Gene Cassette Research Articles

Class 1 and 2 integrons and antibiotic resistance profile in Salmonella spp. from San Cristobal River, Laguna, Philippines

ABSTRACT Infection with multidrug resistant bacteria is a significant public health concern. Bacteria culture of water samples (n=120) collected in San Cristobal River, Philippines, showed that half (n=60) were positive for Salmonella spp. Screening of all isolates (n=179) for susceptibility to antibiotics showed that most (76.4%; n=113) were positive for class 1 integrons, of which one isolate was also positive for the class 2 integron. The presence of class 1 integrons was associated with resistance to antibiotics (p<0.05). Sequencing of class 1 integron variable regions (VRs) differeciated 11 gene cassettes: dfrA1 or dfrA17; aadA1 or aadA2; blaCTX-M-2 or bla-OXA-1; SmdAB; CmlA1 and aaC 3-Id. However, sequencing of class 2 integron VR differenciated estX, sat2, and aadA1. These results provide insights into evolutionary changes within bacterial multidrug resistant cassettes, more accurately to estimate heath risk associated with the river water.

Identification of Huge Phages from Wastewater Metagenomes.

Huge phages have genomes larger than 200 kilobases, which are particularly interesting for their genetic inventory and evolution. We screened 165 wastewater metagenomes for the presence of viral sequences. After identifying over 600 potential huge phage genomes, we reduced the dataset using manual curation by excluding viral contigs that did not contain viral protein-coding genes or consisted of concatemers of several small phage genomes. This dataset showed seven fully annotated huge phage genomes. The phages grouped into distinct phylogenetic clades, likely forming new genera and families. A phylogenomic analysis between our huge phages and phages with smaller genomes, i.e., less than 200 kb, supported the hypothesis that huge phages have undergone convergent evolution. The genomes contained typical phage protein-coding genes, sequential gene cassettes for metabolic pathways, and complete inventories of tRNA genes covering all standard and rare amino acids. Our study showed a pipeline for huge phage analyses that may lead to new enzymes for therapeutic or biotechnological applications.

Distribution and associations for antimicrobial resistance and antibiotic resistance genes of Escherichia coli from musk deer (Moschus berezovskii) in Sichuan, China.

This study aimed to investigate the antimicrobial resistance (AMR), antibiotic resistance genes (ARGs) and integrons in 157 Escherichia coli (E. coli) strains isolated from feces of captive musk deer from 2 farms (Dujiang Yan and Barkam) in Sichuan province. Result showed that 91.72% (144/157) strains were resistant to at least one antimicrobial and 24.20% (38/157) strains were multi-drug resistant (MDR). The antibiotics that most E. coli strains were resistant to was sulfamethoxazole (85.99%), followed by ampicillin (26.11%) and tetracycline (24.84%). We further detected 13 ARGs in the 157 E. coli strains, of which blaTEM had the highest occurrence (91.72%), followed by aac(3')-Iid (60.51%) and blaCTX-M (16.56%). Doxycycline, chloramphenicol, and ceftriaxone resistance were strongly correlated with the presence of tetB, floR and blaCTX-M, respectively. The strongest positive association among AMR phenotypes was ampicillin/cefuroxime sodium (OR, 828.000). The strongest positive association among 16 pairs of ARGs was sul1/floR (OR, 21.667). Nine pairs positive associations were observed between AMR phenotypes and corresponding resistance genes and the strongest association was observed for CHL/floR (OR, 301.167). Investigation of integrons revealed intl1 and intl2 genes were detected in 10.19% (16/157) and 1.27% (2/157) E. coli strains, respectively. Only one type of gene cassettes (drA17-aadA5) was detected in class 1 integron positive strains. Our data implied musk deer is a reservoir of ARGs and positive associations were common observed among E. coli strains carrying AMRs and ARGs.

