Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches.

BACKGROUND: Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. RESULTS: In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. CONCLUSIONS: Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

Identification of New Circulating Recombinant Form of HIV-1 CRF127_07109 in Northern Vietnam.

Some candidates of a new circulating recombinant form (CRF) of HIV-1 were found in northern Vietnam in our previous study. We succeeded in near full-length sequencing using MinION with plasma samples from 12 people living with HIV. Three of the samples were CRF109_0107, which was recently reported in China. Three others were the newly identified CRF127_07109, while six of them were considered to be CRF127_07109-related unique recombinant forms (URFs). The time to the most recent common ancestor of CRF127_07109 was estimated to be between 2015 and 2019. Our findings showed that CRF127_07109 and related URFs were generated recently in northern Vietnam, rather than migrated independently to northern Vietnam.

The entire chloroplast genome sequence of Asparagus setaceus (Kunth) Jessop: Genome structure, gene composition, and phylogenetic analysis in Asparagaceae.

Asparagus setaceus (Kunth) Jessop is a horticultural plant of the genus Asparagus. Herein, the whole chloroplast (cp) genome of A. setaceus was sequenced with PacBio and Illumina sequencing systems. The cp genome shows a characteristic quadripartite structure with 158,076 bp. In total, 135 genes were annotated, containing 89 protein-coding, 38 tRNA, and 8 rRNA genes. Contrast with the previous cp genome of A. setaceus registered in NCBI, we identified 7 single-nucleotide polymorphisms and 15 indels, mostly situated in noncoding areas. Meanwhile, 36 repeat structures and 260 simple sequence repeats were marked out. A bias for A/T-ending codons was shown in this cp genome. Furthermore, we predicted 78 RNA-editing sites in 29 genes, which were all for C-to-U transitions. And it was also proven that positive selection was exerted on the rpoC1 gene of A. setaceus with the K a/K s data. Meanwhile, a conservative gene order and highly similar sequences of protein-coding genes were revealed within Asparagus species. Phylogenetic tree analysis indicated that A. setaceus was a sister to Asparagus cochinchinensis. Taken together, our released genome provided valuable information for the gene composition, genetics comparison, and the phylogeny studies of A. setaceus.

Complete mitogenome and phylogenetic analysis of the tropical rocky shore crab Grapsus albolineatus (Lamarck, 1818) (Crustacea: Grapsoidea).

In this study, the complete mitogenome of Grapsus albolineatus (Lamarck, 1818) (Crustacea: Grapsoidea) was sequenced. The mitogenome of G. albolineatus was a circular molecule with 15,578 bp length. Its nucleotide composition was 26.81% A, 16.37% G, 34.51% T, and 22.31% C. It comprised 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA), and two ribosomal RNA (rRNA). All PCGs were initiated by ATN codons, except for the atp8 and nad1 genes. Ten PCGs used a common stop codon of TAA or TAG, and the other three ended with a truncated stop codon (a single stop nucleotide T). Phylogenetic analysis revealed that G. albolineatus was closely related to species from the genera Pachygrapsus and Metopograpsus.

Epidemiology of Nocardia Species at a Tertiary Hospital in Southern Taiwan, 2012 to 2020: MLSA Phylogeny and Antimicrobial Susceptibility.

The identification and antimicrobial susceptibility of Nocardia spp. are essential for guiding antibiotic treatment. We investigated the species distribution and evaluated the antimicrobial susceptibility of Nocardia species collected in southern Taiwan from 2012 to 2020. A total of 77 Nocardia isolates were collected and identified to the species level using multi-locus sequence analysis (MLSA). The susceptibilities to 15 antibiotics for Nocardia isolates were determined by the broth microdilution method, and the MIC50 and MIC90 for each antibiotic against different species were analyzed. N. cyriacigeorgica was the leading isolate, accounting for 32.5% of all Nocardia isolates, and the prevalence of Nocardia isolates decreased in summer. All of the isolates were susceptible to trimethoprim/sulfamethoxazole, amikacin, and linezolid, whereas 90.9% were non-susceptible to cefepime and imipenem. The phylogenic tree by MLSA showed that the similarity between N. beijingensis and N. asiatica was as high as 99%, 73% between N. niigatensis and N. crassostreae, and 86% between N. cerradoensis and N. cyriacigeorgica. While trimethoprim/sulfamethoxazole, amikacin, and linezolid remained fully active against all of the Nocardia isolates tested, 90.9% of the isolates were non-susceptible to cefepime and imipenem.

