Electrochemical detection of MMP-2 with enhanced sensitivity: Utilizing T7 RNA polymerase and CRISPR-Cas12a for neuronal studies.

This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.

Agrochemical control of gene expression using evolved split RNA polymerase. II.

Agrochemical inducible gene expression system provides cost-effective and orthogonal control of energy and information flow in bacterial cells. However, the previous version of Mandipropamid inducible gene expression system (Mandi-T7) became constitutively active at room temperature. We moved the split site of the eRNAP from position LYS179 to position ILE109. This new eRNAP showed proximity dependence at 23 °C, but not at 37 °C. We built Mandi-T7-v2 system based on the new eRNAP and it worked in both Escherichia coli and Agrobacterium tumefaciens. We also induced GFP expression in Agrobacterium cells in a semi-in vivo system. The modified eRNAP when combined with the leucine zipper-based dimerization system, behaved as a cold inducible gene expression system. Our new system provides a means to broaden the application of agrochemicals for both research and agricultural application. Portions of this text were previously published as part of a preprint (https://www.biorxiv.org/content/10.1101/2024.04.02.587689v1).

Clinical features and prognosis of patients with myeloid neoplasms harboring t(7;11)(p15;p15) translocation: a single-center retrospective study.

BACKGROUND: For myeloid neoplasms with t(7;11)(p15;p15) translocation, the prognosis is quite dismal. Because these tumors are rare, most occurrences are reported as single cases. Clinical results and optimal treatment approaches remain elusive. This study endeavors to elucidate the clinical implications and prognosis of this cytogenetic aberration. METHODS: This study retrospectively analyzed 23 cases of myeloid neoplasm with t(7;11)(p15;p15). Clinicopathological characteristics, genetic alterations, and outcomes were evaluated, and the Kaplan-Meier method was employed to construct survival curves. RESULTS: Of these, nine cases were newly diagnosed acute myeloid leukemia (ND AML), seven presented with relapsed refractory AML (R/R AML), four had myelodysplastic syndrome (MDS), two had secondary AML, and one exhibited a mixed germinoma associated with MDS. Patients with t(7;11)(p15;p15) in AML were primarily younger females who preferred subtype M2. Interestingly, these patients had decreased hemoglobin and red blood cell counts, along with markedly elevated levels of lactic dehydrogenase and interleukin-6, and exhibited the expression of CD117. R/R AML patients exhibited a higher likelihood of additional chromosome abnormalities (ACAs) besides t(7;11). WT1 and FLT3-ITD were the most commonly found mutated genes, and 10 of those instances showed evidence of the NUP98::HOXA9 fusion gene. The composite complete remission rate was 66.7% (12/18), while the cumulative graft survival rate was 100% (4/4). However, the survival outcomes were dismal. Interestingly, the median overall survival for R/R AML patients was 4.0 months (95% CI: 1.7-6.4). Additionally, the type of AML diagnosis or the presence of ACAs or molecular prognostic stratification did not significantly influence clinical outcomes (p = 0.066, p = 0.585, p = 0.570, respectively). CONCLUSION: Myeloid leukemia with t(7;11) exhibits unique clinical features, cytogenetic properties, and molecular genetic characteristics. These survival outcomes were dismal. R/R AML patients have a limited lifespan. For myeloid patients with t(7;11), targeted therapy or transplantation may be an effective course of treatment.

Targeted Delivery of Nanoparticle-Conveyed Neutrophils to the Glioblastoma Site for Efficient Therapy.

Glioblastoma is a common brain tumor that poses considerable challenges in drug delivery. In this study, we investigated the potential of cell-based nanoparticles for targeted drug delivery to the glioblastoma sites. The anticancer drug of temozolomide (TMZ)-loaded T7-cholesterol nanoparticle micelles efficiently delivered nanoparticles to neutrophils and, subsequently, to the tumors. T7 is a cell-penetrating peptide that enhances the delivery of T7/TMZ to the target cells. T7 also serves as a transferrin target peptide, enabling targeted delivery to tumors. T7-conjugated cholesterol can self-assemble into micelles in aqueous solution and attach to the membrane of neutrophils. We confirmed that T7/TMZ nanoparticle micelles were efficiently located inside the neutrophils. Thereafter, T7/TMZ-conveyed neutrophils were administered to a glioblastoma mouse model, enabling neutrophils to penetrate the blood-brain barrier and deliver drugs directly to the tumor site. We evaluated the drug delivery efficiency and therapeutic effects of intravenous injection of T7/TMZ-conveyed neutrophils to a glioblastoma mouse model. These results demonstrate the promising role of neutrophil-based nanoparticle delivery systems in the targeted therapy of glioblastoma.

Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose.

In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition.

Transferring an Adult-Plant Stripe-Rust Resistance Gene Yr7VS from Chromosome 7V of Dasypyrum villosum (L.) to Bread Wheat.

Stripe rust (Puccinia striiformis West. f.sp. tritici, Pst) is a destructive disease that seriously threatens wheat production globally. Exploring novel resistance genes for use in wheat breeding is an urgent need, as continuous Pst evolution frequently leads to a breakdown of host resistance. Here, we identified a set of wheat-Dasypyrum villosum 01I139 (V#6) disomic introgression lines for the purpose of determining their responses to a mixture of Pst isolates CYR32, CYR33 and CYR34 at both seedling and adult-plant stages. The results showed that all introgression lines exhibited high susceptibility at the seedling stage, with infection-type (IT) scores in the range of 6-8, whereas, for chromosomes 5V#6 and 7V#6, disomic addition lines NAU5V#6-1 and NAU7V#6-1 displayed high resistance at the adult-plant stage, indicating that adult-plant resistance (APR) genes were located on them. Further, in order to transfer the stripe-rust resistance on chromosome 7V#6, four new wheat-D. villosum introgression lines were identified, by the use of molecular cytogenetic approaches, from the self-pollinated seeds of 7D and 7V#6, in double monosomic line NAU7V#6-2. Among them, NAU7V#6-3 and NAU7V#6-4 were t7V#6L and t7V#6S monosomic addition lines, and NAU7V#6-5 and NAU7V#6-6 were homozygous T7DS·7V#6L and T7DL·7V#6S whole-arm translocation lines. Stripe-rust tests and genetic analyses of chromosome 7V#6 introgression lines revealed a dominant APR gene designated as Yr7VS on the chromosome arm 7V#6S. Comparison with the homozygous T7DL·7V#6S translocation line and the recurrent parent NAU0686 showed no significant differences in yield-related traits. Thus, T7DL·7V#6S whole-arm translocation with the APR gene Yr7VS provided a valuable germplasm for breeding for resistance.

Introgression of an adult-plant powdery mildew resistance gene Pm4VL from Dasypyrum villosum chromosome 4V into bread wheat.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) seriously threatens wheat production worldwide. It is imperative to identify novel resistance genes from wheat and its wild relatives to control this disease by host resistance. Dasypyrum villosum (2n = 2x = 14, VV) is a relative of wheat and harbors novel genes for resistance against multi-fungal diseases. In the present study, we developed a complete set of new wheat-D. villosum disomic introgression lines through genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and molecular markers analysis, including four disomic substitution lines (2n=42) containing respectively chromosomes 1V#6, 2V#6, 3V#6, and 6V#6, and four disomic addition lines (2n=44) containing respectively chromosomes 4V#6, 5V#6, 6V#6 and 7V#6. These lines were subsequently evaluated for their responses to a mixture Bgt isolates at both seedling and adult-plant stages. Results showed that introgression lines containing chromosomes 3V#6, 5V#6, and 6V#6 exhibited resistance at both seedling and adult-plant stages, whereas the chromosome 4V#6 disomic addition line NAU4V#6-1 exhibited a high level of adult plant resistance to powdery mildew. Moreover, two translocation lines were further developed from the progenies of NAU4V#6-1 and the Ph1b mutation line NAU0686-ph1b. They were T4DL·4V#6S whole-arm translocation line NAU4V#6-2 and T7DL·7DS-4V#6L small-fragment translocation line NAU4V#6-3. Powdery mildew tests of the two lines confirmed the presence of an adult-plant powdery mildew resistance gene, Pm4VL, located on the terminal segment of chromosome arm 4V#6L (FL 0.6-1.00). In comparison with the recurrent parent NAU0686 plants, the T7DL·7DS-4V#6L translocation line NAU4V#6-3 showed no obvious negative effect on yield-related traits, providing a new germplasm in breeding for resistance.