Genomic Diversity, Antimicrobial Resistance, Plasmidome, and Virulence Profiles of Salmonella Isolated from Small Specialty Crop Farms Revealed by Whole-Genome Sequencing.

Salmonella is the leading cause of death associated with foodborne illnesses in the USA. Difficulty in treating human salmonellosis is attributed to the development of antimicrobial resistance and the pathogenicity of Salmonella strains. Therefore, it is important to study the genetic landscape of Salmonella, such as the diversity, plasmids, and presence antimicrobial resistance genes (AMRs) and virulence genes. To this end, we isolated Salmonella from environmental samples from small specialty crop farms (SSCFs) in Northeast Ohio from 2016 to 2021; 80 Salmonella isolates from 29 Salmonella-positive samples were subjected to whole-genome sequencing (WGS). In silico serotyping revealed the presence of 15 serotypes. AMR genes were detected in 15% of the samples, with 75% exhibiting phenotypic and genotypic multidrug resistance (MDR). Plasmid analysis demonstrated the presence of nine different types of plasmids, and 75% of AMR genes were located on plasmids. Interestingly, five Salmonella Newport isolates and one Salmonella Dublin isolate carried the ACSSuT gene cassette on a plasmid, which confers resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline. Overall, our results show that SSCFs are a potential reservoir of Salmonella with MDR genes. Thus, regular monitoring is needed to prevent the transmission of MDR Salmonella from SSCFs to humans.

Dissecting molecular evolution of class 1 integron gene cassettes and identifying their bacterial hosts in suburban creeks via epicPCR.

Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution. We applied a single-cell fusion PCR-based technique-emulsion, paired isolation and concatenation PCR (epicPCR)-to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host. Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, namely the gain of a regulatory 5'-untranslated region (5'-UTR), the loss of attC recombination sites between adjacent gene cassettes, and the invasion of a 5'-UTR by an IS element. Notably, our experimental testing of a novel variant of the aadA11 gene cassette demonstrated that gaining the observed 5'-UTR contributed to a 3-fold increase in the MIC of streptomycin relative to the ancestral reference gene cassette in E. coli. Dissecting the observed signatures of molecular evolution of class 1 integrons allowed us to explain their effects on antibiotic resistance phenotypes, while identifying their bacterial hosts enabled us to make better inferences on the likely origins of novel gene cassettes and IS that invade known gene cassettes.

A facile Agrobacterium-mediated transformation method for the model unicellular green algae Chlamydomonas reinhardtii

A reliable and simple Agrobacterium-mediated transformation system for the unicellular green algae model organism Chlamydomonas reinhardtii has been developed. The protocol has been successfully employed with both neomycin phosphotransferase II (nptII) and the phleomycin resistance (bleI) genes coupled with the selective agents paromomycin and zeocin, respectively. A set of binary vectors were assembled that carry the selectable marker cassettes under control either of the Rbcs2 alone or fused to the HSP270A leader sequence, PsaD, or ß-tubulin2 promoters. The corresponding T-DNA elements also harbored a cassette with a codon-optimized version of yellow fluorescence protein (YFP) under control of the Rbcs2 promoter in which the YFP open reading frame was interrupted with the first intron of Rbcs2 to prevent expression in Agrobacterium tumefaciens. The resultant binary vectors were introduced into A. tumefaciens strain C58C1/pMP90, and the derived transconjugants were used for transformation studies with the walled C. reinhardtii strain CC124. Estimated transformation frequencies ranged from 0.09 to 2.86 colonies per 106 cells inoculated. Molecular characterizations on a subset of the transgenic lineages revealed that most of the transgenic events harbored single locus insertions. Moreover, sequencing of captured junction fragments about the T-DNA insertion site showed that minimal disruption of the C. reinhardtii genome occurred. However, the transgenic lineages often harbored truncated T-DNA regions within the non-selectable marker gene cassettes.

A Wohlfahrtiimonas chitiniclastica with a novel type of blaVEB-1-carrying plasmid isolated from a zebra in China.