The complete chloroplast genome of two traditional medical plants: Asparagus cochinchinensis (Lour.) Merr. and Asparagus dauricus Fisch. ex Link.

Asparagus cochinchinensis (Lour.) Merr. and Asparagus dauricus Fisch. ex Link are two traditional medical plants with therapeutic effects, distributed in mountainous regions of China. In the current study, the complete chloroplast (cp) genomes of A. cochinchinensis and A. dauricus were sequenced on the Illumina Hiseq 2500, and obtained with a length of 157,095 bp and 156,918 bp, respectively, both containing a large single-copy region and a small single-copy region separated by a pair of inverted repeat regions. The cp genome of A. cochinchinensis has 132 annotated genes including 86 protein-coding genes, 38 tRNA, and eight rRNA genes. A. dauricus has 112 annotated genes containing 78 protein genes, 30 tRNA, and four rRNA genes. The maximum-likelihood tree was reconstructed with 17 species, indicating that A. cochinchinensis is a sister group to the clade including A. officinalis to A. racemosa. This clade includes five species of Asparagus.

The complete chloroplast genome of Asparagus densiflorus (Kunth) Jessop L.

Asparagus densiflorus (Kunth) Jessop L. is a horticultural plant widely cultivated in China. Herein, we reported the complete chloroplast (cp) genome of A. densiflorus from the genus Asparagus. The entire cp genome of A. densiflorus was 157,141 bp in length with one large single-copy region of 91,255 bp and one small single-copy region of 20,355 bp, separated by a pair of inverted-repeat regions of 45,531 bp. The GC content is 36.46% in this cp genome. A total of 134 genes were annotated including 90 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The maximum-likelihood phylogenetic analysis showed that A. densiflorus was the most closely related to Asparagus cochinchinensis.

Signature of chronic hepatitis B virus infection in nails and hair.

BACKGROUND: Hepatitis B virus (HBV) is detected in extrahepatic tissues of individuals with HBV infection. Whether nails and hair contain HBV has been unknown. METHODS: We examined two patient groups: those with chronic HBV infection alone (n = 71), and those with both chronic HBV and hepatitis delta virus (HDV) infections (n = 15). HBV DNA in the patients' fingernails and hair were measured by real-time PCR. Hepatitis B surface antigen (HBsAg) of fingernails was evaluated by an enzyme immunoassay. HDV RNA in fingernails was measured by real-time PCR. Immunochemical staining was performed on nails. We used chimeric mice with humanized livers to evaluate the infectivity of nails. RESULTS: Of the 71 pairs of HBV-alone nail and hair samples, 70 (99%) nail and 60 (85%) hair samples were positive for ß-actin DNA. Of those 70 nail samples, 65 (93%) were HBV DNA-positive. Of the 60 hair samples, 49 (82%) were HBV DNA-positive. The serum HBV DNA level of the nail HBV DNA-positive patients was significantly higher than that of the nail HBV DNA-negative patients (p < 0.001). The hair HBV DNA-positive patients' serum HBV DNA level was significantly higher compared to the hair HBV DNA-negative patients (p < 0.001). The nail HBV DNA level was significantly higher than the hair HBV DNA level (p < 0.001). The nails and hair HBV DNA levels were correlated (r = 0.325, p < 0.05). A phylogenetic tree analysis of the complete genome sequence of HBV isolated from nails and hair identified the infection source. Of the 64 nail samples, 38 (59%) were HBsAg-positive. All 15 pairs of chronic HBV/HDV infection nail and hair samples were ß-actin DNA-positive. However, nail HBV DNA was detected in two patients (13%). None of the 15 patients were positive for hair HBV DNA. Nail HDV RNA was detected in three patients (20%). Of the 15 patients, eight (53%) were nail HBsAg-positive. HBsAg and hepatitis delta (HD) antigen were detected in the nails by immunochemical staining. Chimeric mice were not infected with PBS containing HBsAg and HBV DNA elucidated from nails. CONCLUSIONS: Nails and hair were the reservoir of HBV DNA. Moreover, nails can contain HBsAg, HDV RNA, and HD antigen.

Hepatitis B virus DNA in the fingernails and hair of children with acute hepatitis B.