Advances in evolved T7 RNA polymerases for expanding the frontiers of enzymatic nucleic acid synthesis.

In vitro RNA synthesis technologies are crucial in developing therapeutic RNA drugs, such as mRNA vaccines and RNA interference (RNAi) therapies. Enzymatic RNA synthesis, recognized for its sustainability and efficiency, enables the production of extensive RNA sequences under mild conditions. Among the enzymes utilized, T7 RNA polymerase is distinguished by its exceptional catalytic efficiency, enabling the precise and rapid transcription of RNA from DNA templates by recognizing the specific T7 promoter sequence. With the advancement in clinical applications of RNA-based drugs, there is an increasing demand for the synthesis of chemically modified RNAs that are stable and resistant to nuclease degradation. To this end, researchers have applied directed evolution to broaden the enzyme's substrate scope, enhancing its compatibility with non-canonical substrates and reducing the formation of by-products. This review summarizes the progress in engineering T7 RNA polymerase for these purposes and explores prospective developments in the field.

Application of CRISPR/Cas12a in miRNA-155 detection: A novel homogeneous electrochemiluminescence biosensor.

BACKGROUND: MicroRNAs (miRNAs) are important non-coding RNA entities that affect gene expression and function by binding to target mRNAs, leading to degradation of the mRNAs or inhibiting their translation. MiRNAs are widely involved in a variety of biological processes, such as cell differentiation, development, metabolism, and apoptosis. In addition, miRNAs are associated with many diseases, including cancer. However, conventional detection techniques often suffer from shortcomings such as low sensitivity, so we need to develop a rapid and efficient detection strategy for accurate detection of miRNAs. RESULTS: We have developed an innovative homogeneous electrochemiluminescence (ECL) biosensor. This biosensor employs CRISPR/Cas12a gene editing technology for accurate and efficient detection of microRNA (miRNA). Compared to conventional technologies, this biosensor employs a unique homogeneous detection format that eliminates laborious probe fixation steps and greatly simplifies the detection process. By using two amplification techniques - isothermal amplification and T7 RNA polymerase amplification - the biosensor improves the sensitivity and specificity of the assay, providing excellent detection performance in the assay. This makes it possible to evaluate miRNA directly from a variety of biological samples such as cell lysates and diluted human serum. Experimental results convincingly demonstrate the extraordinary performance of this biosensor, including its extremely low detection limit of 1.27 aM, high sensitivity, reproducibility and stability. SIGNIFICANCE: The application of our constructed sensor in distinguishing between cancerous and non-cancerous cell lines highlights its potential for early cancer detection and monitoring. This innovative approach represents a major advancement in the field of miRNA detection, providing a user-friendly, cost-effective, and sensitive solution with broad implications for clinical diagnosis and patient care, especially in point-of-care settings.

[Corrigendum] T7 peptide inhibits angiogenesis via downregulation of angiopoietin­2 and autophagy.

Following the publication of this article, an interested reader drew to the authors' attention that two pairs of protein bands featured in the western blots in Fig. 3A and 5D on p. 679 and 681 respectively appeared to be strikingly similar. After having re­examined their original data, the authors realized that Fig. 5D had been assembled incorrectly. The revised version of Fig. 5, now including the correct data for Fig. 5D, is shown on the next page. Note that the errors made in terms of assembling the data in Fig. 5 did not greatly affect either the results or the conclusions reported in this paper, and all the authors agree to the publication of this corrigendum. The authors regret that these errors went unnoticed prior to the publication of their article, are grateful to the Editor of Oncology Reports for allowing them this opportunity to publish this corrigendum. They also apologize to the readership for any inconvenience caused. [Oncology Reports  33: 675­684, 2015; DOI: 10.3892/or.2014.3653].

Tailored expression of ICCM cutinase in engineered Escherichia coli for efficient polyethylene terephthalate hydrolysis.

