Gene Transfer Research Articles

Enhancement of physiology via adaptive transcription.

The enhancement of complex physiological functions such as cognition and exercise performance in healthy individuals represents a challenging goal. Adaptive transcription programs that are naturally activated in animals to mediate cellular plasticity in response to stimulation can be leveraged to enhance physiological function above wild-type levels in young organisms and counteract complex functional decline in aging. In processes such as learning and memory and exercise-dependent muscle remodeling, a relatively small number of molecules such as certain stimulus-responsive transcription factors and immediate early genes coordinate widespread changes in cellular physiology. Adaptive transcription can be targeted by various methods including pharmaceutical compounds and gene transfer technologies. Important problems for leveraging adaptive transcription programs for physiological enhancement include a better understanding of their dynamical organization, more precise methods to influence the underlying molecular components, and the integration of adaptive transcription into multi-scale physiological enhancement concepts.

Degradation of extracellular antibiotic resistance genes using peracetic acid (PAA) and performic acid (PFA)

Antibiotic resistant bacteria (ARB) associated infections are identified as a major threat to human health in the 21st century. Wastewater contains antibiotic resistance determinants and they are discharged to aquatic environments with treated or untreated effluents. These antibiotic resistance determinants can be taken up by environmental bacteria via horizontal gene transfer (HGT) and they can become antibiotic resistant. Among antibiotic resistance determinants, extracellular DNA containing antibiotic resistance genes can be up taken by environmental bacteria via natural transformation. Therefore, we evaluated the suitability of low-cost disinfectants peracetic acid (PAA) and performic acid (PFA) on inactivating total and functional extracellular ARGs in ultrapure water and secondary wastewater effluent matrices using pUC19 as a model ARG. Performance of PAA and PFA in inactivating extracellular ARGs was compared with the performance of free chlorine disinfection. RT-qPCR results showed that inactivation of ampR gene in pUC19 plasmid suspended in ultrapure water and wastewater secondary effluent by free chlorine was higher compared to PAA and PFA. However, the reduction of functional pUC19 by PAA and PFA were comparable to free chlorine. Transformation frequency of pUC19 treated by PAA and PFA achieved the maximum possible reduction within a shorter time compared to free chlorine treatment, highlighting the possibility of having disinfection chambers with smaller footprints in wastewater treatment plants. These results suggest the suitability of PAA and PFA for inactivation of ARGs in aquatic samples as alternative disinfectants to free chlorine.

Niche-specific evolution and gene exchange of Salmonella in retail pork and chicken

Salmonella exhibits extensive genetic diversity, facilitated by horizontal gene transfer occurring within and between species, playing a pivotal role in this diversification. Nevertheless, most studies focus on clinical and farm animal isolates, and research on the pangenome dynamics of Salmonella isolates from retail stage of the animal food supply chain is limited. Here, we investigated the genomes of 950 Salmonella isolates recovered from retail chicken and pork meats in seven provinces and one municipality of China in 2018. We observed a strong correlation between Salmonella sublineage diversity and the accessory genome with meat type, revealing reduced diversity associated with increased resistance. Importantly, genes associated with antibiotic, biocide, and heavy metal resistance were unevenly distributed in Salmonella from retail chicken and pork. Pork Salmonella isolates showed a higher prevalence of copper and silver resistance genes, while chicken Salmonella isolates displayed a significant predominance of genetic determinants associated with cephalosporin and ciprofloxacin resistance. Moreover, co-occurrence patterns of resistance determinants and their interaction with mobile genetic elements also correlated with meat type. In summary, our findings shed light on how Salmonella achieves their ecological niche success driven by evolution and gene changes in the retail stage of the animal food supply chain.

Microplastic and antibiotics in waters: Interactions and environmental risks.

