Macrogenomics Reveals Effects on Marine Microbial Communities during Oplegnathus punctatus Enclosure Farming.

(1) Background: Laizhou Bay is an important aquaculture area in the north of China. Oplegnathus punctatus is one of the species with high economic benefits. In recent years, the water environment of Laizhou Bay has reached a mild eutrophication level, while microorganisms are an important group between the environment and species. In this study, we evaluated alterations in environmental elements, microbial populations, and antibiotic resistance genes (ARGs) along with their interconnections during Oplegnathus punctatus net culture. (2) Methods: A total of 142 samples from various water layers were gathered for metagenome assembly analysis. Mariculture increases the abundance of microorganisms in this culture area and makes the microbial community structure more complex. The change had more significant effects on sediment than on seawater. (3) Results: Certain populations of cyanobacteria and Candidatus Micrarchaecta in seawater, and Actinobacteria and Thaumarchaeota in sediments showed high abundance in the mariculture area. Antibiotic resistance genes in sediments were more sensitive to various environmental factors, especially oxygen solubility and salinity. (4) Conclusions: These findings highlight the complex and dynamic nature of microorganism-environment-ARG interactions, characterized by regional specificity and providing insights for a more rational use of marine resources.

Complete mitochondrial genome and phylogenetic analysis of Mancinella alouina.

BACKGROUND: The Muricidae family in the Class Gastropoda comprises numerous species with a vast range of morphological features and a worldwide presence. The phylogeny of the Muricidae has been analyzed in previous studies; however, the evolutionary relationships among the main branches of the Muricidae remain unknown. METHODS AND RESULTS: In the present study, the mitochondrial genome of Mancinella alouina was sequenced. The mitochondrial genome was found to be 16,671 bp in length and made up of 37 genes (13 protein-coding genes, 22 transfer RNA and 2 ribosomal RNA genes). The genome has an A-T-rich region (66.5% A + T content) and all of the PCGs use the ATN start codon and the TAG or TAA stop codons. The mitochondrial gene arrangement of Mancinella alouina is similar to that of other Muricidae, except for Ocinebrellus inornatus and Ceratostoma burnetti. On the basis of a flexible molecular clock model, time-calibrated phylogenetic results indicate that the genus Mancinella diverged roughly 18.09 Mya, and that the family Muricidae emerged in the Late Cretaceous. CONCLUSIONS: This study reveals the structural and sequence information features of the mitochondrial genome of Mancinella alouina. This study provides evidence for the relationships within the family Muricidae at the molecular level, and infer the divergence time. The results of phylogenetic analyses strongly support the current classification.

Mitochondrial Genomes of Streptopelia decaocto: Insights into Columbidae Phylogeny.

In this research, the mitochondrial genome of the Streptopelia decaocto was sequenced and examined for the first time to enhance the comprehension of the phylogenetic relationships within the Columbidae. The complete mitochondrial genome of Streptopelia decaocto (17,160 bp) was structurally similar to the recognized members of the Columbidae family, but with minor differences in gene size and arrangement. The structural AT content was 54.12%. Additionally, 150 mitochondrial datasets, representing valid species, were amassed in this investigation. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees and evolutionary time relationships of species were reconstructed based on cytb gene sequences. The findings from the phylogenetic evaluations suggest that the S. decaocto was classified under the Columbinae subfamily, diverging from the Miocene approximately 8.1 million years ago, indicating intricate evolutionary connections with its close relatives, implying a history of species divergence and geographic isolation. The diversification of the Columbidae commenced during the Late Oligocene and extended into the Miocene. This exploration offers crucial molecular data for the S. decaocto, facilitating the systematic taxonomic examination of the Columbidae and Columbiformes, and establishing a scientific foundation for species preservation and genetic resource management.

Unraveling the effects and mechanisms of microplastics on anaerobic fermentation: Exploring microbial communities and metabolic pathways.

