Journal, genetic engineering & biotechnology最新文献

Organoids derived from tissues such as the endometrium and liver represent crucial models for investigating cellular and molecular processes in a three-dimensional framework. These systems enable detailed studies of organ development and disease pathology through various methods, including immunostaining, for precise visualization and localization of specific proteins. This simplified method outlines a systematic protocol for isolating, culturing, and immunostaining individual organoids using transwell inserts. This streamlined method optimizes procedures for fixation, permeabilization, and single or double antibody staining, ensuring robust and reproducible outcomes. Our method allows selecting a single organoid in a temporal manner without disturbing the 3D structure of Matrigel domes. By standardizing these critical steps, researchers can efficiently utilize organoid cultures to advance our understanding of complex biological mechanisms and explore therapeutic interventions targeting diseases affecting the endometrium and liver.

Cancer burden has become a global concern, specifically with hormone-sensitive cancers (HSC). Among the various factors influencing the progress and treatment of cancers, the human microbiota plays a vital role. The current analysis explores the role of microbial metabolites in various regulatory pathways associated with cancer biomarkers and finds the common microbial metabolites associated with HSCs. AR, ESR1, and TP53 in breast cancer; ERBB2, MAPK21, and TP53 in ovarian cancer; AKT1, EGFR, and ERBB2 in endometrial cancer and AR, MYC, and RB1 in prostate cancer were identified as important genes in HSC development. The associated metabolites of these genes were contributing to various regulatory pathways. The metabolites hydrogen peroxide, adenosine triphosphate, adenosine diphosphate, guanosine diphosphate, and guanosine triphosphate produced by microbes within the humans were found to be involved in nucleotide and amino acid metabolism pathways. The purine and pyrimidine metabolism pathways, glyoxylate, and dicarboxylate metabolism pathway, arginine and proline pathway, and glycine, serine, and threonine pathways were found common and significantly enriched between human and microbial metabolites showing close association between the microbial metabolites and the HSCs. The microbes belonging to Bacteroidetes, Proteobacteria, and Firmicutes phylum were found to be significantly associated with HSCs. The expression analysis and the similar pathways enriched between the correlated genes and hub genes validated the significance of the metabolites which can be used as potential biomarkers. Thus, the metabolic biomarkers can help in early diagnosis, targeted therapy based on the microbiome, and in prevention of cancer.

Optimising downstream processes and achieving high product yields are essential for cost-effective and scalable bioproduction, with efficient protein secretion being a critical factor in industrial applications. To address this, we leveraged the widely utilised Sec secretion system of Bacillus subtilis to enhance heterologous protein secretion. Given the lack of reliable in silico tools for predicting optimal combinations of Sec system signal peptides (SPs) with a protein of interest (POI), we aimed to optimise the secretion efficiency of SP-POI pairings by a toolbox approach. We developed an integrative evaluation vector in which the promoter, SPs, and a coding sequence (CDS) of the POI are easily exchangeable. Further, we generated a toolbox containing 74 SPs naturally present in B. subtilis that can easily be integrated into the evaluation vector and thereby fused to the POI. As proof-of-concept, two short peptides from yeast: α-pheromone from Saccharomyces cerevisiae and P-pheromone from Schizosaccharomyces pombe were chosen as POI and secretion efficiency was measured. Successful expression and secretion of both peptides in B. subtilis were verified by an indirect ELISA assay. Eight out of 74 SPs facilitated P-pheromone secretion, while just three effectively enabled the expression of α-pheromone. The maximum observed peptide secretion levels were 43 nM for P-pheromone and 8 nM for α-pheromone. This work demonstrates the necessity of versatile screening approaches to find a matching pairing of the Sec secretion system SP and a POI. We close this gap by providing a robust toolbox with easily exchangeable elements.

Background: Due to the advantages of molecular methods over biochemical methods, the use of molecular methods for diagnosing nosocomial infections such as Pseudomonas can be an appropriate and rapid way to choose the right diagnosis and treatment of infection and prevent further complications caused by the infection. The present article provides a description of the development of a nanoparticle-based detection technique for sensitive and specific deoxyribonucleic acid-based diagnostic of Pseudomonas aeruginosa. Specific thiolated oligonucleotide probes for one of the hypervariable regions of the 16S rDNA gene were designed and applied for colorimetric detection of the bacteria.

Results: The results of gold nanoprobe-nucleic sequence amplification indicated the probe attached to gold nanoparticles in the presence of the target deoxyribonucleic acid. It caused aggregation of gold nanoparticles in the form of connected networks resulting in color change and indicating the presence of the target molecule in the sample, which could be observed by the naked eye. In addition, the wavelength of gold nanoparticles changed from 524 to 558 nm. Multiplex polymerase chain reactions were performed using four specific genes of Pseudomonas aeruginosa (oprL, oprI, toxA, and 16S rDNA). The sensitivity and specificity of the two techniques were assessed. According to the observations, the specificity of both techniques was 100%, and the sensitivity was 0.5 ng/μL and 0.01 ng/μL of genomic deoxyribonucleic acid for multiplex polymerase chain reaction and colorimetric assay, respectively.

