Recombinant Plasmid Research Articles

STAT3-specific nanocarrier for shRNA/drug dual delivery and tumor synergistic therapy

Non-small cell lung cancer (NSCLC) is a major disease with high incidence, low survival rate and prone to develop drug resistance to chemotherapy. The mechanism of secondary drug resistance in NSCLC chemotherapy is very complex, and studies have shown that the abnormal activation of STAT3 (Signal Transducer and Activator of Transcription 3) plays an important role in it. In this study, the pGPU6/GFP/Neo STAT3-shRNA recombinant plasmid was constructed with STAT3 as the precise target. By modifying hydrophilic and hydrophobic blocks onto chitosan, a multifunctional vitamin E succinate-chitosan-polyethylene glycol monomethyl ether histidine (VES-CTS-mPEG-His) micelles were synthesized. The micelles could encapsulate hydrophobic drug doxorubicin through self-assembly, and load the recombinant pGPU6/GFP/Neo STAT3-shRNA (pDNA) through positive and negative charges to form dual-loaded nanoparticles DOX/VCPH/pDNA. The co-delivery and synergistic effect of DOX and pDNA could up-regulate the expression of PTEN (Phosphatase and Tensin Homolog), down-regulate the expression of CD31, and induce apoptosis of tumor cells. The results of precision targeted therapy showed that DOX/VCPH/pDNA could significantly down-regulate the expression level of STAT3 protein, further enhancing the efficacy of chemotherapy. Through this study, precision personalized treatment of NSCLC could be effectively achieved, reversing its resistance to chemotherapy drugs, and providing new strategies for the treatment of drug-resistant NSCLC.

A fragment of the β-glucosidase gene from the rumen fungus Neocallimastix patriciarum J11 encodes a recombinant protein that exhibits activities in β-glucosidase and β-glucanase

Lignocellulose, the most abundant organic waste on Earth, is of economic value because it can be converted into biofuels like ethanol by enzymes such as β-glucosidase. This study involved cloning a β-glucosidase gene named JBG from the rumen fungus Neocallimastix patriciarum J11. When expressed recombinantly in Escherichia coli, the rJBG enzyme exhibited significant activity, hydrolyzing 4-nitrophenyl-β-d-glucopyranoside and cellobiose to release glucose. Surprisingly, the rJBG enzyme also showed hydrolytic activity against β-glucan, breaking it down into glucose, indicating that the rJBG enzyme possesses both β-glucosidase and β-glucanase activities, a characteristic rarely found in β-glucosidases. When the JBG gene was expressed in Saccharomyces cerevisiae and the transformants were inoculated into a medium containing β-glucan as the sole carbon source, the ethanol concentration in the culture medium increased from 0.17 g/L on the first day to 0.77 g/L on the third day, reaching 1.3 g/L on the fifth day, whereas no ethanol was detected in the yeast transformants containing the recombinant plasmid pYES-Sur under the same conditions. These results demonstrate that yeast transformants carrying the JBG gene can directly saccharify β-glucan and ferment it to produce ethanol. This gene, with its dual β-glucosidase and β-glucanase activities, simplifies and reduces the cost of the typical process of converting lignocellulose into bioethanol using enzymes and yeast.

Dye‐based recombinase‐aided amplification assay with enhanced sensitivity and specificity

AbstractObjectiveFluorescent recombinase‐aided amplification (RAA) assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity, compared with real‐time PCR (qPCR) assays, but they require a complex probe design. To eliminate the addition of fluorescent probes for RAA, an EvaGreen dye‐based recombinase‐aided amplification (EvaGreen‐RAA) assay using self‐avoiding molecular recognition system (SAMRS) primers was developed.MethodsThe SAMRS primers effectively avoided the production of primer dimers, thus improving the detection sensitivity, while EvaGreen dye was used to quantitatively measure the amplified products in real time. Using Staphylococcus aureus (SA) and Listeria monocytogenes (LM) as examples, EvaGreen‐RAA with SAMRS primers was developed. As a reference and comparison, a traditional fluorescence probe RAA method and a RAA with SAMRS primers (SAMRS‐RAA) for detecting SA and LM were also investigated. Serial dilutions of recombinant plasmids were used to evaluate the sensitivity of the assays. Unenriched and enriched simulated milk samples were used to evaluate the limits of detection (LOD) of these methods. Using high‐resolution melting (HRM) was used to explore the sensitivity of the dual EvaGreen‐RAA assay.ResultsThe sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS‐RAA and EvaGreen‐RAA for detecting SA and LM plasmids was 1 copies/μL. The LOD values of the EvaGreen‐RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL, respectively, and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL. EvaGreen‐RAA had linear amplification in real time in the range of 1–105 copies/μL of the plasmids of SA and LM. The sensitivity of the dual EvaGreen‐RAA assay for SA and LM was estimated to be 102 CFU/mL.ConclusionA real‐time quantitative EvaGreen‐RAA method for detecting SA and LM was developed, which eliminates the need to design complex RAA probes. This dye‐based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens, which has many potential applications.