To investigate the effects of microplastics (MPs) on hydrolysis, acidification and microbial characteristics during waste activated sludge (WAS) anaerobic fermentation process, five different kinds of MPs were added into the WAS fermentation system and results indicated that, compared to the control group, the addition of polyvinyl chloride (PVC)-MPs exhibited the least inhibition on volatile fatty acids (VFAs), reducing them by 13.49 %. Conversely, polyethylene (PE)-MPs resulted in the greatest inhibition, with a reduction of 29.57 %. MPs, while accelerated the dissolution of WAS that evidenced by an increase of lactate dehydrogenase (LDH) release, concurrently inhibited the activities of relevant hydrolytic enzymes (α-Glucosidase, protease). For microbial mechanisms, MPs addition affected the proliferation of key microorganisms (norank_f_Bacteroidetes_vadinHA17, Ottowia, and Propioniclava) and reduced the abundance of genes associated with hydrolysis and acidification (pfkb, gpmI, ilvE, and aces). Additionally, MPs decreased the levels of key hydrolytic and acidogenic enzymes to inhibit hydrolysis and acidification processes. This research provides a basis for understanding and unveils impact mechanisms of the impact of MPs on sludge anaerobic fermentation.

Chromosome-level genome assembly of Przevalski's partridge (Alectoris magna).

Przevalski's partridge (Alectoris magna) is one of the birds in the genus Alectoris endemic to China. The distribution of A. magna was narrow, and it was only found in parts of the Qinghai, Gansu, and Ningxia provinces. A. magna was considered a monotypic species until it was distinguished into two subspecies. However, external morphological characteristics, rather than genetic differences or evolutionary relationships, are now commonly used as evidence of subspecies differentiation. In this study, a chromosome-level reference genome of A. magna has been constructed by combining Illumina, PacBio and Hi-C sequencing data. The 1135.01 Mb A. magna genome was ultimately assembled. The genome showed 96.9% completeness (BUSCO), with a contig N50 length of 23.34 Mb. The contigs were clustered and oriented on 20 chromosomes, covering approximately 99.96% of the genome assembly. Additionally, altogether 19,103 protein-coding genes were predicted, of which 95.10% were functionally annotated. This high-quality genome assembly could serve as a valuable genomic resource for future research on the functional genomics, genetic protection, and interspecific hybridization of A. magna.

The complete mitochondrial genome and phylogenetic analysis of Lepidozona coreanica (Reeve, 1847).

In the present study, the complete mitochondrial genome of Lepidozona coreanica was sequenced and described. The complete mitogenome sequence of L. coreanica is 16,572 bp long and contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and two ribosomal RNA (rRNA) genes. The base composition was AT biased (70.1%). The 13 PCGs of L. coreanica and the other 15 species of Polyplacophora were used for phylogenetic analysis using maximum-likelihood methods. The results showed that L. coreanica, Ischnochiton hakodadensis, and Chaetopleura apiculata are sister groups of the three lineages.

A Palm Germ-Radar (PaGeR) for rapid and simple COVID-19 detection by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

The current COVID-19 pandemic caused by SARS-CoV-2 is raging, seriously threatening people's lives. The establishment of rapid and accurate pathogen detection technology is not only critical in this epidemic, but also a reminder that we must always be prepared for possible future outbreaks. Therefore, we developed a Palm Germ-Radar (PaGeR) device for rapid and simple detection of COVID-19 from extracted patient sample RNA by RT-LAMP. The whole procedure of rapid COVID-19 detection is based on 4 simple steps: inactivation, extraction, amplification, and detection. SARS-CoV-2 down to 1 copy/µL could be detected selectively with naked-eye. Three detection methods (colorimetric, fluorometric and lateral dipstick readout) could be performed in PaGeR instrument. By employing the PaGeR, we successfully detected SARS-CoV-2 in clinical RNA samples isolated from swab specimens. The results showed that 15 out of 17 COVID-19 patients were diagnosed as positive while all 55 normal samples were diagnosed as negative. Therefore, the developed PaGeR instrument can realize the detection of COVID-19 with easily visualized results, providing a promising instrument for rapid detection in the community as well as at home.

Heterologous Boost Following Mycobacterium bovis BCG Reduces the Late Persistent, Rather Than the Early Stage of Intranasal Tuberculosis Challenge Infection.