Wohlfahrtiimonas chitiniclastica is an emerging fly-borne zoonotic pathogen, which causes infections in immunocompromised patients and some animals. Herein, we reported a W. chitiniclastica BM-Y from a dead zebra in China. The complete genome sequencing of BM-Y showed that this isolate carried one chromosome and one novel type of blaVEB-1-carrying plasmid. Detailed genetic dissection was applied to this plasmid to display the genetic environment of blaVEB-1. Three novel insertion sequence (IS) elements, namely ISWoch1, ISWoch2, and ISWoch3, were found in this plasmid. aadB, aacA1, and gcuG were located downstream of blaVEB-1, composing a gene cassette array blaVEB-1-aadB-aacA1-gcuG bracketed by an intact ISWoch1 and a truncated one, which was named the blaVEB-1 region. The 5'-RACE experiments revealed that the transcription start site of the blaVEB-1 region was located in the intact ISWoch1 and this IS provided a strong promoter for the blaVEB-1 region. The spread of the blaVEB-1-carrying plasmid might enhance the ability of W. chitiniclastica to survive under drug selection pressure and aggravate the difficulty in treating infections caused by blaVEB-1-carrying W. chitiniclastica. To the best of our knowledge, this is the first report of the genetic characterization of a novel blaVEB-1-carrying plasmid with new ISs from W. chitiniclastica.

Development of lycopene-based whole-cell biosensors for the visual detection of trace explosives and heavy metals

Residual explosives in conflicting zones have caused irreversible damage to human safety and the environment. Whole-cell biosensors can to detect remnants of buried explosives, such as 2,4-dinitrotoluene (DNT), a stable and highly volatile compound in explosives. However, all the reported whole-cell biosensors utilize fluorescence or luminescence as the biological markers, making their detection difficult in real minefields. Here, we presented a lycopene-based whole-cell biosensor in Escherichia coli to output visible signals in response to DNT, which can help in the visual detection of buried explosives. To construct the whole-cell biosensor, the DNT-responsive promoter yqjF was used as the sensing element, and the lycopene synthetic gene cassette crtEBI was served as the reporting element. Then, the metabolic flux for lycopene production was enhanced to improve the output signal of the whole-cell biosensor, and a terminator was utilized to reduce the background interference. The optimized biosensor LSZ05 could perceive at least 1 mg/L DNT. The DNT-specificity and robust performance of the biosensor under different environmental factors were confirmed. Our results showed that converting the biosensor into a lyophilized powder was an effective storage method. The biosensor LSZ05 could effectively detect DNT in two kinds of soil samples. The lycopene-based whole-cell biosensor could also be used to visually detect heavy metals. Our findings laid the foundation for visually detecting buried explosives in minefields, which was a valuable supplement to the reported biosensors. The methods used for optimizing the lycopene-based whole-cell biosensor, including the improvement of the output signal and reduction of background interference, were quite effective.

Exploring the role of mobile genetic elements in shaping plant–bacterial interactions for sustainable agriculture and ecosystem health