Antibiotics (ATs) are ubiquitously detected in natural waters worldwide, and their tendency to co-migrate with microplastics (MPs) post-adsorption leads to heightened environmental risk. Research on the adsorption of ATs on MPs and their subsequent effects on the environmental risks is gaining significant attention globally. This adsorption process predominantly occurs through hydrophobic forces, hydrogen bonds, and electrostatic interactions and is influenced by various environmental factors. The interaction between MPs and ATs exhibited varying degrees of efficiency across different pH levels and ionic strengths. Furthermore, this paper outlines the environmental risks associated with the co-presence of MPs and ATs in aquatic environments, emphasizing the potential effect of MPs on the distribution of antibiotic resistance genes (ARGs) and related environmental risks. The potential hazards posed by MPs and ATs in aquatic systems warrant serious consideration. Future research should concentrate on the adsorption of ATs/ARGs on MPs under real environmental conditions, horizontal gene transfer on MPs, as well as biofilm formation and agglomeration behavior on MPs that needs to be emphasized.

Resistome analysis of wastewater treatment plants in Agadir city, Morocco, using a metagenomics approach

Water scarcity has evolved into a pressing global issue, significantly impacting numerous regions worldwide. The use of treated wastewater stands out as a promising solution to this problem. However, the proliferation of various contaminants, primarily Antimicrobial Resistance Genes (ARGs), poses a significant challenge to its safe and sustainable use. In this study, we assessed the composition and abundance of 373 ARGs, corresponding to 31 different classes of antibiotics, in six wastewater treatment plants (WWTP) in Agadir city of Morocco. Influent and effluent samples were collected during the months of February and July in 2020, in addition to samples from the Atlantic Ocean. In total, 223 ARGs were uncovered, highlighting in particular resistance to aminoglycoside, macrolide lincosamide, beta-lactamase, chloramphenicol, sulfonamide, tetracycline, and other antibiotics. The mechanisms of action of these ARGs were mainly antibiotic inactivation, antibiotic target alteration, efflux pump and cellular protection. Mobile genetic elements (MGEs) were detected at high levels their co-occurrence with ARGs highlights their involvement in the acquisition and transmission of ARGs in microbial communities through horizontal gene transfer. While many wastewater treatment methods effectively reduce a large proportion of gene material and pathogens, a substantial fraction of ARGs and other contaminants persist in treated wastewater. This persistence poses potential risks to both human health and the environment, warranting the need of more effective treatment strategies.

MTOR maintains endothelial cell integrity to limit lung vascular injury

The functional and structural integrity of the endothelium is essential for vascular homeostasis. Loss of barrier function in quiescent and migratory capacity in proliferative endothelium causes exuberant vascular permeability, a cardinal feature of many inflammatory diseases including acute lung injury (ALI). However, the signals governing these fundamental endothelial cell (EC) functions are poorly understood. Here, we identify Mechanistic Target of Rapamycin (MTOR) as an important link in preserving the barrier integrity and migratory/angiogenic responses in EC and preventing lung vascular injury and mortality in mice. Knockdown of MTOR in EC altered cell morphology, impaired proliferation and migration, and increased endocytosis of cell surface VE-Cadherin leading to disrupted barrier function. MTOR-depleted EC also exhibited reduced VE-Cadherin and VEGFR2 levels mediated in part by autophagy. Similarly, lungs from mice with EC-specific MTOR deficiency displayed spontaneous vascular leakage marked by decreased VE-Cadherin and VEGFR2 levels, indicating that MTOR deficiency in EC is sufficient to disrupt lung vascular integrity and may be a key pathogenic mechanism of ALI. Indeed, MTOR as well as VEGFR2 and VE-Cadherin levels were markedly reduced in injured mouse lungs or EC. Importantly, EC-targeted gene transfer of MTOR cDNA, either prophylactically or therapeutically, mitigated inflammatory lung injury, and improved lung function and survival in mouse models of ALI. These findings reveal an essential role of MTOR in maintaining EC function, identify loss of endothelial MTOR as a key mechanism of lung vascular injury, and show the therapeutic potential of EC-targeted MTOR expression in combating ALI and mortality in mice.

