analysis was performed using Mega 6.0. Results: From 2018 to 2021, the overall detection rate of the above five common viruses was 37.6%(281/748)in children under 5 years old in Beijing. NoV, Enteric AdV and RV were still the top three diarrhea-related viruses, followed by AstV and SaV, accounting for 41.6%, 29.2%, 27.8%, 8.9% and 7.5%, respectively. The detection rate of co-infections with two or three diarrhea-related viruses was 4.7% (35/748). From the perspective of annual distribution, the detection rate of Enteric AdV was the highest in 2021, while NoV was predominant in the other 4 years. From the perspective of genetic characteristics, NoV was predominant by GⅡ.4, and after the first detection of GⅡ.4[P16] in 2020, it occupied the first two gene groups together with GⅡ.4[P31]. Although the predominant RV was G9P[8], the rare epidemic strain G8P[8] was first detected in 2021. The predominant genotypes of Enteric AdV and AstV were Ad41 and HAstV-1. SaV was sporadic spread with a low detection rate. Conclusion: Among the diarrhea-related viruses infected children under 5 years of age in Beijing, the predominant strains of NoV and RV have changed and new sub-genotypes have been detected for the first time, while the predominant strains of AstV and Enteric AdV are relatively stable.

Pathogenic characterization of Klebsiella pneumoniae resistant to carbapenems and polymyxin / 中华预防医学杂志

Objective: Analysis and investigation of pathogenic characteristics of polymyxin-and carbapenem-resistant Klebsiella pneumoniae (PR-CRKP). Methods: A total of 23 PR-CRKP strains isolated from clinical specimens from the General Hospital of Southern Theater Command from March 2019 to July 2021 were retrospectively collected, Whole-genome sequencing was performed on 23 PR-CRKP strains, resistance genes were identified by comparison of the CARD and the ResFinder database, high-resolution typing of PR-CRKP strains was analyzed by core genomic multilocus sequencing (cgMLST) and single nucleotide polymorphism (SNP); polymyxin resistance genes were determined by PCR and sequencing. Results: All PR-CRKP strains were KPC-2 producing ST11 types. cgMLST results showed that the evolutionary distance between the PR-CRKP strains and Klebsiella pneumoniae in mainland China was 66.44 on average, which is more closely related than foreign strains; the 23 PR-CRKP strains were divided into 3 main subclusters based on SNP phylogenetic trees, with some aggregation among Clade 2-1 in the isolation department and date. The two-component negative regulatory gene mgrB has seven mutation types including point mutations, different insertion fragments and different insertion positions. Conclusion: The close affinity of PR-CRKP strains indicate the possibility of nosocomial clonal transmission and the need to strengthen surveillance of PR-CRKP strains to prevent epidemic transmission of PR-CRKP.

Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro / 中国中药杂志

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus / 中国中药杂志

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

Analysis of WRKY transcription factor family based on full-length transcriptome sequencing in Polygonatum cyrtonema / 中国中药杂志

WRKY transcription factor family plays an important role in plant growth and development, secondary metabolite synthesis, and biotic and abiotic stress responses. The present study performed full-length transcriptome sequencing of Polygonatum cyrtonema by virtue of the PacBio SMRT high-throughput platform, identified the WRKY family by bioinformatics methods, and analyzed the physicochemical properties, subcellular localization, phylogeny, and conserved motifs. The results showed that 30.69 Gb nucleotide bases and 89 564 transcripts were obtained after redundancy removal. These transcripts had a mean length of 2 060 bp and an N50 value of 3 156 bp. Based on the full-length transcriptome sequencing data, 64 candidate proteins were selected from the WRKY transcription factor family, with the protein size of 92-1 027 aa, the relative molecular mass of 10 377.85-115 779.48 kDa, and the isoelectric point of 4.49-9.84. These WRKY family members were mostly located in the nucleus and belonged to the hydrophobic proteins. According to the phylogenetic analysis of WRKY family in P. cyrtonema and Arabidopsis thaliana, all WRKY family members were clustered into seven subfamilies and WRKY proteins from P. cyrtonema were distributed in different numbers in these seven subgroups. Expression pattern analysis confirmed that 40 WRKY family members had distinct expression patterns in the rhizomes of 1-and 3-year-old P. cyrtonema. Except for PcWRKY39, the expression of 39 WRKY family members was down-regulated in 3-year-old samples. In conclusion, this study provides abundant reference data for genetic research on P. cyrtonema and lays a foundation for the in-depth investigation of the biological functions of the WRKY family.

Identification of terpene synthase gene family in Gynostemma pentaphyllum and expression pattern analysis under abiotic stresses / 中国中药杂志

The present study aimed to investigate the composition of the terpene synthase(TPS) gene family in Gynostemma pentaphyllum and its role in abiotic stresses. The G. pentaphyllum TPS gene family was identified and analyzed at the genome-wide level using bioinformatics analysis, and the expression patterns of these family members were analyzed in different tissues of G. pentaphyllum as well as under various abiotic stresses. The results showed that there were 24 TPS gene family members in G. pentaphyllum with protein lengths ranging from 294 to 842 aa. All of them were localized in the cytoplasm or chloroplasts and unevenly distributed on the 11 chromosomes of G. pentaphyllum. The results of the phylogenetic tree showed that the G. pentaphyllum TPS gene family members could be divided into five subfamilies. As revealed by the analysis of promoter cis-acting elements, TPS gene family members in G. pentaphyllum were predicted to respond to a variety of abiotic stresses such as salt, low temperature, and dark stress. The analysis of gene expression patterns in different tissues of G. pentaphyllum revealed that nine TPS genes were tissue-specific in expression. The qPCR results showed that GpTPS16, GpTPS17, and GpTPS21 responded to a variety of abiotic stresses. This study is expected to provide references in guiding the further exploration of the biological functions of G. pentaphyllum TPS genes under abiotic stresses.

Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province / 中国中药杂志

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.

Identification and biological characteristics of violet root rot pathogen of Pseudostellaria heterophylla in Fujian province / 中国中药杂志

Violet root rot is one of the main root diseases in the production process of Pseudostellaria heterophylla. To clarify the pathogenic species that cause the violet root rot of P. heterophylla in Fujian province, the roots and the sclerotia with violet root rot symptoms were collected from the main producing areas of P. heterophylla(Fujian province) from 2017 to 2021, and the pathogens were isolated by tissue separation method and identified by morphology and multi-gene phylogenetic analysis. Additionally, the biological characteristics of the pathogens were studied and the fungicides were determined. The results showed that 78 strains of violet root rot were isolated from the collected root samples, which belonged to one type after preliminary morphological identification. Two represen-tative strains were selected from the pathogens for multi-gene phylogenetic analysis, and they were clustered with Helicobasidium mompa together. The suitable culture conditions for the mycelium were OA medium, 25 ℃, pH 6, and ammonium oxalate as the nitrogen source. The lethal temperature of the mycelium was 50 ℃ for 10 minutes. Moreover, 99.1% propiconazole and 98.7% azoxystrobin had the optimal bacteriostatic effect, and the concentrations with the 50% bacteriostatic rate were 16.85 and 12.24 μg·mL~(-1), respectively. On the basis of the above results, the pathogen causing violet root rot of P. heterophylla in Fujian province was H. mompa. The medium type, growth temperature, pH value, nitrogen source, etc. had significant effect on the growth of mycelium.