Societal Impact StatementPlants and bacteria interact in complex ways that are crucial to the health and productivity of native vegetation and croplands. While the range of characterised plant‐beneficial bacterial traits continues to grow, key questions remain regarding the distribution and mobility of genes associated with these traits. This work explores the diversity of mobile genetic elements carried by bacteria associated with plant root surfaces, assessing their capacity to help shape plant–bacterial interactions. The significance of this work lies in the potential to contribute to new strategies for enhancing plant health, promoting sustainable agriculture and managing plant diseases in an era when we must respond to environmental change.Summary Integrons are gene capture and expression systems that contribute to bacterial adaptation. Integron research has mainly focused on the role that these elements play in spreading antimicrobial resistance. However, their contribution to niche adaptation is potentially much broader because integrons can sample the vast repertoire of diverse functions encoded by integron gene cassettes. Integrons and gene cassettes have been identified in many bacterial lineages residing in soil and water across varied ecosystems, but there has been little investigation of integrons in plant‐associated bacteria. Bacteria and plants have complex, dynamic relationships that influence plant health and productivity. To investigate whether integrons contribute to adaptative processes in plant microbiomes, we examined gene cassette and microbial taxonomic profiles in rhizoplanes of four important crop species grown under controlled glasshouse conditions. We identified 38,546 unique gene cassettes, including elements carrying genes associated with antibiotic resistance, type II toxin–antitoxin systems and genes with putative functions associated with plant growth promotion, along with a larger set encoding genes of unknown functions. Rhizoplane microbiomes of different plant species showed more similarity in their community composition profiles than in their gene cassette profiles, with complex and distinct suites of gene cassettes associated with each plant species, suggesting that gene cassettes might have a role in specific plant–bacterial interactions. We show that rhizoplane microbiomes carry diverse integron gene cassettes that could play a role in establishing and maintaining rhizoplane communities.

Functional enrichment of integrons: Facilitators of antimicrobial resistance and niche adaptation

Integrons are genetic elements, found among diverse bacteria and archaea, that capture and rearrange gene cassettes to rapidly generate genetic diversity and drive adaptation. Despite their broad taxonomic and geographic prevalence, and their role in microbial adaptation, the functions of gene cassettes remain poorly characterized. Here, using a combination of bioinformatic and experimental analyses, we examined the functional diversity of gene cassettes from different environments. We find that cassettes encode diverse antimicrobial resistance (AMR) determinants, including those conferring resistance to antibiotics currently in the developmental pipeline. Further, we find a subset of cassette functions is universally enriched relative to their broader metagenomes. These are largely involved in (a)biotic interactions, including AMR, phage defense, virulence, biodegradation, and stress tolerance. The remainder of functions are sample-specific, suggesting that they confer localised functions relevant to their microenvironment. Together, they comprise functional profiles different from bulk metagenomes, representing niche-adaptive components of the prokaryotic pangenome.

Iminoctadine tris (albesilate) resistance in Geotrichum citri-aurantii and its management

The bis-guanidine fungicide iminoctadine tris (albesilate) (ITA) is widely used to control citrus sour rot pathogen Geotrichum citri-aurantii but its resistance status in the orchards is still unclear. In the present study, ITA resistance in G. citri-aurantii was assessed and resistance mechanisms and its alternative fungicides were explored. ITA resistance frequency in field isolates of G. citri-aurantii collected between 2018 and 2020 from orchards in China was 6.2%. Consecutively culturing on potato dextrose agar (PDA) for 10 generations showed that ITA resistance for field resistant isolates was relatively stable, whereas resistance for the laboratory induced resistant isolates declined by 53.9%. The competitive ability on citrus fruit for resistant isolates was substantially lower than that of the sensitive isolates. Resistance mechanism studies showed that an ATP-binding cassette (ABC) gene (EVM0003677) and two major facilitator superfamily (MFS) genes (EVM0000087 and EVM0003101) might be involved in ITA resistance only in the induced resistant isolates but not in the field resistant isolates. Bioassay of 26 fungicides found that the demethylation inhibitor fungicides metconazole and propiconazole, and the dinitro-aniline fungicide fluazinam were highly effective to ITA-sensitive isolates of G. citri-aurantii and their EC50 values were 0.33, 0.56, and 0.54 mg/L, respectively. The protective control efficacies of ITA, propiconazole, metconazole, and fluazinam all at 400 mg/L were 100%, 60.4%, 33.9%, and 18.5%, respectively. No positive cross resistance was detected between ITA and metconazole, propiconazole or fluazinam. The mixture of ITA and propiconazole at the mass ratio of 9:8 had a synergistic co-toxicity factor of 26. These results have significant implications for our awareness of ITA resistance in orchards and its management.