Genomic Epidemiology of Antimicrobial Resistance: Tracking the Global Spread of Resistant Pathogens

The rapid development and spread of antimicrobial resistance have been one of the most prominent health crises globally, which significantly challenges treating infectious diseases. This paper will address the contribution that genomic epidemiology makes to track and understand the global spread of antimicrobial-resistant pathogens. Genomic epidemiology, an area combining pathogen genomics with epidemiological data, allows for an extremely powerful approach in the identification of resistance mechanisms and tracing transmission routes for guiding public health interventions. This review Emphasizes major bacterial pathogens, such as Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae, which harbor major resistance genes: blaCTX-M, mecA, and blaKPC. These genes contribute to the worldwide dissemination of resistance, mainly through mechanisms involving horizontal gene transfer and selective antibiotic pressure. Thus, genomic surveillance, phylogenetic analysis, and bioinformatics approaches allow for the pinpointing of transmission hotspots, mapping of resistance gene distribution, and tracking of emerging resistant strains in real time. It also provides information on how genomic data are being used to improve antimicrobial stewardship programs and inform clinical treatment decisions. Public health responses can further be tailored by recognizing regional resistance patterns, hence enhancing interventions. However, several challenges persist—for example, the need for standardized sharing of data, interpretation of genomic data, and integration of these insights into public health systems, especially in low-resource settings. This abstract highlights the transformative impact of genomic epidemiology in the management of the AMR crisis while emphasizing the continued need for global cooperation, enhanced data infrastructures, and policy initiatives to ensure the effective use of genomic data in public health.

African Horned Melon (Cucumis metuliferus): Climate‐Resilient Crop and Gene Donor in the Breeding of Major Cucurbits

ABSTRACTAfrican horned melon (Cucumis metuliferus E. Meyer ex Naudin, 2n = 2x = 24) is an under‐researched and under‐utilised cucurbit crop primarily grown for its nutritious fruit. In its centre of diversity, the crop is valued for its relatively high tolerance to insect pests, diseases, drought and heat stress. It is a potential opportunity crop and a valuable source of genes to major Cucumis species, including cucumber (Cucumis sativas L.) and melon (C. melo L.). Its climate resilience and nutrient‐rich fruit provide niche market opportunities. Hence, production and value‐adding will make African horned melon a crop of choice globally. There is a need for an in‐depth investigation into the genetic diversity, breeding and food composition of the crop. Therefore, the objective of this review was to provide perspectives on the production and breeding status of African horned melon to appraise its genetic value for human welfare, strategic production, genetic conservation and breeding of market‐preferred varieties, including closely related Cucumis species. The first section described the botany, production status, germplasm resources and characterisation based on phenotypic and genetic markers. This is followed by breeding progress for biotic and abiotic stress tolerance, utilities and challenges of gene transfer and potential rootstock to Cucumis species, especially cucumber and melon. The review summarised the main breeding goals and approaches, including mutation breeding to fast‐track the development of new varieties. Information presented in the review will guide cultivar design in African horned melon or related cucurbits, aiming for superior agronomic and nutritional quality traits and tolerance to biotic and abiotic stresses.

Perceptions of Cancer Through the Ages—From Hippocratic Oncology to Precision Cancer Medicine

The unravelling of the human genome created new perceptions of the origin and evolution of diseases, and for cancer in particular, it established the notion that neoplasia has been a companion of life since its appearance on Earth. It is not surprising that neoplasms, in various forms, develop in numerous species of animals and even in plants. Unmistakable accounts of cancer with clinical features as are understood today begin in the 5th c. B.C. The principles and practice of the Hippocratic and Galenic tradition dominated cancer care virtually into the 20th century. Advanced sequencing technologies at the dawn of the 21st century generated new therapeutic opportunities with immunotherapy, oncolytic virotherapy, and gene transfer, with the latter especially being used in cases of hereditary cancer.