Conclusions: The sensitivity of colorimetric detection was about 50 times higher than the polymerase chain reaction using the 16SrDNA gene. The results of our study proved to be highly specific with potential use for early detection of Pseudomonas aeruginosa.

Background: The forming, blending, and characterization of materials at a size of one billionth of a meter or less is referred to as nanotechnology. The objective of the current study was to synthesize ecologically friendly gold nanoparticles (AuNPs) from Gymnosporia montana L. (G. montana) leaf extract, characterize them, assess their interaction with different types of deoxyribonucleic acid (DNA), and investigate their antioxidant and toxic capabilities.

Results: The biosynthesized AuNPs presence was validated by a color change from yellow to reddish pink as well as using UV-visible spectrophotometer. Fourier transform infrared (FTIR) spectroscopy analysis showed the presence of phytoconstituents like, alcohols, phenols, and nitro compounds responsible for the reduction of AuNPs. Zeta sizer and zeta potential of 559.6 d. nm and - 4.5 mV, respectively, demonstrated potential stability. With an average size between 10 and 50 nm, X-ray diffraction (XRD), and high-resolution transmission electron microscope (HR-TEM), revealed the crystalline formation of AuNPs. Surface topology with 3D characterization, irregular spherical shape, and size with 6.48 nm of AuNPs was determined with the help of an atomic force microscope (AFM). AuNPs with some irregular and spherical shapes, and sizes between 2 and 20 nm, were revealed by field emission scanning electron microscope (FESEM) investigation. Shifts in the spectrum were visible when the bioavailability of AuNPs with calf-thymus DNA (CT-DNA) and Herring sperm DNA (HS-DNA) was tested. Additionally, the DNA nicking assay's interaction with pBR322 DNA confirmed its physiochemical and antioxidant properties. The same was also found by using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which showed a 70-80% inhibition rate. Finally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay revealed that viability decreased with increasing dosage, going from 77.74 to 46.99% on MCF-7 cell line.

Conclusion: Synthesizing AuNPs through biogenic processes and adopting G. montana for the first time revealed potential DNA interaction, antioxidant, and cytotoxicity capabilities. Thus, opening new possibilities in the turf of therapeutics as well as in other areas.

Background: Thottea siliquosa (Lamk.) Ding Hou., an important medicinal shrub, is widely used in both ayurvedic and indigenous systems of medicine. Root being the most useful part, the plant is constantly uprooted and thus puts pressure on the natural population. Until date, no micropropagation study is available in this plant. The objective of the study is to develop an efficient in vitro propagation protocol and assessment of clonal fidelity of T. siliquosa.

Results: Media browning was a serious issue during micropropagation, and the addition of 40.0 mg/L ascorbic acid reduced the media browning. For direct shoot regeneration, the optimum response (92% frequency with 20.9 shoots per explant) was obtained when 7-day-old cotyledons were cultured on WPM supplemented with 1.0 mg/L thidiazuron and 0.25 mg/L α-naphthalene acetic acid. The cultures were transferred to WPM augmented with 0.4 mg/L thidiazuron for shoot elongation and growth. On this medium, 100% of cultures responded with a mean number of 27.6 shoots. For callus induction, MS medium with 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L N₆-benzylaminopurin was used. Shoot organogenesis was initiated on the same medium, and calli with minute shoots were transferred to MS medium fortified with 0.5 mg/L N₆-benzylaminopurin and 0.25 mg/L α-naphthalene acetic acid for highest shoot regeneration (100% cultures responded with a mean number of 26.5 shoots per explant). Maximum rooting frequency (82%) and number (20.8) were obtained on half-strength MS medium with 1.0 mg/L indole-3-butyric acid. The rooted plants were acclimatized and transferred to the field. The HPTLC and SCoT analysis revealed the phytochemical and clonal similarity between the in vitro propagated plants and mother plant.

Conclusions: In this study, it is confirmed that cotyledon is an excellent explant for direct and indirect shoot organogenesis in T. siliquosa. For direct shoot induction WPM and indirect organogenesis, MS medium was found to give better response. The true-to-type nature of in vitro-derived plants were confirmed by phytochemical and SCoT analysis. The protocol described here could be used for the large-scale propagation of elite clones of T. siliquosa.

Background: The root system is vital to plant growth and survival. Therefore, genetic improvement of the root system is beneficial for developing stress-tolerant and improved plant varieties. This requires the identification of proteins that significantly contribute to root development. Analyzing protein-protein interaction (PPI) networks is vastly beneficial in studying developmental phenotypes, such as root development, because a phenotype is an outcome of several interacting proteins. PPI networks can be analyzed to identify modules and get a global understanding of important proteins governing the phenotypes. PPI network analysis for root development in rice has not been performed before and has the potential to yield new findings to improve stress tolerance.

