SurfPro-NN: A 3D point cloud neural network for the scoring of protein-protein docking models based on surfaces features and protein language models.

Protein-protein interactions (PPI) play a crucial role in numerous key biological processes, and the structure of protein complexes provides valuable clues for in-depth exploration of molecular-level biological processes. Protein-protein docking technology is widely used to simulate the spatial structure of proteins. However, there are still challenges in selecting candidate decoys that closely resemble the native structure from protein-protein docking simulations. In this study, we introduce a docking evaluation method based on three-dimensional point cloud neural networks named SurfPro-NN, which represents protein structures as point clouds and learns interaction information from protein interfaces by applying a point cloud neural network. With the continuous advancement of deep learning in the field of biology, a series of knowledge-rich pre-trained models have emerged. We incorporate protein surface representation models and language models into our approach, greatly enhancing feature representation capabilities and achieving superior performance in protein docking model scoring tasks. Through comprehensive testing on public datasets, we find that our method outperforms state-of-the-art deep learning approaches in protein-protein docking model scoring. Not only does it significantly improve performance, but it also greatly accelerates training speed. This study demonstrates the potential of our approach in addressing protein interaction assessment problems, providing strong support for future research and applications in the field of biology.

Combination therapy with oncolytic virus and T cells or mRNA vaccine amplifies antitumor effects.

Antitumor therapies based on adoptively transferred T cells or oncolytic viruses have made significant progress in recent years, but the limited efficiency of their infiltration into solid tumors makes it difficult to achieve desired antitumor effects when used alone. In this study, an oncolytic virus (rVSV-LCMVG) that is not prone to induce virus-neutralizing antibodies was designed and combined with adoptively transferred T cells. By transforming the immunosuppressive tumor microenvironment into an immunosensitive one, in B16 tumor-bearing mice, combination therapy showed superior antitumor effects than monotherapy. This occurred whether the OV was administered intratumorally or intravenously. Combination therapy significantly increased cytokine and chemokine levels within tumors and recruited CD8+ T cells to the TME to trigger antitumor immune responses. Pretreatment with adoptively transferred T cells and subsequent oncolytic virotherapy sensitizes refractory tumors by boosting T-cell recruitment, down-regulating the expression of PD-1, and restoring effector T-cell function. To offer a combination therapy with greater translational value, mRNA vaccines were introduced to induce tumor-specific T cells instead of adoptively transferred T cells. The combination of OVs and mRNA vaccine also displays a significant reduction in tumor burden and prolonged survival. This study proposed a rational combination therapy of OVs with adoptive T-cell transfer or mRNA vaccines encoding tumor-associated antigens, in terms of synergistic efficacy and mechanism.

Orchestrated codelivery of peptide antigen and adjuvant to antigen-presenting cells by using an engineered chimeric peptide enhances antitumor T-cell immunity.

The intrinsic pharmacokinetic limitations of traditional peptide-based cancer vaccines hamper effective cross-presentation and codelivery of antigens and adjuvants, which are crucial for inducing robust antitumor CD8+ T-cell responses. Here, we report the development of a versatile strategy that simultaneously addresses the different pharmacokinetic challenges of soluble subunit vaccines composed of antigens and CpG to modulate vaccine efficacy via translating an engineered chimeric peptide, eTAT, as an intramolecular adjuvant. Linking antigens to eTAT enhanced cytosolic delivery of the antigens. This, in turn, led to improved activation and lymph node-trafficking of antigen-presenting cells and antigen cross-presentation, thus promoting antigen-specific T-cell immune responses. Simple mixing of eTAT-linked antigens and CpG significantly enhanced codelivery of antigens and CpG to the antigen-presenting cells, and this substantially augmented the adjuvant effect of CpG, maximized vaccine immunogenicity and elicited robust and durable CD8+ T-cell responses. Vaccination with this formulation altered the tumor microenvironment and exhibited potent antitumor effects, with generally further enhanced therapeutic efficacy when used in combination with anti-PD1. Altogether, the engineered chimeric peptide-based orchestrated codelivery of antigen and adjuvant may serve as a promising but simple strategy to improve the efficacy of peptide-based cancer vaccines.

Wa-VP4* as a candidate rotavirus vaccine induced homologous and heterologous virus neutralizing antibody responses in mice, pigs, and cynomolgus monkeys.