Genetic Relationships and Population Structure among Nigerian Ethnic Groups (Ibibio, Igbo, Hausa, Tiv and Yoruba) Based on Nine DNA Loci

Abstract Context: Population-specific attributes typically determine the behavior of human genotypes and traits in individuals. Examining the genetic relationships between populations presents an opportunity to observe patterns of genetic changes over time. By comparing populations, it is possible to see factors that may have sparked the evolution of a trait or genotype as well as the genetic diversity of the populations. Genetic diversity in the groups in Nigeria is still largely undefined. This can be achieved with the use of genetic markers. Aims: To determine the genetic relationships between Igbo, Ibibio, Yoruba, Tiv, and Hausa ethnic groups using nine short tandem repeat DNA markers. Settings and Design: Observational studies of 250 consenting participants. Methods and Material: Individuals of Igbo, Yoruba, Hausa, Ibibio, and Tiv were randomly sampled from their ethnic homes in Nigeria at participating institutions. DNA was extracted from blood samples using commercial DNA kits, and tested for nine autosomal short tandem repeat loci alleles using PCR and electrophoresis. The allele types and sizes were read and recorded for each locus for every individual. Statistical Analysis Used: Allelic frequencies, population pairwise genetic distances (FST and RST, analysis of molecular variance (AMOVA), and principal component analysis (PCA) were calculated using GenAlEx v6.502. Results: The fixation index (FST) was between 0.001 and 0.500 in the five populations. The estimated variation was 99.98% within individuals and 0.10% among populations. Four heterogeneous clusters were observed with three axes accounting for 32.86% variation. Conclusions: The genetic relationships closely mirrored linguistic groupings showing that language may have shaped early interaction and ultimately gene transfer between these groups.

Large-scale genomic analysis of Elizabethkingia anophelis

The recent emergence of Elizabethkingia anophelis as a human pathogen is a major concern for global public health. This organism has the potential to cause severe infections and has inherent antimicrobial resistance. The potential for widespread outbreaks and rapid global spread highlights the critical importance of understanding the biology and transmission dynamics of this infectious agent. We performed a large-scale analysis of available 540 E. anophelis, including one novel strain isolated from raw milk and sequenced in this study. Pan-genome analysis revealed an open and diverse pan-genome in this species, characterized by the presence of many accessory genes. This suggests that the species has a high level of adaptability and can thrive in a variety of environments. Phylogenetic analysis has also revealed a complex population structure, with limited source-lineage correlation. We identified diverse antimicrobial resistance factors, including core-genome and accessory ones often associated with mobile genetic elements within specific lineages. Mobilome analysis revealed a dynamic landscape primarily composed of genetic islands, integrative and conjugative elements, prophage elements, and small portion of plasmids emphasizing a complex mechanism of horizontal gene transfer. Our study underscores the adaptability of E. anophelis, characterized by a diverse range of antimicrobial resistance genes, putative virulence factors, and genes enhancing fitness. This adaptability is also supported by the organism’s ability to acquire genetic material through horizontal gene transfer, primarily facilitated by mobile genetic elements such as integrative and conjugative elements (ICEs). The potential for rapid evolution of this emerging pathogen poses a significant challenge to public health efforts.

Evaluating Sequence Alignment Tools for Antimicrobial Resistance Gene Detection in Assembly Graphs

Antimicrobial resistance (AMR) is an escalating global health threat, often driven by the horizontal gene transfer (HGT) of resistance genes. Detecting AMR genes and understanding their genomic context within bacterial populations is crucial for mitigating the spread of resistance. In this study, we evaluate the performance of three sequence alignment tools—Bandage, SPAligner, and GraphAligner—in identifying AMR gene sequences from assembly and de Bruijn graphs, which are commonly used in microbial genome assembly. Efficiently identifying these genes allows for the detection of neighboring genetic elements and possible HGT events, contributing to a deeper understanding of AMR dissemination. We compare the performance of the tools both qualitatively and quantitatively, analyzing the precision, computational efficiency, and accuracy in detecting AMR-related sequences. Our analysis reveals that Bandage offers the most precise and efficient identification of AMR gene sequences, followed by GraphAligner and SPAligner. The comparison includes evaluating the similarity of paths returned by each tool and measuring output accuracy using a modified edit distance metric. These results highlight Bandage’s potential for contributing to the accurate identification and study of AMR genes in bacterial populations, offering important insights into resistance mechanisms and potential targets for mitigating AMR spread.