Adults are the leading population affected by tuberculosis (TB) epidemic and death. Developing an effective vaccine against adult TB is urgently needed. Mycobacterium bovis Bacillus Calmette-Guerin (BCG) prime-heterologous boost strategy has been explored extensively to protect adults against primary TB infection, but the majority of experimental regimens have not improved the protection primed by the BCG vaccine. The reason attributed to the failure remains unknown. In this study, CTT3H-based vaccines, namely DMT adjuvanted CTT3H subunit or DNA vaccine (pCTT3H-DMT), and recombinant adenovirus rAdCTT3H were constructed. Protective efficacy and immunogenicity of BCG prime-CTT3H based boosters were compared in C57BL/c mice models of primary or late persistent TB infection. Similar protective efficacy against early intranasal infection was provided by different CTT3H-based vaccines alone in vaccinated mice, and their protection was inferior to that of the BCG vaccine. In addition, CTT3H-based heterologous boosters did not enhance the protection conferred by the BCG vaccine against primary infection. However, all of these three boosters provided stronger protection against late persistent TB infection than BCG alone, regardless of vaccine types. Although BCG prime-boosters elicited Th1-biased responses to the antigen CTT3H, the number of CTT3H-sepcific IFN-γ-expressing TEM (CD62LloCD44hi) and IL-2-expressing TCM (CD62LhiCD44hi) cells in the spleen was not improved before exposure to Mycobacterium tuberculosis infection. In contrast, IFN-γ+ TEM and IL-2+ TCM cells in spleens, especially in lungs were significantly increased in BCG prime-boosters after exposure vaccination. Our results indicate that BCG prime-boost strategy might be a promising measure for the prevention against late persistent TB infection by induction of IFN-γ+ TEM and IL-2+ TCM cells in the lung, which can be used as alternative biomarkers for guiding the clinical practice and future development of TB vaccine for adults.

Immunogenicity of heparin-binding hemagglutinin expressed by Pichia pastoris GS115 strain.

OBJECTIVES: Heparin-binding hemagglutinin (HBHA), a mycobacterial cell surface protein, mediates adhesion to nonphagocytic cells and the dissemination of Mycobacterium tuberculosis (M. tuberculosis) from the site of primary infection. Superior expression systems are required to obtain abundant M. tuberculosis proteins for the purpose of diagnosing M. tuberculosis infection or for the immunization. Here, HBHA was expressed by Pichia pastoris (P. pastoris) GS115 strain , and the immunogenicity of HBHA was evaluated. MATERIALS AND METHODS: The HBHA gene of M. tuberculosis was cloned into the pPIC9K plasmid, which was good for electroporation into P. pastoris GS115 strain. Unlabeled HBHA protein was purified using a Sepharose CL-6B column, and its expression was confirmed using anti-HBHA polyclonal antibody from mouse serum. We injected C57BL/6 mice with HBHA/ dimethyldioctadecylammonium/trehalose 6,6'-dibehenate (HBHA/DDA/TDB) to investigate the immunogenicity of this potential vaccine. RESULTS: The results demonstrated that HBHA/DDA/TDB has the ability to induce high levels of HBHA-specific IgG antibody and its subclasses, as well as interferon-gamma, compared with injection of phosphate-buffered saline, DDA/TDB alone and Bacillus Calmette-Guérin (BCG) controls (P<0.05). Moreover, the ratio of IgG2a/IgG1 of the HBHA/DDA/TDB group was higher than that of the BCG group (P<0.05). CONCLUSION: HBHA with no label has excellent immunogenicity, and is suitable for evaluating the effectiveness to prevent M. tuberculosis infection.

A Multistage Subunit Vaccine Effectively Protects Mice Against Primary Progressive Tuberculosis, Latency and Reactivation.

Adult tuberculosis (TB) is the main cause of TB epidemic and death. The infection results mainly by endogenous reactivation of latent TB infection and secondarily transmitted by exogenous infection. There is no vaccine for adult TB. To this end, we first chose antigens from a potential antigenic reservoir. The antigens strongly recognized T cells from latent and active TB infections that responded to antigens expressed by Mycobacterium tuberculosis cultured under different metabolic states. Fusions of single-stage polyprotein CTT3H, two-stage polyprotein A1D4, and multistage CMFO were constructed. C57BL/6 mice vaccinated with DMT adjuvant ed CMFO (CMFO-DMT) were protected more significantly than by CTT3H-DMT, and efficacy was similar to that of the only licensed vaccine, Bacillus Calmette-Guérin (BCG) and A1D4-DMT in the M. tuberculosis primary infection model. In the setting of BCG priming and latent TB infection, M. tuberculosis in the lung and spleen was eliminated more effectively in mice boosted with CMFO-DMT rather than with BCG, A1D4-DMT, or CTT3H-DMT. In particular, sterile immunity was only conferred by CMFO-DMT, which was associated with expedited homing of interferon-gamma+CD4+ TEM and interleukin-2+ TCM cells from the spleen to the infected lung. CMFO-DMT represents a promising candidate to prevent the occurrence of adult TB through both prophylactic and therapeutic methods, and warrants assessment in preclinical and clinical trials.