Conversion of polyploid and alloploid Saccharomyces sensu stricto strains to leu2 mutants by genome DNA editing

A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2− mutants produced in this study will be deposited in several repository organizations.Key points• All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains• Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains• The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 geneGraphical

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses.

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/μL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

Moniezia benedeni drives the SNAP-25 expression of the enteric nerves in sheep's small intestine

BackgroundThe neuroimmune network plays a crucial role in regulating mucosal immune homeostasis within the digestive tract. Synaptosome-associated protein 25 (SNAP-25) is a presynaptic membrane-binding protein that activates ILC2s, initiating the host's anti-parasitic immune response.MethodsTo investigate the effect of Moniezia benedeni (M. benedeni) infection on the distribution of SNAP-25 in the sheep's small intestine, the recombinant plasmid pET-28a-SNAP-25 was constructed and expressed in BL21, yielding the recombinant protein. Then, the rabbit anti-sheep SNAP-25 polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of SNAP-25 in the intestines of normal and M. benedeni-infected sheep were detected by ELISA.ResultsThe results showed that the SNAP-25 recombinant protein was 29.3 KDa, the titer of the prepared immune serum reached 1:128,000. It was demonstrated that the rabbit anti-sheep SNAP-25 polyclonal antibody could bind to the natural protein of sheep SNAP-25 specifically. The expression levels of SNAP-25 in the sheep's small intestine revealed its primary presence in the muscular layer and lamina propria, particularly around nerve fibers surrounding the intestinal glands. Average expression levels in the duodenum, jejunum, and ileum were 130.32 pg/mg, 185.71 pg/mg, and 172.68 pg/mg, respectively. Under conditions of M. benedeni infection, the spatial distribution of SNAP-25-expressing nerve fibers remained consistent, but its expression level in each intestine segment was increased significantly (P < 0.05), up to 262.02 pg/mg, 276.84 pg/mg, and 326.65 pg/mg in the duodenum, jejunum, and ileum, and it was increased by 101.06%, 49.07%, and 89.16% respectively.ConclusionsThese findings suggest that M. benedeni could induce the SNAP-25 expression levels in sheep's intestinal nerves significantly. The results lay a foundation for further exploration of the molecular mechanism by which the gastrointestinal nerve-mucosal immune network perceives parasites in sheep.

Mechanism of IRF5-regulated CXCL13/CXCR5 Signaling Axis in CCI-induced Neuropathic Pain in Rats.

Neuropathic pain is chronic and affects the patient's life. Studies have shown that IRF5 and CXCL13/CXCR5 are involved in neuropathic pain; however, their interactions are unknown. In this study, a rat neuropathic pain model was constructed by inducing chronic compression injury (CCI). IRF5 recombinant lentiviral vector and CXCL13 neutralizing antibody were administered to investigate their action mechanisms in neuropathic pain. Consequently, the new strategies for disease treatment could be evolved. The CCI rats were intrathecally injected with recombinant lentivirus plasmid LV-IRF5 (overexpression), LV-SH-IRF5 (silencing), and CXCL13 neutralizing antibody. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured. The tumor necrosis factor (TNF)-alpha, interleukin (IL)-1β, and IL-6 levels were recorded via the enzyme-linked immunosorbent assay (ELISA). The spinal cord was stained using hematoxylin-eosin (HE). The binding of IRF5 to CXCL13 was analyzed by chromatin immunoprecipitation (ChIP) and dual luciferase reporter assay. The IRF5, neuronal nuclei (NeuN), CXCL13, and CXCR5 expressions were detected through quantitative real-time polymerase chain reaction and Western blot. The MWT and TWL values in the CCI group were lower than in the Sham group. The expressions of CXCL13, CXCR5, and IRF5 in CCI rats were gradually increased with the modeling time. IRF5 silencing suppressed the expression of NeuN and lumbar enlargement in CCI rats and promoted MWT and TWL. Moreover, IRF5 silencing inhibited the expressions of CXCR5 and CXCL13 genes and down-regulated the expression levels of inflammatory factors. IRF5 was directly and specifically bound with the endogenous CXCL13 promoter and thus regulated it. IRF5 overexpression exacerbated the disease phenotype of CCI-induced neuropathic pain in rats. Administration of CXCL13 neutralizing antibodies reversed the IRF5 overexpression effects. The IRF5 silencing alleviated neuropathic pain in CCI rats by downregulating the pain threshold, inflammatory cytokine levels, and CXCL13/CXCR5 signaling. IRF5 overexpression exacerbated the disease parameters of CCI-induced neuropathic pain in rats; however, they were reversed by neutralizing antibodies against CXCL13.