Comprehensive analysis on Wnt signaling pathway to develop a novel signal modifier

Wnt signals play a key role in organ formation and function. Abnormalities in organ function or genetic diseases and carcinogenesis could point to issues with these signals, they are therefore an effective target for drug delivery. Dr Yuki Ikebuchi, Department of Pharmacy, The University of Tokyo Hospital, Japan, is heading up a team of researchers working to shed greater light on the Wnt signalling pathway. This will facilitate the development and delivery of more targeted drug therapies, helping patients. One element of Ikebuchi’s research is a detailed analysis on the Wnt signalling pathway to develop a novel signal modifier. As previous studies have found that Wnt molecules exert their physiological activities through glycosylation and lipid modification, a possible method to input signals downstream of the Wnt-Fzd complex is to crosslink and interact with Fzd molecules or co-receptors using antibody-like molecules. The researchers sought to obtain high-definition signal activation profiles of 10 types of Fzd molecules; something that hasnâ–™t been done before. At first, the team established the cell line, derived from mouse osteoblast-like MC3T3-E1 cells, in which endogenously expressed all Fzd and co-receptor genese were knocked out using CRISPR-Cas9 system. And then, they used adenoviruses containing the gene sequences of Wnt, Fzd and the co-receptor to transiently express them in knocked-out cells. The researchers will assess the resulting activation of downstream pathways by Luciferase assays using various reporter sequences. They will adopt a dual-glo assay that can correct the gene transfer efficiency and further enhance accuracy using NanoLuc.

Strategy to develop and validate digital droplet PCR methods for global antimicrobial resistance wastewater surveillance.

According to World Health Organization (WHO), antimicrobial resistance (AMR) is currently one of the world's top 10 health threats, causing infections to become difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability, and death. Accurate surveillance is a key component in the fight against AMR. Wastewater is progressively becoming a new player in AMR surveillance, with the promise of a cost-effective real-time tracking of global AMR profiles in specific regions. One of the most useful analytical methods for wastewater surveillance is currently based on real-time PCR (qPCR) and digital droplet PCR (ddPCR) technologies. As stated in the EU Wastewater Treatment Directive proposal, methodological standardization, including a workflow for method development and validation, will play a crucial role in global monitoring of AMR in wastewater. However, according to our knowledge, there are currently no qPCR and ddPCR methods for AMR surveillance available that have been validated according to international standard performance criteria. Therefore, this study proposes a workflow for the development and validation of PCR-based methods for a harmonized and global AMR surveillance, including the construction of specific sequence databases and microbial collections for an efficient method development and method specificity evaluation. Following this strategy, we have developed and validated four duplex ddPCR methods responding to international standard performance criteria, focusing on seven AMR genes (ARG's), including extended spectrum beta-lactam (blaCTX-M), carbapenem (blaKPC-2/3), tetracycline (tet(M)), erythromycin (erm(B)), vancomycin (vanA), sulfonamide (sul2), and aminoglycoside (aac(3)-IV), as well as one indicator of antibiotic (multi-) resistance and horizontal gene transfer, named the class I integron (intl1). The performance of these ddPCR methods was successfully assessed for their specificity, as no false-positive and false-negative results were observed. These ddPCR methods were also considered to be highly sensitive as showing a limit of detection below 25 copies of the targets. In addition, their applicability was confirmed using 14 wastewater samples collected from two Belgian water resource recovery facilities. The proposed study represents therefore a step forward to reinforce method harmonization in the context of the global AMR surveillance in wastewater. PRACTITIONER POINTS: In the context of wastewater surveillance, no PCR-based methods for global AMR monitoring are currently validated according to international standards. Consequently, we propose a workflow to develop and validate PCR-based methods for a harmonized and global AMR surveillance. This workflow resulted here in four duplex ddPCR methods targeting seven ARGs and one general indicator for mobilizable resistance genes. The applicability of these validated ddPCR methods was confirmed on 14 wastewater samples from two Belgian water resource recovery facilities.