Group A rotavirus (RVA) is the primary etiological agent of acute gastroenteritis (AGE) in children under 5 years of age. Despite the global implementation of vaccines, rotavirus infections continue to cause over 120,000 deaths annually, with a majority occurring in developing nations. Among infants, the P[8] rotavirus strain is the most prevalent and can be categorized into four distinct lineages. In this investigation, we expressed five VP4(aa26-476) proteins from different P[8] lineages of human rotavirus in E. coli and assessed their immunogenicity in rabbits. Among the different P[8] strains, the Wa-VP4 protein, derived from the MT025868.1 strain of the P[8]-1 lineage, exhibited successful purification in a highly homogeneous form and significantly elicited higher levels of neutralizing antibodies (nAbs) against both homologous and heterologous rotaviruses compared to other VP4 proteins derived from different P[8] lineages in rabbits. Furthermore, we assessed the immunogenicity of the Wa-VP4 protein in mice, pigs, and cynomolgus monkeys, observing that it induced robust production of nAbs in all animals. Interestingly, there was no significant difference between in nAb titers against homologous and heterologous rotaviruses in pigs and mankeys. Collectively, these findings suggest that the Wa-VP4* protein may serve as a potential candidate for a rotavirus vaccine.

A non-viral DNA delivery system consisting of multifunctional chimeric peptide fused with zinc-finger protein.

Non-viral gene delivery systems have received sustained attention as a promising alternative to viral vectors for disease treatment and prevention in recent years. Numerous methods have been developed to enhance gene uptake and delivery in the cytoplasm; however, due to technical difficulties and delivery efficiency, these systems still face challenges in a range of biological applications, especially in vivo. To alleviate this challenge, we devised a novel system for gene delivery based on a recombinant protein eTAT-ZF9-NLS, which consisted of a multifunctional chimeric peptide and a zinc-finger protein with sequence-specific DNA-binding activity. High transfection efficiency was observed in several mammalian cells after intracellular delivery of plasmid containing ZF9-binding sites mediated by eTAT-ZF9-NLS. Our new approach provides a novel transfection strategy and the transfection efficiency was confirmed both in vitro and in vivo, making it a preferential transfection reagent for possible gene therapy.

Design of a recombinant asparaginyl ligase for site-specific modification using efficient recognition and nucleophile motifs.

Asparaginyl ligases have been extensively utilized as valuable tools for site-specific bioconjugation or surface-modification. However, the application is hindered by the laborious and poorly reproducible preparation processes, unstable activity and ambiguous substrate requirements. To address these limitations, this study employed a structure-based rational approach to obtain a high-yield and high-activity protein ligase called OaAEP1-C247A-aa55-351. It was observed that OaAEP1-C247A-aa55-351 exhibits appreciable catalytic activities across a wide pH range, and the addition of the Fe3+ metal ion effectively enhances the catalytic power. Importantly, this study provides insight into the recognition and nucleophile peptide profiles of OaAEP1-C247A-aa55-351. The ligase demonstrates a higher recognition ability for the "Asn-Ala-Leu" motif and an N-terminus "Arg-Leu" as nucleophiles, which significantly increases the reaction yield. Consequently, the catalytic activity of OaAEP1-C247A-aa55-351 with highly efficient recognition and nucleophile motif, "Asn-Ala-Leu" and "Arg-Leu" under the buffer containing Fe3+ is 70-fold and 2-fold higher than previously reported OaAEP1-C247A and the most efficient butelase-1, respectively. Thus, the designed OaAEP1-C247A-aa55-351, with its highly efficient recognition and alternative nucleophile options, holds promising potential for applications in protein engineering, chemo-enzymatic modification, and the development of drugs.