Enzymatic depolymerization of PET waste emerges as a crucial and sustainable solution for combating environmental pollution. Over the past decade, PET hydrolytic enzymes, such as PETase from Ideonella sakaiensis (IsPETases), leaf compost cutinases (LCC), and lipases, have been subjected to rational mutation to enhance their enzymatic properties. ICCM, one of the best LCC mutants, was selected for overexpression in Escherichia coli BL21(DE3) for in vitro PET degradation. However, overexpressing ICCM presents challenges due to its low productivity. A new stress-inducible T7RNA polymerase-regulating E. coli strain, ASIAhsp, which significantly enhances ICCM production by 72.8 % and achieves higher enzyme solubility than other strains. The optimal cultural condition at 30 °C with high agitation, corresponding to high dissolved oxygen levels, has brought the maximum productivity of ICCM and high PET-hydrolytic activity. The most effective PET biodegradation using crude or pure ICCM occurred at pH 10 and 60 °C. Moreover, ICCM exhibited remarkable thermostability, retaining 60 % activity after a 5-day reaction at 60 °C. Notably, crude ICCM eliminates the need for purification and efficiently degrades PET films.

Split T7 switch-mediated cell-free protein synthesis system for detecting target nucleic acids.

Cell-free protein synthesis (CFPS) reactions can be used to detect nucleic acids. However, most CFPS systems rely on a toehold switch and exhibit the following critical limitations: (i) off-target signals due to leaky translation in the absence of target nucleic acids, (ii) a suboptimal detection limit of approximately 30 nM without pre-amplification, and (iii) labor-intensive screening processes due to sequence constraints for the target nucleic acids. To overcome these shortcomings, we developed a new split T7 switch-mediated CFPS system in which the split T7 promoter was applied to a three-way junction structure to selectively initiate transcription-translation only in the presence of target nucleic acids. Both fluorescence and colorimetric detection systems were constructed by employing different reporter proteins. Notably, we introduced the self-complementation of split fluorescent proteins to streamline preparation of the proposed system, enabling versatile applications. Operation of this one-pot approach under isothermal conditions enabled the detection of target nucleic acids at concentrations as low as 10 pM, representing more than a thousand times improvement over previous toehold switch-based approaches. Furthermore, the proposed system demonstrated high specificity in detecting target nucleic acids and compatibility with various reporter proteins encoded in the expression region. By eliminating issues associated with the previous toehold switch system, our split T7 switch-mediated CFPS system could become a core platform for detecting various target nucleic acids.

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription.

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

NASBA Coupled to Paper Microfluidics for RNA Detection.

The analysis of RNA sequences is crucial to obtain invaluable insights into disease prognosis. Reliable and rapid diagnostic solutions at the site of sample collection contribute toward optimal delivery of medical treatment. For this reason, the development of more sensitive and portable RNA detection techniques are expected to advance current point-of-care (POC) diagnostic capabilities. Advancements of POC diagnostic technologies will also contribute to counter the spread of emerging viruses. Reverse transcriptase polymerase chain reaction (RT-PCR) is the most commonly used technique to identify etiological organisms of infections. However, the need for thermocycler and fluorescent measurement renders RT-PCR less suitable for POC applications. Here, we provide a step-by-step protocol of Nucleic Acid Sequence-Based Amplification (NASBA), a robust isothermal RNA amplification technique, coupled with a portable paper microfluidics detection format.

Strategies to Reduce Promoter-Independent Transcription of DNA Nanostructures and Strand Displacement Complexes.

Bacteriophage RNA polymerases, in particular T7 RNA polymerase (RNAP), are well-characterized and popular enzymes for many RNA applications in biotechnology both in vitro and in cellular settings. These monomeric polymerases are relatively inexpensive and have high transcription rates and processivity to quickly produce large quantities of RNA. T7 RNAP also has high promoter-specificity on double-stranded DNA (dsDNA) such that it only initiates transcription downstream of its 17-base promoter site on dsDNA templates. However, there are many promoter-independent T7 RNAP transcription reactions involving transcription initiation in regions of single-stranded DNA (ssDNA) that have been reported and characterized. These promoter-independent transcription reactions are important to consider when using T7 RNAP transcriptional systems for DNA nanotechnology and DNA computing applications, in which ssDNA domains often stabilize, organize, and functionalize DNA nanostructures and facilitate strand displacement reactions. Here we review the existing literature on promoter-independent transcription by bacteriophage RNA polymerases with a specific focus on T7 RNAP, and provide examples of how promoter-independent reactions can disrupt the functionality of DNA strand displacement circuit components and alter the stability and functionality of DNA-based materials. We then highlight design strategies for DNA nanotechnology applications that can mitigate the effects of promoter-independent T7 RNAP transcription. The design strategies we present should have an immediate impact by increasing the rate of success of using T7 RNAP for applications in DNA nanotechnology and DNA computing.

Analyzing the impact of T7L variants overexpression on the metabolic profile of Escherichia coli.

INTRODUCTION: Exploring metabolic changes within host E. coli through an untargeted metabolomic study of T7L variants overexpression to optimize engineered endolysins for clinical/therapeutic use. AIM AND OBJECTIVE: This study aims to assess the impact of overexpressing T7L variants on the metabolic profiles of E. coli. The two variants considered include T7L-H37A, which has enhanced lytic activity compared to its wild-type protein, and T7L-H48K, a dead mutant with no significant activity. METHODS: 1H NMR-based metabolomics was employed to compare the metabolic profiles of E. coli cells overexpressing T7L wild-type protein and its variants. RESULTS: Overexpression of the T7L wild-type (T7L-WT) protein and its variants (T7L-H48K and T7L-H37A) was compared to RNAP overexpression in E. coli cells using 1H NMR-based metabolomics, analyzing a total of 75 annotated metabolites, including organic acids, amino acids, sugars, and nucleic acids. The results showed distinct clustering patterns for the two T7L variant groups compared with the WT, in which the dead mutant (H48K) group showed clustering close to that of RNAP. Pathway impact analysis revealed different effects of T7L variants on E. coli metabolic profiles, with T7L-H48K showing minimal alterations in energy and amino acid pathways linked to osmotic stress compared to noticeable alterations in these pathways for both T7L-H37A and T7L-WT. CONCLUSIONS: This study uncovered distinct metabolic fingerprints when comparing the overexpression of active and inactive mutants of T7L lytic enzymes in E. coli cells. These findings could contribute to the optimization and enhancement of suitable endolysins as potential alternatives to antibiotics.

Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western.

Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires <10 µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.

T7 peptide-mediated co-delivery platform overcoming multidrug-resistant breast cancer: In vitro and in vivo evaluation.

P-glycoprotein (P-gp) overexpressed mutidrug resistance (MDR) is currently a key factor limiting the effectiveness of breast cancer chemotherapy. Systemic administration based on P-gp-associated mechanism leads to severe toxic side effects. Here, we designed a T7 peptide-modified mixed liposome (T7-MLP@DTX/SchB) that, by active targeting co-delivering chemotherapeutic agents and P-gp inhibitors, harnessed synergistic effects to improve the treatment of MDR breast cancer. This study established drug-resistant cell models and animal models. Subsequently, comprehensive evaluations involving cell uptake, cell apoptosis, cellular toxicity assays, in vivo tumor-targeting capability, and anti-tumor activity assays were conducted to assess the drug resistance reversal effects of T7-MLP@DTX/SchB. Additionally, a systematic assessment of the biosafety profile of T7-MLP@DTX/SchB was executed, including blood profiles, biochemical markers, and histopathological examination. It was found that this co-delivery strategy successfully exerted the synergistic effects, since there was a significant tumor growth inhibitory effect on multidrug-resistant breast cancer. Targeted modification with T7 peptide enhanced the therapeutic efficacy remarkably, while vastly ameliorating the biocompatibility compared to free drugs. The intriguing results supported the promising potential use of T7-MLP@DTX/SchB in overcoming MDR breast cancer treatment.

Assessing the synergistic potential of bacteriophage endolysins and antimicrobial peptides for eradicating bacterial biofilms.

Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.

Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism.

Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10 -2 to 10 -5. The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase 1. 3 gene, which may be involved in the MMEJ event, and the frequency of MMEJ at 4. 3 is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ 1. 3 increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1. 3 can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.