An ultrasensitive strip sensor for rapid detection of African swine fever virus

African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) infecting pigs, which has caused huge economic losses in countries around the world. Currently, there is no effective vaccine, and the prevention and control of ASF is mainly through rapid detection, so it is particularly important to carry identify and develop rapid detection methods for ASFV. In this study, recombinant plasmid PET-28a(+)-p30 was constructed, and the recombinant protein was obtained by inducing expression and Ni+ resin affinity column purification. Mice were immunized with recombinant p30 protein, and after three immunizations, ten strains of hybridoma cells that stably secreted monoclonal antibodies (mAbs) against p30 protein were obtained by cell fusion and subcloning. A colloidal gold immunochromatography assay (GICA) based on double antibody sandwich technology was established to screen the paired antibodies, and the trapping and detecting antibodies were mAb-11F11 and mAb-7A8, respectively, with a detection limit of 1 ng/mL, which laid an important material foundation for the early detection of ASF in the future.

Roles of transcriptional factor PsrA in the regulation of quorum sensing in Pseudomonas aeruginosa PAO1.

The transcription factor PsrA regulates fatty acid metabolism, the type III secretion system, and quinolone signaling quorum sensing system in Pseudomonas aeruginosa. To explore additional roles of PsrA in P. aeruginosa, this study engineered a P. aeruginosa PAO1 strain to carry a recombinant plasmid with the psrA gene (pMMBpsrA) and examined the impact of elevated psrA expression to the bacterium. Transcriptomic analysis revealed that PsrA significantly downregulated genes encoding the master quorum-sensing regulators, RhlR and LasR, and influenced many quorum-sensing-associated genes. The role of PsrA in quorum sensing was further corroborated by testing autoinducer synthesis in PAO1 [pMMBpsrA] using two reporter bacteria strains Chromobacterium violaceum CV026 and Escherichia coli [pSB1075], which respond to short- and long-chain acyl homoserine lactones, respectively. Phenotypic comparisons of isogenic ΔpsrA, ΔlasR, and ΔpsrAΔlasR mutants revealed that the reduced elastase, caseinase, and swarming activity in PAO1 [pMMBpsrA] were likely mediated through LasR. Additionally, electrophoretic mobility shift assays demonstrated that recombinant PsrA could bind to the lasR promoter at a 5'-AAACGTTTGCTT-3' sequence, which displays moderate similarity to the previously reported consensus PsrA binding motif. Furthermore, the PsrA effector molecule oleic acid inhibited PsrA binding to the lasR promoter and restored several quorum sensing-related phenotypes to wild-type levels. These findings suggest that PsrA regulates certain quorum-sensing phenotypes by negatively regulating lasR expression, with oleic acid acting as a crucial signaling molecule.