Hepatitis B virus (HBV) DNA is detectable in the nails and hair of patients with chronic HBV infection. However, it remains unclear whether HBV DNA can be detectable in the nails and hair of patients with acute HBV infection. We encountered two cases of children with acute HBV infection. HBV DNA in the nails and hair from the two children was evaluated by real-time PCR. To clarify the characteristics of HBV DNA, full-length HBV genome sequencing and phylogenetic tree analysis were performed. The levels of serum HBV DNA in children of cases 1 and 2 at day 0 were 7.6 Log IU/mL and 7.4 Log IU/mL, respectively. Nail HBV DNA was detected in both children (case 1: 4.6 Log IU/mL at day 0, case 2: 5.5 Log IU/mL at day 14). Moreover, hair HBV DNA was detectable in case 2 (4.0 Log IU/mL at day 14). Serum HBV DNA became undetectable within approximately 3-4 months after the first hospital visit. After the resolution of HBV viremia, nail and hair HBV DNA became undetectable. The sequence analysis of serum, nail and hair HBV DNA showed the same HBV genotype in each case (case1: genotype C, case 2: genotype A). In case 1, 3 nucleotides were different in the full-genome HBV sequence between the serum and nails. In case 2, the full-genome HBV sequences were identical among the serum, nails and hair. In conclusion, HBV DNA was detectable in nails and hair of children with acute HBV infection.

Comparative Genome Analysis of Streptococcus suis Serotype 9 Isolates from China, The Netherland, and the U.K.

Streptococcus suis (S. suis) is an important swine pathogen and an emerging zoonotic agent worldwide. Serotype 9 is the most prevalent serotype in several European countries but it is relatively rare in China. In this study, through the investigation of the serotypes of 279 S. suis strains isolated from China from 2015 to 2017, it was found that serotype 9 is the second most prevalent serotype (43 out of 279), behind serotype 2 (83 out of 279). Next, the 43 serotype 9 isolates were sequenced and compared with those from the Netherland (28) and the U.K. (eight). For the purpose of comparison, the strain D12 (GCA_000231905), which has completed genome sequences, was also incorporated. Phylogenetic tree analysis showed that the strains from China and the U.K. were heterogeneous. In contrast, all but one from the Netherland belonged to the same clade. The dominant clades of Chinese strains (33) and strains from the Netherland (27) were very similar. Both of them may have originated from the same strain about 70 years ago. Then, the distributions of virulence-associated genes and antibiotic resistance genes among different clades and sources were analyzed. By comparison, strains from the Netherland carried more virulence-associated genes and those from the U.K. had more antibiotic resistance genes. Additionally, some virulence-associated genes (salK and salR) and antibiotic resistance genes (lincomycin and spectinomycin) existed only in several Chinese strains. In conclusion, our data displayed the population characteristics and differences of S. suis serotype 9 between China and Europe, suggesting that they have taken different evolutionary paths.

Genetic epidemiology using whole genome sequencing and haplotype networks revealed the linkage of SARS-CoV-2 infection in nosocomial outbreak.

BACKGROUND: A characteristic feature of SARS-CoV-2 is its ability to transmit from pre- or asymptomatic patients, complicating the tracing of infection pathways and causing outbreaks. Despite several reports that whole genome sequencing (WGS) and haplotype networks are useful for epidemiologic analysis, little is known about their use in nosocomial infections. AIM: We aimed to demonstrate the advantages of genetic epidemiology in identifying the link in nosocomial infection by comparing single nucleotide variations (SNVs) of isolates from patients associated with an outbreak in Showa University Hospital. METHODS: We used specimens from 32 patients in whom COVID-19 had been diagnosed using clinical reverse transcription-polymerase chain reaction tests. RNA of SARS-CoV-2 from specimens was reverse-transcribed and analysed using WGS. SNVs were extracted and used for lineage determination, phylogenetic tree analysis, and median-joining analysis. FINDINGS: The lineage of SARS-CoV-2 that was associated with outbreak in Showa University Hospital was B.1.1.214, which was consistent with that found in the Kanto metropolitan area during the same period. Consistent with canonical epidemiological observations, haplotype network analysis was successful for the classification of patients. Additionally, phylogenetic tree analysis revealed three independent introductions of the virus into the hospital during the outbreak. Further, median-joining analysis indicated that four patients were directly infected by any of the others in the same cluster. CONCLUSION: Genetic epidemiology with WGS and haplotype networks is useful for tracing transmission and optimizing prevention strategies in nosocomial outbreaks.

The complete chloroplast genome of Bupleurum euphorbioides, a traditional medicinal plant.