Long-term efficacy of a recombinant hepatitis E vaccine in adults: 10-year results from a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Hepatitis E virus (HEV) is a frequently overlooked causative agent of acute hepatitis. Evaluating the long-term durability of hepatitis E vaccine efficacy holds crucial importance. METHODS: This study was an extension to a randomised, double-blind, placebo-controlled, phase-3 clinical trial of the hepatitis E vaccine conducted in Dontai County, Jiangsu, China. Participants were recruited from 11 townships in Dongtai County. In the initial trial, a total of 112 604 healthy adults aged 16-65 years were enrolled, stratified according to age and sex, and randomly assigned in a 1:1 ratio to receive three doses of hepatitis E vaccine or placebo intramuscularly at month 0, month 1, and month 6. A sensitive hepatitis E surveillance system including 205 clinical sentinels, covering the entire study region, was established and maintained for 10 years after vaccination. The primary outcome was the per-protocol efficacy of hepatitis E virus vaccine to prevent confirmed hepatitis E occurring at least 30 days after administration of the third dose. Throughout the study, the participants, site investigators, and laboratory staff remained blinded to the treatment assignments. This study is registered with ClinicalTrials.gov (NCT01014845). FINDINGS: During the 10-year study period from Aug 22, 2007, to Oct 31, 2017, 90 people with hepatitis E were identified; 13 in the vaccine group (0·2 per 10 000 person-years) and 77 in the placebo group (1·4 per 10 000 person-years), corresponding to a vaccine efficacy of 83·1% (95% CI 69·4-91·4) in the modified intention-to-treat analysis and 86·6% (73·0 to 94·1) in the per-protocol analysis. In the subsets of participants assessed for immunogenicity persistence, of those who were seronegative at baseline and received three doses of hepatitis E vaccine, 254 (87·3%) of 291 vaccinees in Qindong at the 8·5-year mark and 1270 (73·0%) of 1740 vaccinees in Anfeng at the 7·5-year mark maintained detectable concentrations of antibodies. INTERPRETATION: Immunisation with this hepatitis E vaccine offers durable protection against hepatitis E for up to 10 years, with vaccine-induced antibodies against HEV persisting for at least 8·5 years. FUNDING: National Natural Science Foundation of China, Fujian Provincial Natural Science Foundation, Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, and the Fundamental Research Funds for the Central Universities.

Numerical and experimental investigation on the performance of rapid ultrasonic-assisted nucleic acid extraction based on dispersive two-phase flow.

BACKGROUND: Nucleic acid extraction (NAE) is an essential step in the whole process of nucleic acid detection (NAT). Traditional manual extraction methods are time-consuming and laborious, unfavorable to the point-of-care testing of nucleic acids. Ultrasound has been emphasized due to its noncontact and easy-to-manipulate characteristics, and integration with microfluidic chip can realize rapid NAE through acoustic streaming effect. The uniformity of magnetic bead mixing in this process is a critical factor affecting the extraction effect. In this study, we developed an ultrasound-assisted NAE technique based on the magnetic bead method and optimized the chip structure to achieve rapid NAE. RESULT: We use ultrasonic-assisted coupled with magnetic bead method for ultra-fast NAE. The mixing process of magnetic beads driven by acoustic streaming is simulated by a dispersive two-phase flow model, and the ultrasonic incidence angle (θin), cone structure aspect ratio (Dc/Hc) and sheet structure thickness (Hp) are optimized to enhance the mixing performance. Furthermore, the effectiveness of NAE is validated by utilizing quantitative real-time PCR (qPCR) detection. The findings reveal that a θin value of 10° yields superior mixing performance compared to other incidence angles, resulting in a maximum increase of 84 % in mixing intensity. When Dc/Hc = 0.5 and Hp = 0.5 mm, the maximum mixing index in the localized region of the chamber after 1 s of ultrasound action can reach 83.6 % and 92.5 %, respectively. Compared to the original chamber, the CT values extracted after 5 s of ultrasound action shifted forward by up to 1.9 ct and 4.1 ct, respectively. SIGNIFICANCE: The dispersed two-phase flow model can effectively simulate the mixing process of magnetic beads, which plays an important role in assisting the structural design of chip extraction chambers. The single-step mixing of ultrasound-assisted NAE takes only 15s to achieve an extraction performance comparable to manual extraction. The extraction process can be completed within 7 min after integrating this technology with microfluidic chips and automated equipment, providing a solution for automated and efficient NAE.

Tpgen: a language model for stable protein design with a specific topology structure.