Construction of the RNAi plasmids to suppress the expression of chitin synthase-encoding genes (&lt;i&gt;chs&lt;/i&gt;) in fungus &lt;i&gt;Mucor lusitanicus&lt;/i&gt;

Mucormycosis is an emerging fungal infection caused by many species belonging to the order Mucorales. The lack of effective therapies to treat this disease is due to our limited understanding of its pathogenesis. Chitin is one of the fungal-specific components of the cell wall. In fungi, chitin synthase and chitin deacetylase are the key enzymes involved in chitin biogenesis. The RNAi mechanisms have been well-studied in the fungus Mucor-lusitanicus, a model organism for mucormycosis studies. The RNAi technique has been previously applied to study gene function. In this study, two chitin synthase-encoding genes (chs1 and chs2) were identified using transcriptomic data. Approximately 1 kb of DNA fragments of the target genes were amplified and cloned into the pMAT1812 vector to generate the corresponding RNAi plasmids. Initial screening by colony PCR identified 3/10 and 1/10 potential colonies that might contain the RNAi plasmids. One plasmid for each clone was selected and named pAT63 and pAT64, corresponding to the chs1 and chs2 genes, respectively. The validation of these recombinant RNAi plasmids using different restriction enzymes (NotI - PvuII and SalI - PstI for plasmids pAT63 and pAT64, respectively) showed the expected results. The DNA sequencing of the plasmids pAT63 and pAT64 using specific primers validated the results of the screening process. These RNAi plasmids can be used to suppress the expression of the target genes. The silencing mechanism of the target genes will be triggered by the expression of the RNAi plasmids. This approach allows us to study the function of the chitin synthase-encoding genes in this fungus.

Capture of armA by a novel ISCR element, ISCR28

ISCR28 is a fully functional and active member of the IS91-like family of insertion sequences. ISCR28 is 1,708-bp long and contains a 1,293-bp long putative open reading frame that codes a transposase. Sixty ISCR28-containing sequences from GenBank generated 27 non-repeat genetic contexts, all of which represented naturally occurring biological events that had occurred in a wide range of gram-negative organisms. Insertion of ISCR28 into target DNA preferred the presence of a 5′-GXXT-3′ sequence at its terIS (replication terminator) end. Loss of the first 4 bp of its oriIS (origin of replication) likely caused ISCR28 to be trapped in ISApl1-based transposons or similar structures. Loss of terIS and fusion with a mobile element upstream likely promoted co-transfer of ISCR28 and the downstream resistance genes. ArmA and its downstream intact ISCR28 can be excised from recombinant pKD46 plasmids forming circular intermediates, further elucidating its activity as a transposase.

Preparation and preliminary application of the polyclonal antibody against Toxoplasma gondii dense granule protein 24

To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications. The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA). SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells. The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

Fitness cost of tet(A) type I variant-mediated tigecycline resistance in Klebsiella pneumoniae

ObjectiveThe aim of the present study is to explore the impact of the tet(A) type I variant (tetA-v1) on its fitness effect in Klebsiella pneumoniae. MethodsClinical K. pneumoniae strains were utilized as parental strains to generate strains carrying only the plasmid vector (pBBR1MCS-5) or the tetA-v1 recombinant plasmid (ptetA-v1). Antimicrobial susceptibility testing was conducted to estimate the contribution of tetA-v1 to drug resistance. Plasmid stability was evaluated by serial passage over 10 consecutive days in the absence of tigecycline. Biological fitness was examined through growth curve analysis, in vitro competition assays and a neutropenic mouse thigh infection model. ResultsA 2–4-fold increase in tigecycline MIC was observed following the acquisition of tetA-v1. Without tigecycline treatment, the stability of ptetA-v1 plasmids has been decreasing since day 1. The ptetA-v1 plasmid in Kp89, Kp91, and Kp93 exhibited a decrease of about 20% compared to the pBBR1MCS-5 plasmid. The acquisition of the tetA-v1 gene could inhibit the growth ability of K. pneumoniae strains both in vitro and in vivo. tetA-v1 gene imposed a fitness cost in K. pneumoniae, particularly in the CRKP strain Kp51, with a W value of approximately 0.56. ConclusionThe presence of tetA-v1 is associated with a significant fitness cost in K. pneumoniae in the absence of tigecycline, both in vitro and in vivo.

