How to report and make sense of a new HIV-1 circulating recombinant form?

Co-circulation of multiple HIV-1 subtypes in the same high-risk groups leads to the on-going generation of various inter-subtype recombinants, including unique (URFs) and circulating (CRFs) recombinant forms, which brings a new challenge for the prevention and eradication of HIV/AIDS. Identification and prompt reporting of new CRFs will provide not only new insights into the understanding of genetic diversity and evolution of HIV-1, but also an early warning of potential prevalence of these variants. Currently, 140 HIV-1 CRFs have been described; however, their prevalence and clinical importance are less concerned. Apart from the mosaic genomic maps, less other valuable information, including the clinical and demographic data, genomic sequence characteristics, origin and evolutionary dynamics, as well as representative genomic fragments for determining the variants, are available for most of these CRFs. Accompanied with the growing increase of HIV-1 full-length genomic sequences, more and more CRFs will be identified in the near future due to the high recombination potential of HIV-1. Here, we discuss the prevalence and clinical importance of various HIV-1 CRFs and propose how to report and make sense of a new HIV-1 CRF

Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology

Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology

A Mismatch-Tolerant Reverse Transcription Loop-Mediated Isothermal Amplification Method and Its Application on Simultaneous Detection of All Four Serotype of Dengue Viruses

Loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens causing infectious diseases. However, mismatches between primers (especially in the 3′-end) and templates significantly reduced the amplification efficiency of LAMP, and limited its application to genetically diverse viruses. Here, we reported a novel mismatch-tolerant LAMP assay and its application in the detection of dengue viruses (DENV). The novel method features the addition of as little as 0.15 U of high-fidelity DNA polymerase to the standard 25 μl LAMP reaction mixture. This amount was sufficient to remove the mismatched bases at the 3′-end of primers, thereby resulting in excellent tolerance for various mismatches occurring at the 3′-end of the LAMP primers during amplification. This novel LAMP assay has a markedly improved amplification efficiency especially for the mutants forming mismatches with internal primers (FIP/BIP) and loop primers (FLP/BLP). The reaction time of the novel method was about 5.6–22.6 min faster than the conventional LAMP method regardless of the presence or absence of mismatches between primers and templates. Using the novel method, we improved a previously established pan-serotype assay for DENV, and demonstrated greater sensitivity for detection of four DENV serotypes than the previous one. The limit of detection (LOD) of the novel assay was 74, 252, 78, and 35 virus RNA copies per reaction for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. Among 153 clinical samples from patients with suspected DENV infection, the novel assay detected 94.8% samples being DENV positive, higher than that detected by the commercial NS1 antigen assay (92.2%), laboratory-based RT-PCR method (78.4%), and the conventional RT-LAMP assay (86.9%). Furthermore, the novel RT-LAMP assay has been developed into a visual determination method by adding colorimetric dyes. Because of its simplicity, all LAMP-based diagnostic assays may be easily updated to the newly improved version. The novel mismatch-tolerant LAMP method represents a simple, sensitive and promising approach for molecular diagnosis of highly variable viruses, and it is especially suited for application in resource-limited settings

Highly efficient glucose production from raw non-pretreated Chinese medicinal herbal residues via the synergism of cellulase and amylolytic enzymes

Available literature on Chinese medicinal herbal residues (CMHRs) bioconversion highlights pretreatment prior to saccharification with cellulase without considering the presence of starch constituent. Herein, four commonly found CMHRs were tested for starch content, and it was found they all contained starch with content ranging from 4.74% to 16.78%. Hydrolysis of raw CMHRs with combined cellulase and amylolytic enzymes yielded increments of 16.85% to 26.51% in 48-h glucan conversion compared to cellulase alone. Further study showed 48-h glucan conversion of raw CMHRs outperformed that pretreated by water-ethanol successive extraction, ultrasound and acid, but underperformed alkali-pretreated CMHRs. Although increasing 48-h glucan conversion in the range of 7.40% to 24.10% compared to raw CMHRs, alkaline pretreatment demonstrated low glucose recovery and incurred additional cost, making it unfavorable. Saccharification of the four raw CMHRs with combined enzymes seems like a preferred option considering the elimination of high-cost pretreatment step

Highly efficient glucose production from raw non-pretreated Chinese medicinal herbal residues via the synergism of cellulase and amylolytic enzymes

Available literature on Chinese medicinal herbal residues (CMHRs) bioconversion highlights pretreatment prior to saccharification with cellulase without considering the presence of starch constituent. Herein, four commonly found CMHRs were tested for starch content, and it was found they all contained starch with content ranging from 4.74% to 16.78%. Hydrolysis of raw CMHRs with combined cellulase and amylolytic enzymes yielded increments of 16.85% to 26.51% in 48-h glucan conversion compared to cellulase alone. Further study showed 48-h glucan conversion of raw CMHRs outperformed that pretreated by water-ethanol successive extraction, ultrasound and acid, but underperformed alkali-pretreated CMHRs. Although increasing 48-h glucan conversion in the range of 7.40% to 24.10% compared to raw CMHRs, alkaline pretreatment demonstrated low glucose recovery and incurred additional cost, making it unfavorable. Saccharification of the four raw CMHRs with combined enzymes seems like a preferred option considering the elimination of high-cost pretreatment step

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance

Ultrasound treatment of herbal extraction residue to enhance enzymatic saccharification

The utilization of herbal extraction residues (HERs) for production of valuable products has gained significant momentum in recent years. In this study, Lianhua Qingwen residue (LQR) was selected as a representative HER for enzymatic saccharification. By subjecting starch-containing raw unpretreated LQR to direct hydrolysis using a combination of cellulolytic and amylolytic enzyme, a glucan conversion of around 60 % was achieved within 48 h. To further enhance the hydrolysis rate and yield, ultrasound treatment of LQR samples was conducted. Investigation into the effect of ultrasound treatment on subsequent enzymatic saccharification revealed a noteworthy acceleration in the saccharification rate of LQR, exceeding 20 % within 10 h of hydrolysis time. Notably, this enhancement was achieved even when the substrate concentration was high at 10 % under optimized conditions of 5 min of sonication at a power intensity of 1.20 kW/cm2 and a temperature of 50 °C. The improvement in efficiency can be attributed to the disruptive effect of ultrasound treatment on the structure of LQR, leading to a reduction in particle size. Consequently, the LQR particles exhibited better contact with the enzyme, promoting a more efficient saccharification process. These findings suggest that ultrasound technology holds considerable potential for enhancing the enzymatic saccharification of HERs

Sandwich structured membrane adsorber with metal organic frameworks for aflatoxin B1 removal

Aflatoxins as toxic substances produced by microorganisms widely existed in food crops and detoxification of aflatoxins is highly desired to avoid their leaking into the environment. Herein, a novel combination strategy of metal organic frameworks (MOFs) and porous membrane was used to prepare membrane adsorbent for aflatoxin B1 (AFB1) removal. Firstly, the MOFs particles were readily fixed on a porous membrane by reverse filtration. Subsequently, polydopamine (PDA) coating and further charged polymer grafting were employed for sealing the MOFs particles. By systematically studying four kinds of MOFs particles for AFB1 adsorption, the MIL-100 and the resultant MIL-100 loaded membrane outperformed the other MOFs materials. Through comprehensively evaluating five different sealing approaches, it was found that the charge of the grafted polymers on the membrane was critical for AFB1 adsorption. The PDA/alginate dialdehyde (ADA) modified MIL-100 loaded membrane prepared with PDA coating and ADA grafting for 30 min, could achieve the highest AFB1 removal efficiency of 76.4% and the membrane stability was greatly improved, meanwhile the water permeate flux was barely affected by the additional layers. A good reusability could be realized by a facile desorption process and the AFB1 removal efficiency kept at 61.8% after five reuse cycles. When dealing with real corn juice, the AFB1 removal efficiency in flow-through mode was lower than that obtained in dipping incubation mode, because severer membrane fouling occurred during dynamic adsorption which blocked the adsorption sites on the membrane. The present work not only demonstrated a reliable and effective approach to synthesize robust MOFs-loaded membrane for AFB1 removal, but also offered new insights into micropollutant removal with MOFs derived materials