BACKGROUND: Natural proteins occupy a small portion of the protein sequence space, whereas artificial proteins can explore a wider range of possibilities within the sequence space. However, specific requirements may not be met when generating sequences blindly. Research indicates that small proteins have notable advantages, including high stability, accurate resolution prediction, and facile specificity modification. RESULTS: This study involves the construction of a neural network model named TopoProGenerator(TPGen) using a transformer decoder. The model is trained with sequences consisting of a maximum of 65 amino acids. The training process of TopoProGenerator incorporates reinforcement learning and adversarial learning, for fine-tuning. Additionally, it encompasses a stability predictive model trained with a dataset comprising over 200,000 sequences. The results demonstrate that TopoProGenerator is capable of designing stable small protein sequences with specified topology structures. CONCLUSION: TPGen has the ability to generate protein sequences that fold into the specified topology, and the pretraining and fine-tuning methods proposed in this study can serve as a framework for designing various types of proteins.

An Inhibitor-Monitorable Single-Tube Duplex Quantitative Real-Time PCR Assay for the Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is a citrus infectious disease caused by 'Candidatus Liberibacter' spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3'-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of 'Candidatus Liberibacter asiaticus' (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.

Reconsideration of maternal serological testing for predicting congenital CMV infection.

BACKGROUND: The value of the widely applied maternal cytomegalovirus (CMV) serological testing approach in predicting intrauterine transmission in highly seroprevalent regions remains unknown. METHODS: A nested case‒control study was conducted based on a maternal-child cohort study. Newborns with congenital CMV (cCMV) infection were included, and each of them was matched to 3 newborns without cCMV infection. Retrospective samples were tested for immunoglobulin G (IgG) avidity and immunoglobulin M (IgM) antibodies in maternal serum and CMV DNA in maternal blood and urine to analyse their associations with cCMV infection. RESULTS: Forty-eight newborns with cCMV infection and 144 matched newborns without infection were included in the study. Maternal IgM antibodies and IgG avidity during pregnancy were not statistically associated with intrauterine transmission. The presence of CMV DNAemia indicated a higher risk of cCMV infection, with the OR values as 5.7, 6.5 and 13.0 in early, middle and late pregnancy, respectively. However, the difference in CMV shedding rates in transmitters and nontransmitters was not significant in urine. CONCLUSION: The value of current maternal CMV serological testing in regions with high seropositivity rates is very limited and should be reconsidered. The detection of DNAemia would be helpful in assessing the risk of intrauterine transmission.

Anti-Epstein-Barr Virus BNLF2b for Mass Screening for Nasopharyngeal Cancer.

BACKGROUND: Population screening of asymptomatic persons with Epstein-Barr virus (EBV) DNA or antibodies has improved the diagnosis of nasopharyngeal carcinoma and survival among affected persons. However, the positive predictive value of current screening strategies is unsatisfactory even in areas where nasopharyngeal carcinoma is endemic. METHODS: We designed a peptide library representing highly ranked B-cell epitopes of EBV coding sequences to identify novel serologic biomarkers for nasopharyngeal carcinoma. After a retrospective case-control study, the performance of the novel biomarker anti-BNLF2b total antibody (P85-Ab) was validated through a large-scale prospective screening program and compared with that of the standard two-antibody-based screening method (EBV nuclear antigen 1 [EBNA1]-IgA and EBV-specific viral capsid antigen [VCA]-IgA). RESULTS: P85-Ab was the most promising biomarker for nasopharyngeal carcinoma screening, with high sensitivity (94.4%; 95% confidence interval [CI], 86.4 to 97.8) and specificity (99.6%; 95% CI, 97.8 to 99.9) in the retrospective case-control study. Among the 24,852 eligible participants in the prospective cohort, 47 cases of nasopharyngeal carcinoma (38 at an early stage) were identified. P85-Ab showed higher sensitivity than the two-antibody method (97.9% vs. 72.3%; ratio, 1.4 [95% CI, 1.1 to 1.6]), higher specificity (98.3% vs. 97.0%; ratio, 1.01 [95% CI, 1.01 to 1.02]), and a higher positive predictive value (10.0% vs. 4.3%; ratio, 2.3 [95% CI, 1.8 to 2.8]). The combination of P85-Ab and the two-antibody method markedly increased the positive predictive value to 44.6% (95% CI, 33.8 to 55.9), with sensitivity of 70.2% (95% CI, 56.0 to 81.4). CONCLUSIONS: Our results suggest that P85-Ab is a promising novel biomarker for nasopharyngeal carcinoma screening, with higher sensitivity, specificity, and positive predictive value than the standard two-antibody method. (Funded by the National Key Research and Development Program of China and others; ClinicalTrials.gov number, NCT04085900.).