Bupleurum euphorbioides is a rare native plant attributed with analgesic, gallbladder-supportive, and other functions in China and the Republic of Korea. However, the complete chloroplast genome sequence of the native plant B. euphorbioides has not been determined. In this study, we sequenced the complete chloroplast genome sequence, and examined the molecular phylogeny and genetic information of B. euphorbioides. The total chloroplast genome of B. euphorbioides was 154,871 bp in length with a large single-copy region (85,089 bp), small single-copy region (17,714 bp), and pair of inverted repeats regions (26,034 bp). The chloroplast genome encoded a total of 176 genes, including 131 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The phylogenetic tree indicated that B. euphorbioides was most closely related to B. latissimum.

The complete mitochondrial genome and phylogenetic analysis of Upogebia major (De Haan, 1841).

The complete mitochondrial (mt) genome of Upogebia major (De Haan, 1841) is 16,131 bp in length, comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The nucleotide composition for U. major is 34.3% of A, 36.6% of T, 10.9% of C, and 18.3% of G. All PCGs are initiated by ATN codons, except for the cox1 gene, which was not determined. Nine PCGs use a common stop codon of TAA or TAG, the other four end with an incomplete stop codon (a single stop nucleotide T). Phylogenetic tree analysis showed that U. major had a close relationship with the species from the family Thalassinidae. The study will provide an important theoretical basis for further analysis of mt genome evolution and the phylogenetic relationships of the order Decapoda.

The complete chloroplast genome of Bupleurum euphorbioides, a traditional medicinal plant.

Bupleurum euphorbioides is a rare native plant attributed with analgesic, gallbladder-supportive, and other functions in China and the Republic of Korea. However, the complete chloroplast genome sequence of the native plant B. euphorbioides has not been determined. In this study, we sequenced the complete chloroplast genome sequence, and examined the molecular phylogeny and genetic information of B. euphorbioides. The total chloroplast genome of B. euphorbioides was 154,871 bp in length with a large single-copy region (85,089 bp), small single-copy region (17,714 bp), and pair of inverted repeats regions (26,034 bp). The chloroplast genome encoded a total of 176 genes, including 131 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic tree indicated that B. euphorbioides was the most closely related to B. latissimum.

Characteristics and phylogenetic analysis of the complete chloroplast genome of Paeonia japonica (Paeoniaceae).

Paeonia japonica, distributed throughout Asia, is a traditional medicinal herb in Korea, with many potential beneficial effects including pain-relieving, anti-inflammatory, and anti-cancer activities. Despite its high pharmacological value, the genetic information on Paeonia japonica remains limited. In this study, the chloroplast genome of P. japonica was sequenced using next-generation sequencing (NGS) technology and genome and phylogeny were analyzed using multiple tools. The chloroplast genome of P. japonica was 152,731 bp in length with an inverted repeat region of 26,656 bp, including a large single-copy region of 84,389 bp and a small single copy region of 17,030 bp. The P. japonica chloroplast genome included 113 genes comprising 80 protein-coding genes, 27 tRNA, and 5 rRNA genes. Phylogenetic analysis indicated that P. japonica and P. obovata share a close evolutionary relationship.

Source identification of HIV-1 transmission in three lawsuits Using Ultra-Deep pyrosequencing and phylogenetic analysis.

BACKGROUND/PURPOSE: Intentional transmission of HIV-1 is a crime. Identifying the source of transmission between HIV-1 infected cases using phylogenetic analysis has limitations, including delayed examinations after the initiation of infection and ambiguity of phyletic relationships. This study was the first to introduce phylogenetic tree Results as forensic evidence in a trial in Taiwan. METHODS: Three lawsuit cases from different district courts in Taiwan were chosen for this study. We identified the source of transmission between individuals in each lawsuit based on the maximum likelihood and Bayesian phylogenetic tree analyses using the HIV-1 sequences from molecular cloning and ultra-deep pyrosequencing (UDPS). Two gene regions of the HIV genome, env and gag, were involved. RESULTS: The results of phylogenetic analysis using sequences from molecular cloning were clear and evidential enough in lawsuits 1 and 3. Due to the delayed sampling time, the result of sequences from molecular cloning in lawsuit 2 was ambiguous. Combined with the analyzed result of sequences from UDPS and epidemiological information, the source of transmission in lawsuit 2 was further identified. CONCLUSION: Hence phylogenetic analyses cannot exclude the possibility of unsampled intermediaries, the data interpretation should be more careful and conservative, and it should not be considered as the only evidence for the source identification in a trial without epidemiological or serological information. The evaluation of the introduced UDPS method in the identification of transmission source has shown that the validity and evidential effects were still limited and need further optimization.