Extended Spectrum β-Lactamase: Tackling Antibiotic Resistance and Overcoming Treatment Challenges

Antibiotics, also known as antibacterials, kill or inhibit bacterial growth but are ineffective against viruses, fungi, or parasites, often leading to misuse. They are categorized by molecular structure, mode of action, and spectrum of activity. Antimicrobial Resistance (AMR) occurs when pathogens no longer respond to antimicrobial drugs, arising naturally or through acquisition. Resistance mechanisms include enzymatic (most common), genetic and physical. Bacteria produce various β-lactamases, such as Extended Spectrum β-lactamases (ESBLs), AmpC enzymes, and carbapenemase to exert resistance to Beta-Lactam (βL) class of antibiotics. ESBL families include TEM, SHV, and CTX-M, with E. coli being the most prevalent host. Any Gram-Negative Bacteria (GNB) can be an ESBL producer, but most common ones are the Enterobacteriaceae including Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Proteus mirabilis. ESBL-producing Enterobacteriaceae (ESBL-E) resist penicillin, aztreonam, and cephalosporins except cephamycins and carbapenems, posing a significant public health risk. Genetic resistance mechanisms involve random mutations and horizontal gene transfer through either of the following processes namely conjugation, transformation, transduction. Physical mechanisms include efflux pump production and decreased porin channels. In some microbiological laboratories, ESBL production are often not determined, rather resistance based on MIC values to third generation Cephalosporins are considered as resistance due to ESBL production. Antibiotic use in agriculture and medicine has increased Multi-drug resistant (MDR) ESBL-producing E. coli and evidenced in retail meat and among meat shop employees. Community-acquired ESBL-E infections are a growing concern, with hospital transmission primarily occurring among patients sharing rooms with ESBL carriers. Empirical and definitive therapies for ESBL-E infections must be adjusted based on Antibiotic Susceptibility Testing (AST). The MERINO trial identified urinary tract infections as the most common source of ESBL-E bacteremia, with E. coli being predominant. For critically ill patients with non-urinary tract infections, Meropenem or Imipenem-cilastatin are recommended. For uncomplicated UTIs, Nitrofurantoin, Cotrimoxazole, and Piperacillin-Tazobactam (Pip-Taz) are effective, while Cotrimoxazole, Fluoroquinolones, and Ceftolozane-tazobactam are suitable for complicated UTIs. New β-lactamase inhibitors like avibactam, vaborbactam, and relebactam are promising for treatment. Misuse of antibiotics, such as inappropriate dosing and duration, contributes to AMR, a growing global challenge. Deaths from AMR, estimated at 1.27 million in 2019, could reach 10 million by 2050. ESBLs drive the use of broad-spectrum antibiotics, accelerating resistance development. Inadequate therapy exacerbates infections, leading to prolonged hospital stays, complications, and increased mortality. Balancing new drug development with resistance emergence is crucial to combat AMR.

PIPdb: a comprehensive plasmid sequence resource for tracking the horizontal transfer of pathogenic factors and antimicrobial resistance genes.

Plasmids, as independent genetic elements, carrying resistance or virulence genes and transfer them among different pathogens, posing a significant threat to human health. Under the 'One Health' approach, it is crucial to control the spread of plasmids carrying such genes. To achieve this, a comprehensive characterization of plasmids in pathogens is essential. Here we present the Plasmids in Pathogens Database (PIPdb), a pioneering resource that includes 792964 plasmid segment clusters (PSCs) derived from 1 009571 assembled genomes across 450 pathogenic species from 110 genera. To our knowledge, PIPdb is the first database specifically dedicated to plasmids in pathogenic bacteria, offering detailed multi-dimensional metadata such as collection date, geographical origin, ecosystem, host taxonomy, and habitat. PIPdb also provides extensive functional annotations, including plasmid type, insertion sequences, integron, oriT, relaxase, T4CP, virulence factors genes, heavy metal resistance genesand antibiotic resistance genes. The database features a user-friendly interface that facilitates studies on plasmids across diverse host taxa, habitats, and ecosystems, with a focus on those carrying antimicrobial resistance genes (ARGs). We have integrated online tools for plasmid identification and annotation from assembled genomes. Additionally, PIPdb includes a risk-scoring system for identifying potentially high-risk plasmids. The PIPdb web interface is accessible at https://nmdc.cn/pipdb.