A live attenuated BCG vaccine overexpressing multistage antigens Ag85B and HspX provides superior protection against Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains one of the most menacing infectious diseases, although attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine has been widely used to protect children against primary TB. There are increasing evidences that rapid growing and dormant Mycobacterium tuberculosis (M. tuberculosis) coexist in vivo after infection. However, BCG vaccine only elicits cell-mediated immune responses to secretory antigens expressed by rapid growing pathogen. BCG vaccine is thus unable to thwart the reactivation of latent tuberculosis infection (LTBI), and its protection wanes over age after neonatal immunization. In order to extend its ability for a durable protection, a novel recombinant BCG (rBCG) strain, named rBCG::XB, was constructed by overexpressing immunodominant multistage antigens of Ag85B and HspX, which are expressed by both rapid replicating and dormant M. tuberculosis. Long-term protective effect and immunogenicity of rBCG::XB were compared with the parental BCG in vaccinated C57BL/6 mice. Our results demonstrated that rBCG::XB provided the stronger and long-lasting protection against M. tuberculosis H37Rv intranasal infection than BCG. The rBCG::XB not only elicited the more durable multistage antigen-specific CD4(+)Th1-biased immune responses and specific polyfunctional CD4(+)T cells but also augmented the CD8(+) CTL effects against Ag85B in vivo. In particular, higher levels of CD4(+) TEM and CD8(+) TCM cells, dominated by IL2(+) CD4(+) and CD8(+) TCM cells, were obtained in the spleen of rBCG::XB vaccinated mice. Therefore, our findings indicate that rBCG::XB is a promising candidate to improve the efficacy of BCG.

Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

High level of IFN-γ released from whole blood of human tuberculosis infections following stimulation with Rv2073c of Mycobacterium tuberculosis.

More efficacious and specific biomarkers are urgently needed for better control of tuberculosis (TB), the second leading infectious cause of mortality worldwide. The region of difference 9 (RD9) presents the genome of the causative pathogen Mycobacterium tuberculosis rather than other species of the genus Mycobacterium, which might be promising targets for specific diagnosis, vaccine development and pathogenesis. In this study, two proteins Rv2073c and Rv2074, encoded by the RD9 were expressed and purified from Escherichia coli system. Following stimulation with both proteins, the levels of IFN-γ secreted by T cells from a total of 49 whole blood samples obtained from clinically diagnosed active TB patients, patients with latent TB infections (LTBIs), and healthy donors, were compared with those of the incubation with recombinant fusion protein of CFP21 and MPT64 (rCM). Our results demonstrated that only Rv2073c could induce a higher level of IFN-γ in TB infections than healthy controls and there was a positive correlation between Rv2073c- and rCM-specific IFN-γ levels in TB infections and healthy donors, respectively. These findings indicate that Rv2073c might be a promising antigen for specific diagnostic reagents and vaccine candidates of TB.

Immunogenicity and protective efficacy of DMT liposome-adjuvanted tuberculosis subunit CTT3H vaccine.

Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8(+) epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6'-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4(+) Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ(+) CD8(+) T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8(+) T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine.

Protection against Mycobacterium tuberculosis infection offered by a new multistage subunit vaccine correlates with increased number of IFN-γ+ IL-2+ CD4+ and IFN-γ+ CD8+ T cells.

Protein subunit vaccines present a compelling new area of research for control of tuberculosis (TB). Based on the interaction between Mycobacterium tuberculosis and its host, five stage-specific antigens of M. tuberculosis that participate in TB pathogenesis--Rv1813, Rv2660c, Ag85B, Rv2623, and HspX--were selected. These antigens were verified to be recognized by T cells from a total of 42 whole blood samples obtained from active TB patients, patients with latent TB infections (LTBIs), and healthy control donors. The multistage polyprotein A1D4 was developed using the selected five antigens as a potentially more effective novel subunit vaccine. The immunogenicity and protective efficacy of A1D4 emulsified in the adjuvant MTO [monophosphoryl lipid A (MPL), trehalose-6,6'-dibehenate (TDB), components of MF59] was compared with Bacillus Calmette-Guerin (BCG) in C57BL/6 mice. Our results demonstrated that A1D4/MTO could provide more significant protection against M. tuberculosis infection than the PBS control or MTO adjuvant alone judging from the A1D4-specific Th1-type immune response; however, its efficacy was inferior to BCG as demonstrated by the bacterial load in the lung and spleen, and by the pathological changes in the lung. Antigen-specific single IL-2-secreting cells and different combinations with IL-2-secreting CD4+ T cells were beneficial and correlated with BCG vaccine-induced protection against TB. Antigen-specific IFN-γ+ IL-2+ CD4+ T cells were the only effective biomarker significantly induced by A1D4/MTO. Among all groups, A1D4/MTO immunization also conferred the highest number of antigen-specific single IFN-γ+ and IFN-γ+ TNF-α+ CD4+ T cells, which might be related to the antigen load in vivo, and single IFN-γ+ CD8+ T cells by mimicking the immune patterns of LTBIs or curable TB patients. Our strategy seems promising for the development of a TB vaccine based on multistage antigens, and subunit antigen A1D4 suspended in MTO adjuvant warrants preclinical evaluation in animal models of latent infection and may boost BCG vaccination.

Comparison of BCG prime-DNA booster and rBCG regimens for protection against tuberculosis.

Developing an effective adult prophylaxis vaccine is a high priority in the global control of tuberculosis (TB), because TB remains an important public health problem and the current widely used BCG vaccine provides effective protection only for children but variable protection against adult TB. BCG priming-heterologous vaccines booster and recombinant BCG technologies have been thought as two important regimens for inducing effective protection against adult TB. Obviously, defining the protective efficacy of the two regimens would benefit more rational design of the future adult TB vaccines. In this study, a recombinant BCG strain (rBCG::685A) expressing the fusion protein of ESAT-6 and Ag85A (r685A) of Mycobacterium tuberculosis was constructed successfully and the secretion of r685A protein from rBCG strain was confirmed by western blotting with anti-ESAT-6 and anti-Ag85A polyclonal antibodies, respectively. The immune responses and protective effects in rBCG::685A vaccinated C57BL/6 mice were compared with that of our previous reported BCG prime-pcD685A booster regimen. Boosting BCG with pcD685A DNA elicited higher level of r685A protein specific IFN-γ secreted by splenocytes and a more significant increase of both TNF-α and iNOS responses in the lung, thus providing better control of bacterial growth in both lung and spleen of immunized mice challenged with virulent M. tuberculosis, compared with mice vaccinated with rBCG::685A or BCG alone. Our results have implications for development of more effective adult TB vaccines for improved control of TB.

Immunogenicity and protective efficacy of a novel recombinant BCG strain overexpressing antigens Ag85A and Ag85B.

Recombinant Bacillus Calmette-Guérin (rBCG) strain is the promising vaccine candidate for tuberculosis (TB) prevention, which aims at providing more enduring and enhanced protection than the parental BCG vaccine. In this study, three rBCG strains overexpressing immunodominant antigens Ag85B (rBCG::85B), Ag85A (rBCG::85A), or both (rBCG::AB) of Mycobacterium tuberculosis were constructed, respectively. rBCG strains showed higher level of overexpression of Ag85A and/or Ag85B proteins than BCG containing empty vector pMV261(rBCG::261), which had low levels of endogenous expression of both proteins as expected. rBCG::AB strain could provide the strongest short-term and long-term protection in the lung against intravenous infection with virulent M. tuberculosis than rBCG::261 control and other two rBCG strains overexpressing single antigen. The stronger and longer-lasting protection provided by rBCG::AB than rBCG::261 was correlated with systemic in vitro antigen-specific IFN-γ responses. Therefore, our results indicate that rBCG::AB could be a very promising TB vaccine candidate and should be further evaluated for the preclinical test.