16S rRNA molecular profiling of heavy metal tolerant bacterial communities isolated from soil contaminated by electronic waste.

Electronic waste is an evolving source of harmful pollutants in our surrounding environments and considered to be perilous as it contains toxic metals such as chromium, cadmium, lead, mercury, zinc, and nickel in huge quantities. Heavy metals are harmful contaminants and accumulated in the environment due to various anthropogenic activities. The present study was conducted to isolate and characterize different heavy metal tolerant bacterial species, based on molecular techniques from soil contaminated by electronic waste. The contaminated soil samples were analyzed for various physicochemical properties such as pH, electrical conductivity, soil moisture, water holding capacity, organic carbon, organic matter, available phosphorus, total nitrogen, and potassium using standard procedures. The soil samples were found to contain a higher amount of different heavy metals such as copper, chromium, lead, iron, cadmium, and nickel. Serial dilution and spread plate techniques have been used for bacterial isolation. The identification and molecular characterization of isolated bacterial species were done by biochemical tests and 16S rRNA gene sequencing technique. The 16S rRNA sequencing analysis confirmed the presence of different bacterial species as, Micrococcus aloeverae, Kocuria turfanensis, Bacillus licheniformis, Bacillus jeotgali, Bacillus velezensis, and Bacillus haikouensis. The findings indicated that the e-waste dumping sites are the storehouse of elite bacterial species. The present research study offers a platform for systematic analysis of e-waste sites by microbial profiling that may help in the innovation of novel microorganisms of scientific importance and better biotechnological potential.

Natural co-infection with two virulent wild strains of Marek's disease virus in a commercial layer flock.

Marek's disease (MD) is a highly contagious lymphoproliferative poultry disease caused by the oncogenic herpesvirus, Marek's disease virus (MDV). MDV strains have shown a continued evolution of virulence leading to immune failure, and MD cases continue to occur. Co-infection of virulent MDV strains is an important factor leading to viral evolution and host immune failure. This study conducted a laboratory diagnosis and analysis of a MDV infected flock. Testing showed that all samples were MDV positive. PCR detection identified a variable 132-base pair repeat (132-bpr) sequence copy number. This indicated that two virulent strains of MDV were co-infecting the flock. Therefore, we performed homology, sequence alignment, and phylogenetic tree analysis of MDV variant genes including meq, pp38, and RLORF4. Two MDV strains had co-infected the flock; one was the 132bpr two-copy characteristic strain (AH2C) and the other was a 132bpr three-copy characteristic strain (AH3C). Specific mutations in AH3C were found, suggesting that it is a new variant strain. Furthermore, the viral load of the two strains in vivo indicated that both strains had high and similar replication ability. There was no significant difference in the proportion of positive samples of the two strains causing disease. In the whole flock, neither strain displayed an obvious advantage. However, there was a dominant strain in individual chickens, with the exception of one sample. This study reported the co-infection regularity of two virulent MDV strains in the same flock, and even in the same chicken in field conditions. In the context of overall epidemiology, this study is a useful reference.

The complete mitochondrial genome of Asparagus officinalis L.

Asparagus officinalis L. is a horticultural plant in nature and belongs to the genus Asparagus of Asparagaceae family. The complete mitochondrial (mt) genome of A. officinalis was 492,062 bp in length with a 45.9% GC content. A total of 60 genes were annotated including 37 protein-coding genes, 17 tRNA genes, and 6 rRNA genes. Phylogenetic tree analysis using the neighbor-joining method demonstrated that A. officinalis was most closely related to Allium cepa and separated from Beta vulgaris subsp. Vulgaris based on the complete mt genome sequence.

Characterization of the complete chloroplast genome of Liriope platyphylla (Asparagaceae: Nolinoideae) isolated in Korea.

Liriope platyphylla is used as an important medicinal plant for fatigue, cough, and inflammation in South Korea. Here, we report the complete chloroplast genome of L. platyphylla. The total genome size of the chloroplast is 157,076 bp with a large single-copy region (LSC: 85,374 bp), a small single-copy region (SSC: 18,748 bp), and inverted repeat regions (IRa and IRb: 26,477 bp). The GC content of the L. platyphylla chloroplast was 37.6%. The cp genome encoded a set of 129 genes, including 83 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The phylogenetic tree analysis indicated that L. platyphylla is closely related to L. spicata.