How Gut Microbiome Perturbation Caused by Antibiotic Pre-Treatments Affected the Conjugative Transfer of Antimicrobial Resistance Genes

The global spread of antimicrobial resistance genes (ARGs) poses a significant threat to public health. While antibiotics effectively treat bacterial infections, they can also induce gut dysbiosis, the severity of which varies depending on the specific antibiotic treatment used. However, it remains unclear how gut dysbiosis affects the mobility and dynamics of ARGs. To address this, mice were pre-treated with streptomycin, ampicillin, or sulfamethazine, and then orally inoculated with Salmonella enterica serovar Typhimurium and S. Heidelberg carrying a multi-drug resistance IncA/C plasmid. The streptomycin pre-treatment caused severe microbiome perturbation, promoting the high-density colonization of S. Heidelberg and S. Typhimurium, and enabling an IncA/C transfer from S. Heidelberg to S. Typhimurium and a commensal Escherichia coli. The ampicillin pre-treatment induced moderate microbiome perturbation, supporting only S. Heidelberg colonization and the IncA/C transfer to commensal E. coli. The sulfamethazine pre-treatment led to mild microbiome perturbation, favoring neither Salmonella spp. colonization nor a conjugative plasmid transfer. The degree of gut dysbiosis also influenced the enrichment or depletion of the ARGs associated with mobile plasmids or core commensal bacteria, respectively. These findings underscore the significance of pre-existing gut dysbiosis induced by various antibiotic treatments on ARG dissemination and may inform prudent antibiotic use practices.

Current Progress on Antibiotic Resistance Genes Removal by Composting in Sewage Sludge: Influencing Factors and Possible Mechanisms

Antibiotic resistance genes (ARGs) present in sewage sludge pose significant environmental and public health challenges. Composting has emerged as a promising method to mitigate these risks by reducing ARGs. This review paper evaluated the current progress in the removal of ARGs through composting, incorporating a bibliometric analysis of 228 publications from January 2010 to January 2024. This review highlights the increasing scholarly interest in this field, with a notable rise in publications since 2010. Key mechanisms identified include the denaturation of proteins and DNA at high temperatures, the adsorption of antibiotics and heavy metals by additives like biochar, and shifts in microbial communities, all contributing to the reduction of ARGs during composting. Despite these findings, challenges remain in achieving consistent ARG removal rates, addressing the potential for ARG regrowth, and understanding horizontal gene transfer post-composting. This review suggests further research into optimizing composting conditions and integrating additional treatment methods to enhance ARG removal and minimize associated risks.

Adeno-Associated Virus Gene Transfer Ameliorates Progression of Skeletal Lesions in Mucopolysaccharidosis IVA Mice.

Mucopolysaccharidosis type IVA (MPS IVA) is an autosomal congenital metabolic lysosomal disease caused by a deficiency of the N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) gene, leading to severe skeletal dysplasia. The available therapeutics for patients with MPS IVA, enzyme replacement therapy and hematopoietic stem cell transplantation, revealed limitations in the impact of skeletal lesions. Our previous study, a significant leap forward in MPS IVA research, showed that liver-targeted adeno-associated virus (AAV) gene transfer of human GALNS (hGALNS) restored GALNS enzymatic activity in blood and multiple tissues and partially improved the aberrant accumulation of storage materials. This promising approach was further validated in our current study, where we delivered AAV8 vectors expressing hGALNS, under the control of a liver-specific or ubiquitous promoter, into MPS IVA murine disease models. The results were highly encouraging, with both AAV8 vectors leading to supraphysiological enzymatic activity in plasma and improved cytoplasmic vacuolization of chondrocytes in bone lesions of MPS IVA mice. Notably, the ubiquitous promoter constructs, a potential game-changer, resulted in significantly greater enzyme activity levels in bone and improved pathological findings of cartilage lesions in these mice than in a liver-specific one during the 12-week monitoring period, reinforcing the positive outcomes of our research in MPS IVA treatment.

Plasmid-free production of the plant lignan pinoresinol in growing Escherichia coli cells

BackgroundThe high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use.ResultsIn this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells.ConclusionsFor efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol.