Advanced DNA Amplification for Efficient Data Storage.

DNA amplification technologies have significantly advanced biotechnology, particularly in DNA storage. However, adaptation of these technologies to DNA storage poses substantial challenges. Key bottlenecks include achieving high throughput to manage large data sets, ensuring rapid and efficient DNA amplification, and minimizing bias to maintain data fidelity. This perspective begins with an overview of natural and artificial amplification strategies, such as polymerase chain reaction and isothermal amplification, highlighting their respective advantages and limitations. It then explores the prospective applications of these techniques in DNA storage, emphasizing the need to optimize protocols for scalability and robustness in handling diverse digital data. Concurrently, we identify promising avenues, including advancements in enzymatic processes and novel amplification methodologies, poised to mitigate existing constraints and propel the field forward. Ultimately, we provide insights into how to utilize advanced DNA amplification strategies poised to revolutionize the efficiency and feasibility of data storage, ushering in enhanced approaches to data retrieval in the digital age.

Spatial Engineering of Heterotypic Antigens on a DNA Framework for the Preparation of Mosaic Nanoparticle Vaccines with Enhanced Immune Activation against SARS-CoV-2 Variants.

Mosaic nanoparticle vaccines with heterotypic antigens exhibit broad-spectrum antiviral capabilities, but the impact of antigen proportions and distribution patterns on vaccine-induced immunity remains largely unexplored. Here, we present a DNA nanotechnology-based strategy for spatially assembling heterotypic antigens to guide the rational design of mosaic nanoparticle vaccines. By utilizing two aptamers with orthogonal selectivity for the original SARS-CoV-2 spike trimer and Omicron receptor-binding domain (RBD), along with a DNA soccer-ball framework, we precisely manipulate the spacing, stoichiometry, and overall distribution of heterotypic antigens to create mosaic nanoparticles with average, bipolar, and unipolar antigen distributions. Systematic in vitro and in vivo immunological investigations demonstrate that 30 heterotypic antigens in equivalent proportions, with an average distribution, lead to higher production of broad-spectrum neutralizing antibodies compared to the bipolar and unipolar distributions. Furthermore, the precise assembly utilizing our developed methodology reveals that a mere increment of five Omicron RBD antigens on a nanoparticle (from 15 to 20) not only diminishes neutralization against the Omicron variant but also triggers excessive inflammation. This work provides a unique perspective on the rational design of mosaic vaccines by highlighting the significance of the spatial placement and proportion of heterotypic antigens in their structure-activity mechanisms.

Efficient genome engineering in Mycolicibacterium neoaurum using Cas9 from Streptococcus thermophilus.

Non-pathogenic mycobacteria, including Mycolicibacterium neoaurum, can directly utilize phytosterols for large-scale industrial production of steroid medicine intermediates due to their natural steroid metabolism pathway. The targeted genetic modification of M. neoaurum is conducive to the selection of high-yield engineering bacteria with high-value-added product, such as Pregnadien-20-carboxylic acid (PDC), which is an important precursor for synthesizing some corticosteroids. Based on heterologous type II CRISPR/sth1Cas9 system, a simple strategy was developed to genetic engineer M. neoaurum genome. Here, a customizable plasmid tool pMSC9 was constructed from pMV261 with integration of sth1Cas9 protein and corresponding sgRNA scaffold. Subsequently, the pMSC9 was inserted with spacer sequences corresponding to different targeted genes, generating editing plasmids, and then transformed into M. neoaurum. As a result, the targeted genes were introduced with DNA double stand breaks (DSBs) by CRISPR/sth1Cas9 system and then repaired by innate non-homologous end-joining (NHEJ) mechanism. Finally, editing plasmids were cured from correctly edited M. neoaurum mutants by means of no resistance cultivation, and the resulting mutant deleting the one target gene was used as the host to which another target gene could be deleted via the same process. This study demonstrated that the CRISPR/sth1Cas9 tool allowed M. neoaurum strains to be rapidly edited. And the editing mode of CRISPR/sth1Cas9 system indicated that this tool was an important supplement to the gene editing toolbox of M. neoaurum.

Effect of a real-time automatic nosocomial infection surveillance system on hospital-acquired infection prevention and control.

BACKGROUND: The systematic collection of valid data related to hospital-acquired infections (HAIs) is considered effective for nosocomial infection prevention and control programs. New strategies to reduce HAIs have recently fueled the adoption of real-time automatic nosocomial infection surveillance systems (RT-NISSs). Although RT-NISSs have been implemented in some hospitals for several years, the effect of RT-NISS on HAI prevention and control needs to be further explored. METHODS: A retrospective, descriptive analysis of inpatients from January 2017 to December 2019 was performed. We collected hospital-acquired infection (HAI) cases and multidrug resistant organism (MDRO) infection cases by traditional surveillance in period 1 (from January 2017 to December 2017), and these cases were collected in period 2 (from January 2018 to December 2018) and period 3 (from January 2019 to December 2019) using a real-time nosocomial infection surveillance system (RT-NISS). The accuracy of MDRO infection surveillance results over the 3 periods was examined. The trends of antibiotic utilization rates and pathogen culture rates in periods 2 and 3 were also analysed. RESULTS: A total of 114,647 inpatients, including 2242 HAI cases, were analysed. The incidence of HAIs in period 2 was significantly greater than that in period 1 (2.28% vs. 1.48%, χ2 = 61.963, p < 0.001) and period 3 (2.28% vs. 2.05%, χ2 = 4.767, p = 0.029). The incidence of five HAI sites, including respiratory infection, urinary tract infection (UTI), surgical site infection (SSI), bloodstream infection (BSI) and skin and soft tissue infection, was significantly greater in period 2 compared with period 1 (both p < 0.05) but was not significantly different from that in period 3. The incidence of hospital-acquired MDRO infections in period 3 was lower than that in period 2. The identification of MDRO infection cases using the RT-NISS achieved a high level of sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV), especially in period 3 (Se = 100%, Sp = 100%, PPV = 100% and NPV = 100%). CONCLUSION: The adoption of a RT-NISS to adequately and accurately collect HAI cases is useful to prevent and control HAIs. Furthermore, RT-NISSs improve accuracy in MDRO infection case reporting, which can timely and accurately guide and supervise clinicians in implementing MDRO infection prevention and control measures.

Multiplexed site-specific genome engineering in Mycolicibacterium neoaurum by Att/Int system.

Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories. For some uncommon microbial hosts, such as Mycolicibacterium and Mycobacterium species, however, it is a challenge. Here, we present a multiplexed integrase-assisted site-specific recombination (miSSR) method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories. First, a single-step multi-copy integration method was established in M. neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy, the efficiencies of which were ∼100% for no more than three-copy integration events and decreased sharply to ∼20% for five-copy integration events. Second, the R4, Bxb1 and ΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events. Third, a reconstructed mycolicibacterial Xer recombinases (Xer-cise) system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation. As a proof of concept, the biosynthetic pathway of ergothioneine (EGT) in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system. With six copies of the biosynthetic gene clusters (BGCs) of EGT and pentose phosphate isomerase (PRT), the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L, which was 3.77 times of that in the wild strain. The improvements indicated that the miSSR system was an effective, flexible, and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.