MiR-34a-5p suppresses cutaneous squamous cell carcinoma progression by targeting SIRT6

Cutaneous squamous cell carcinoma (cSCC) is a malignant tumor originating from epidermal or appendageal keratinocytes, with a rising incidence in recent years. Understanding the molecular mechanism driving its development is crucial. This study aims to investigate whether miR-34a-5p is involved in the pathogenesis of cSCC by targeting Sirtuin 6 (SIRT6).The expression levels of miR-34a-5p and SIRT6 were determined in 15 cSCC tissue specimens, 15 normal tissue specimens and cultured cells via real-time polymerase chain reaction (RT-qPCR). Pearson’s correlation analysis was conducted to evaluate the relationship between miR-34a-5p and SIRT6 expression levels in cSCC tissues. A431 and SCL-1 cells were transfected with miR-34a-5p mimic, negative control or miR-34a-5p mimic together with recombinant plasmids containing SIRT6 gene. Cell counting kit-8, clone formation assay, wound healing assay, and flow cytometry were employed to assess the effects of these transfections on proliferation, migration, and apoptosis, respectively. The interaction between miR-34a-5p and SIRT6 was characterized using a dual-luciferase reporter assay.MiR-34a-5p expression was down-regulated in cSCC tissues significantly, while the SIRT6 expression was the opposite. A negative correlation was observed between the expression of miR-34a-5p and SIRT6 in cSCC tissues. Furthermore, overexpression of miR-34a-5p led to a significant reduction in the proliferation and migration abilities of A431 and SCL-1 cells, accompanied by an increase in apoptosis levels and a decrease in SIRT6 expression levels. MiR-34a-5p was identified as a direct target of SIRT6. Importantly, overexpression of SIRT6 effectively counteracted the inhibitory effect mediated by miR-34a-5p in cSCC cells.Our findings suggest that miR-34a-5p functions as a tumor suppressor in cSCC cells by targeting SIRT6.

Tumor Pre-Targeting System Using Streptavidin-Expressing Bacteria.

A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe. We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-arabinose 4days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed. Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin. We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.

Knockdown the moyamoya disease susceptibility gene, RNF213, upregulates the expression of basic fibroblast growth factor and matrix metalloproteinase-9 in bone marrow derived mesenchymal stem cells.

Moyamoya disease (MMD) is a chronic, progressive cerebrovascular occlusive disease. Ring finger protein 213 (RNF213) is a susceptibility gene of MMD. Previous studies have shown that the expression levels of angiogenic factors increase in MMD patients, but the relationship between the susceptibility gene RNF213 and these angiogenic mediators is still unclear. The aim of the present study was to investigate the pathogenesis of MMD by examining the effect of RNF213 gene knockdown on the expression of matrix metalloproteinase-9 (MMP-9) and basic fibroblast growth factor (bFGF) in rat bone marrow-derived mesenchymal stem cells (rBMSCs). Firstly, 40 patients with MMD and 40 age-matched normal individuals (as the control group) were enrolled in the present study to detect the levels of MMP-9 and bFGF in serum by ELISA. Secondly, Sprague-Dawley male rat BMSCs were isolated and cultured using the whole bone marrow adhesion method, and subsequent phenotypic analysis was performed by flow cytometry. Alizarin red and oil red O staining methods were used to identify osteogenic and adipogenic differentiation, respectively. Finally, third generation rBMSCs were transfected with lentivirus recombinant plasmid to knockout expression of the RNF213 gene. After successful transfection was confirmed by reverse transcription-quantitative PCR and fluorescence imaging, the expression levels of bFGF and MMP-9 mRNA in rBMSCs and the levels of bFGF and MMP-9 protein in the supernatant of the culture medium were detected on the 7th and 14th days after transfection. There was no significant difference in the relative expression level of bFGF among the three groups on the 7th day. For the relative expression level of MMP-9, there were significant differences on the 7th day and 14th day. In addition, there was no statistically significant difference in the expression of bFGF in the supernatant of the RNF213 shRNA group culture medium, while there was a significant difference in the expression level of MMP-9. The knockdown of the RNF213 gene affects the expression of bFGF and MMP-9. However, further studies are needed to determine how they participate in the pathogenesis of MMD. The findings of the present study provide a theoretical basis for clarifying the pathogenesis and clinical treatment of MMD.

Long non-coding RNA UCA1 Knockdown Assisted by CRISPR/Cas9 in Female Cancer Cell Lines Increases Mir-143 Tumor-Suppressor.