Sophisticated and precise: design and implementation of a real-time optical detection system for ultra-fast PCR.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) has become indispensable in the realm of disease nucleic acid screening and diagnostics, owing to its remarkable precision and sensitivity, in which the real-time fluorescence detection system plays an extremely critical role. To solve the problems of long time and slow speed of traditional nucleic acid detection, PCR systems are evolving towards ultra-rapid configurations. Nonetheless, most extant ultra-rapid PCR systems either depend on endpoint detection for qualitative assessments due to inherent structural or heating constraints or circumvent the challenge of adapting optical systems to expeditious amplification systems, resulting in potential shortcomings in assay efficacy, volume, or expense. Consequently, this study proposed a design of a real-time fluorescence detection system for ultra-fast PCR, capable of executing six channels of real-time fluorescence detection. Through the meticulous calculation of the optical pathway within the optical detection module, effective regulation of system dimensions and the cost was accomplished. By devising an optical adaptation module, the signal-to-noise ratio was enhanced by approximately 307% without compromising the PCR temperature alteration rate. Ultimately, by employing a fluorescence model that accounted for the spatial attenuation effect of excitation light, as proposed herein, fluorescent dyes were arranged to evaluate the repeatability, channel interference, gradient linearity, and limit of detection of the system, which proved that the system had good optical detection performance. Finally, the real-time fluorescence detection of human cytomegalovirus (CMV) under 9 min ultra-fast amplification was achieved by a complete ultra-fast amplification experiment, which further validated the potential of the system to be applied to rapid clinical nucleic acid detection.

Intranasal influenza-vectored COVID-19 vaccine restrains the SARS-CoV-2 inflammatory response in hamsters.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants and "anatomical escape" characteristics threaten the effectiveness of current coronavirus disease 2019 (COVID-19) vaccines. There is an urgent need to understand the immunological mechanism of broad-spectrum respiratory tract protection to guide broader vaccines development. Here we investigate immune responses induced by an NS1-deleted influenza virus vectored intranasal COVID-19 vaccine (dNS1-RBD) which provides broad-spectrum protection against SARS-CoV-2 variants in hamsters. Intranasal delivery of dNS1-RBD induces innate immunity, trained immunity and tissue-resident memory T cells covering the upper and lower respiratory tract. It restrains the inflammatory response by suppressing early phase viral load post SARS-CoV-2 challenge and attenuating pro-inflammatory cytokine (Il6, Il1b, and Ifng) levels, thereby reducing excess immune-induced tissue injury compared with the control group. By inducing local cellular immunity and trained immunity, intranasal delivery of NS1-deleted influenza virus vectored vaccine represents a broad-spectrum COVID-19 vaccine strategy to reduce disease burden.

Ultra-fast, sensitive and low-cost real-time PCR system for nucleic acid detection.

Nucleic acid detection directly identifies the presence of pathogenic microorganisms and has various advantages, such as high sensitivity, commendable specificity and a short window period, and has been widely used in many fields, such as early tumor screening, prenatal diagnosis and infectious disease detection. Real-time PCR (polymerase chain reaction) is the most commonly used method for nucleic acid detection in clinical practice, but it always takes about 1-3 hours, severely limiting its application in particular scenarios such as emergency testing, large-scale testing and on-site testing. To solve the time-consuming problem, a real-time PCR system based on multiple temperature zones was proposed, which realized the speed of temperature change of biological reagents from 2-4 °C s-1 to 13.33 °C s-1. The system integrates the advantages of fixed microchamber-type and microchannel-type amplification systems, including a microfluidic chip capable of fast heat transfer and a real-time PCR device with a temperature control strategy based on the temperature difference. The detection of HCMV biological samples using the real-time PCR system in this research took only 15 min, which was 75% shorter compared to the commercial qPCR instrument such as BIO-RAD, and the detection sensitivity remained essentially the same. The system could complete nucleic acid detection within 9 min under extreme conditions, characterized by fast detection speed and high sensitivity, providing a promising solution for ultra-fast nucleic acid detection.

Hepatitis B Core Antibody Level: A Surrogate Marker for Host Antiviral Immunity in Chronic Hepatitis B Virus Infections.