Results: Here, the network module for root development was extracted from the global Oryza sativa PPI network retrieved from the STRING database. Novel protein candidates were predicted, and hub proteins and sub-modules were identified from the extracted module. The validation of the predictions yielded 75 novel candidate proteins, 6 sub-modules, 20 intramodular hubs, and 2 intermodular hubs.

Conclusions: These results show how the PPI network module is organized for root development and can be used for future wet-lab studies for producing improved rice varieties.

Background: Vaccination is the one of the agendas of many countries to reduce cervical cancer caused by the Human papillomavirus. Currently, VLP-based vaccine is the most potent vaccine against HPV, which could be produced by a variety of expression systems. Our study focuses on a comparison of recombinant protein expression L1 HPV52 using two common yeasts, Pichia pastoris and Hansenula polymorpha that have been used for vaccine production on an industrial scale. We also applied bioinformatics approach using reverse vaccinology to design alternative multi-epitope vaccines in recombinant protein and mRNA types.

Results: Our study found that P. pastoris relatively provided higher level of L1 protein expression and production efficiency compared to H. polymorpha in a batch system. However, both hosts showed self-assembly VLP formation and stable integration during protein induction. The vaccine we have designed exhibited high immune activation and safe in computational prediction. It is also potentially suitable for production in a variety of expression systems.

Conclusion: By monitoring the overall optimization parameter assessment, this study can be used as the basis reference for large-scale production of the HPV52 vaccine.

Background: Date palm, oasis pivot, plays a vital socio-economic part in the southern area of Morocco. However, with climate change and drought intensity and frequency increasing, the Moroccan palm grove is threatened with significant genetic degradation. Genetic characterization of this resource is key element for the development of effective conservation and management strategies in the current circumstances of climate change and various biotic and abiotic stresses. To evaluate the genetic diversity of date palm populations collected from different Moroccan oases, we used simple sequence repeats (SSR) and directed amplification of mini-satellite DNA (DAMD) markers. Our results showed that used markers could efficiently assess genetic diversity in Phoenix dactylifera L.

Results: A total of 249 and 471 bands were respectively scored for SSR and DAMD, of which 100% and 92.9% were polymorphic. The polymorphic information content (PIC = 0.95), generated by the SSR primer was nearly identical to that generated by the DAMD primer (PIC = 0.98). The resolving power (Rp) was higher in DAMD than SSR (29.46 and 19.51, respectively). Analysis of the molecular variance (AMOVA) based on the combined data sets for both markers revealed a higher variance within populations (75%) than among populations (25%). Principal coordinate analysis (PCoA) and the ascendant hierarchical classification showed that the population of Zagora and Goulmima regions were the closest populations. The STRUCTURE analysis clustering of the 283 tested samples into seven clusters based on their genetic composition.

Conclusion: The results drawn from this study will orient genotypes selection strategies for a successful future breeding and conservation program, particularly under climate change context.

Background: SARS-CoV-2 infection involves disturbing multiple molecular pathways related to immunity and cellular functions. PIM1 is a serine/threonine-protein kinase found to be involved in the pathogenesis of several viral infections. One PIM1 substrate, Myc, was reported to interact with TMPRSS2, which is crucial for SARS-CoV-2 cell entry. PIM1 inhibitors were reported to have antiviral activity through multiple mechanisms related to immunity and proliferation. This study aimed to evaluate the antiviral activity of 2-pyridone PIM1 inhibitor against SARS-CoV-2 and its potential role in hindering the progression of COVID-19. It also aimed to assess PIM1 inhibitor's effect on the expression of several genes of Notch signaling and Wnt pathways. In vitro study was conducted on Vero-E6 cells infected by SARS-CoV-2 "NRC-03-nhCoV" virus. Protein-protein interaction of the study genes was assessed to evaluate their relation to cell proliferation and immunity. The effect of 2-pyridone PIM1 inhibitor treatment on viral load and mRNA expression of target genes was assessed at three time points.

Results: Treatment with 2-pyridone PIM1 inhibitor showed potential antiviral activity against SARS-CoV-2 (IC₅₀ of 37.255 µg/ml), significantly lowering the viral load. Functional enrichments of the studied genes include negative regulation of growth rate, several biological processes involved in cell proliferation, and Interleukin-4 production, with interleukin-6 as a predicted functional partner. These results suggest an interplay between study genes with relation to cell proliferation and immunity. Following in vitro SARS-CoV-2 infection, Notch pathway genes, CTNNB1, SUMO1, and TDG, were found to be overexpressed compared to uninfected cells. Treatment with 2-pyridone PIM1 inhibitor significantly lowers the expression levels of study genes, restoring Notch1 and BCL9 to the control level while decreasing Notch2 and CTNNB1 below control levels.

Conclusion: 2-pyridone PIM1 inhibitor could hinder cellular entry of SARS-CoV-2 and modulate several pathways implicated in immunity, suggesting a potential benefit in the development of anti-SARS-CoV-2 therapeutic approach.