The lncRNAs has been linked to several malignancies, including breast cancer. Our objective was to investigate the impact of urothelial carcinoma associated 1 (UCA1) on cellular growth and death by a CRISPR/Cas9 knockdown technique. In 2020, the CHOPCHOP program was utilized to design two sgRNAs targeting the UCA gene. sgRNA1 and sgRNA2 were inserted into two different CRISPR plasmids to produce two recombinant plasmids. These recombinant plasmids were simultaneously transfected into MCF-7 and MDA-MB 231 carcinoma of the breast cells. Proliferation and apoptosis were compared using the MTT test, CCK-8 assay, and flow cytometry evaluation. RNA-hybrid software, quantitative reverse transcription PCR, and luciferase assays were utilized to confirm the relationship between UCA1 and miR-143. Proliferated cells were less active in MTT and CCK-8 tests and fellow cytometry analysis. The PX459-sgRNA1,2 group had elevated levels of the cancer biomarker Caspase-3 gene expression (P<0.001). When WT-UCA1 and miR-143 were co-transfected, the luciferase activity was drastically decreased. One very effective method of regulating cellular proliferation in vitro is the deletion of UCA1, which CRISPR/Cas9 accomplishes.

NSP4 promotes replication of porcine reproductive and respiratory syndrome virus-2

Porcine reproductive and respiratory syndrome (PRRS) is one of the most detrimental contagious swine ailments worldwide. Currently, no effective drugs are available for its treatment. Targeting the structural and non-structural proteins (NSP) of the type 2 PRRS virus (PRRSV-2) with small interfering RNA (siRNA) is an effective approach to inhibit PRRSV replication. NSP4, which is highly conserved and possesses 3 C-like serine protease activity (3CLSP), can cleave PRRSV self-proteins, thereby contributing to viral replication. To investigate the mechanism by which NSP4 regulates PRRSV-2 replication and screen for effective siRNA inhibitors of PRRSV-2 replication, the recombinant plasmid pEGFP-C1-NSP4 was constructed, and a control siRNA pair and two siRNA pairs targeting the PRRSV-2 NSP4 gene (shRNA-ctr, shRNA-150, and shRNA-536) were synthesized and cloned into the pSilencer4.1-CMV vector. After 24 h of incubation, Marc-145 cells were transfected with recombinant plasmids, and subsequently infected with different PRRSV-2 (XH-GD, ZQ-GD, GDr180, and JXA1-R). Subsequently, the effects of NSP4 overexpression, shRNA on PRRSV-2 replication were evaluated by assessing cytopathic effects (CPE), TCID50, quantitative real-time PCR (qPCR), immunofluorescence assays (IFA), and Western blotting. The data from these CPE, TCID50, qPCR, and IFA experiments revealed that NSP4 overexpression significantly enhanced PRRSV-2 replication and shRNA targeting NSP4 can inhibit PRRSV-2 replication in Marc-145 cells, indicating that shRNA could serve as candidate molecules for fundamental research on PRRSV-2.

DNA assembly by multi-fragment digestion/ligation and homologous recombination

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.

CTNNAL1 promotes the structural integrity of bronchial epithelial cells through the RhoA/ROCK1 pathway.

Adhesion molecules play critical roles in maintaining the structural integrity of the airway epithelium in airways under stress. Previously, we reported that catenin alpha-like 1 (CTNNAL1) is downregulated in an asthma animal model and upregulated at the edge of human bronchial epithelial cells (HBECs) after ozone stress. In this work, we explore the potential role of CTNNAL1 in the structural adhesion of HBECs and its possible mechanism. We construct a CTNNAL1 ‒/‒ mouse model with CTNNAL1-RNAi recombinant adeno-associated virus (AAV) in the lung and a CTNNAL1-silencing cell line stably transfected with CTNNAL1-siRNA recombinant plasmids. Hematoxylin and eosin (HE) staining reveals that CTNNAL1 ‒/‒ mice have denuded epithelial cells and structural damage to the airway. Silencing of CTNNAL1 in HBECs inhibits cell proliferation and weakens extracellular matrix adhesion and intercellular adhesion, possibly through the action of the cytoskeleton. We also find that the expressions of the structural adhesion-related molecules E-cadherin, integrin β1, and integrin β4 are significantly decreased in ozone-treated cells than in vector control cells. In addition, our results show that the expression levels of RhoA/ROCK1 are decreased after CTNNAL1 silencing. Treatment with Y27632, a ROCK inhibitor, abolished the expressions of adhesion molecules induced by ozone in CTNNAL1-overexpressing HBECs. Overall, the findings of the present study suggest that CTNNAL1 plays a critical role in maintaining the structural integrity of the airway epithelium under ozone challenge, and is associated with epithelial cytoskeleton dynamics and the expressions of adhesion-related molecules via the RhoA/ROCK1 pathway.