The hepatitis B virus core protein (HBcAg) is a highly immunogenic particulate antigen. Nearly all patients with persistent or resolved hepatitis B virus (HBV) infection show seropositivity for hepatitis B core antibody (anti-HBc), which appears in the early stage of infection and is mostly present for life. Traditionally, the anti-HBc is regarded as an evidential serological marker of HBV infections. In the last ten years, several studies revealed the predictive value of quantitative anti-HBc (qAnti-HBc) level in the treatment response and clinical outcome of chronic HBV infections, implying new insights into this classic marker. Overall, qAnti-HBc should be regarded as an indicator of the host's immune response specific to HBV, which correlates with HBV-related hepatitis activity and liver pathology. This review summarized the latest understanding of the clinical values of qAnti-HBc for differentiating the CHB phase, predicting treatment response, and providing disease prognosis. Moreover, we also discussed the possible mechanism of qAnti-HBc regulation during different courses of HBV infection.

PointDE: Protein Docking Evaluation Using 3D Point Cloud Neural Network.

Protein-protein interactions (PPIs) play essential roles in many vital movements and the determination of protein complex structure is helpful to discover the mechanism of PPI. Protein-protein docking is being developed to model the structure of the protein. However, there is still a challenge to selecting the near-native decoys generated by protein-protein docking. Here, we propose a docking evaluation method using 3D point cloud neural network named PointDE. PointDE transforms protein structure to the point cloud. Using the state-of-the-art point cloud network architecture and a novel grouping mechanism, PointDE can capture the geometries of the point cloud and learn the interaction information from the protein interface. On public datasets, PointDE surpasses the state-of-the-art method using deep learning. To further explore the ability of our method in different types of protein structures, we developed a new dataset generated by high-quality antibody-antigen complexes. The result in this antibody-antigen dataset shows the strong performance of PointDE, which will be helpful for the understanding of PPI mechanisms.

Ultrafast Microfluidic PCR Thermocycler for Nucleic Acid Amplification.

The polymerase chain reaction (PCR) is essential in nucleic acid amplification tests and is widely used in many applications such as infectious disease detection, tumor screening, and food safety testing; however, most PCR devices have inefficient heating and cooling ramp rates for the solution, which significantly limit their application in special scenarios such as hospital emergencies, airports, and customs. Here, we propose a temperature control strategy to significantly increase the ramp rates for the solution temperature by switching microfluidic chips between multiple temperature zones and excessively increasing the temperature difference between temperature zones and the solution; accordingly, we have designed an ultrafast thermocycler. The results showed that the ramp rates of the solution temperature are a linear function of temperature differences within a range, and a larger temperature difference would result in faster ramp rates. The maximum heating and cooling ramp rates of the 25 µL solution reached 24.12 °C/s and 25.28 °C/s, respectively, and the average ramp rate was 13.33 °C/s, 6-8 times higher than that of conventional commercial PCR devices. The thermocycler achieved 9 min (1 min pre-denaturation + 45 PCR cycles) ultrafast nucleic acid amplification, shortening the time by 92% compared to the conventional 120 min nucleic acid amplification, and has the potential to be used for rapid nucleic acid detection.

Bacterial-Artificial-Chromosome-Based Genome Editing Methods and the Applications in Herpesvirus Research.

Herpesviruses are major pathogens that infect humans and animals. Manipulating the large genome is critical for exploring the function of specific genes and studying the pathogenesis of herpesviruses and developing novel anti-viral vaccines and therapeutics. Bacterial artificial chromosome (BAC) technology significantly advanced the capacity of herpesviruses researchers to manipulate the virus genomes. In the past years, advancements in BAC-based genome manipulating and screening strategies of recombinant BACs have been achieved, which has promoted the study of the herpes virus. This review summarizes the advances in BAC-based gene editing technology and selection strategies. The merits and drawbacks of BAC-based herpesvirus genome editing methods and the application of BAC-based genome manipulation in viral research are also discussed. This review provides references relevant for researchers in selecting gene editing methods in herpes virus research. Despite the achievements in the genome manipulation of the herpes viruses, the efficiency of BAC-based genome manipulation is still not satisfactory. This review also highlights the need for developing more efficient genome-manipulating methods for herpes viruses.