Characterization of integrons, extended spectrum beta lactamases and genetic diversity among uropathogenic Escherichia coli isolates from Kerman, south east of Iran.

The study aimed to investigate the distribution of genes encoding integrons, extended spectrum beta-lactamase (ESBL) in E. coli isolated from UTIs, as well as the genetic diversity among the isolates. E. coli isolates were recovered from the patients with UTI in Kerman Iran. Antibiotic susceptibility was done according to CLSI guidelines. The presence of ESBL genes and integrons was evaluated using PCR. PCR and sequencing were applied for the evaluation of cassette content of integrons. Genotyping of the isolates was performed by multiple-locus variable-number tandem repeat analysis (MLVA). Imipenem was the most effective antibiotic, while the highest resistance was observed to streptomycin. In total 40.2% of isolates were ESBL producers. Of 69 integron-positive isolates, 59 only had class I integrons, 4 only had class II integrons and 6 had both types. The most common gene cassette found within class I integrons was dfrA17-aadA5 (n=27). The E. coli isolates were divided into 16 MLVA clusters. The current study demonstrated the simultaneous presence of class I integrons and ESBLs involved in the resistance of UPEC isolates to antibacterial agents. Our finding also revealed that the E. coli isolates belonged to diverse clones.

A Class 4-like Chromosomal Integron Found in Aeromonas sp. Genomospecies paramedia Isolated from Human Feces.

Integrons are genetic elements that store, express and exchange gene cassettes. These elements are characterized by containing a gene that codes for an integrase (intI), a cassette integration site (attI) and a variable region holding the cassettes. Using bioinformatics and molecular biology methods, a functional integron found in Aeromonas sp. 3925, a strain isolated from diarrheal stools, is described. To confirm the integron class, a phylogenetic analysis with amino acid sequences was conducted. The integrase was associated to class 4 integrases; however, it is clearly different from them. Thus, we classified the associated element as a class 4-like integron. We found that the integrase activity is not under the control of the SOS or catabolic repression, since the expression was not increased in the presence of mitomycin or arabinose. The class-4-like integron is located on the chromosome and contains two well-defined gene cassettes: aadA1 that confers resistance to streptomycin and lpt coding for a lipoprotein. It also includes eight Open Reading frames (ORFs) with unknown functions. The strain was characterized through a Multilocus Phylogenetic Analyses (MLPA) of the gyrB, gyrA, rpoD, recA, dnaJ and dnaX genes. The phylogenetic results grouped it into a different clade from the species already reported, making it impossible to assign a species. We resorted to undertaking complete genome sequencing and a phylogenomic analysis. Aeromonas sp. 3925 is related to A. media and A. rivipollensis clusters, but it is clearly different from these species. In silico DNA-DNA hybridization (isDDH) and Average Nucleotide Identity (ANI) analyses suggested that this isolate belongs to the genomospecies paramedia. This paper describes the first class 4-like integron in Aeromonas and contributes to the establishment of genomospecies paramedia.

Occurrence and characterization of plasmids carrying tmexCD1-toprJ1, blaDHA-1, and blaCTX-M-127, in clinical Klebsiella pneumoniae strains.

Today, the emergence of Klebsiella pneumoniae with the tmexCD1-toprJ1 gene cassette in patients has presented a significant clinical challenge. To present the detailed genetic features of the tmexCD1-toprJ1 gene cassette of K. pneumoniae strain F4_plasmid pA, the whole bacterial genome was sequenced by Illumina and nanopore platforms, and mobile genetic elements related to antibiotic resistance genes were analyzed with a series of bioinformatics methods. K. pneumoniae strain F4 was determined to be a class A+C beta-lactamase, and was resistant to routinely used antibiotics, especially tigecycline, because of the oqxAB gene localized on the F4_chromosome and tmexCD1-toprJ1 on F4_plasmid A. After plasmid transfer assays, the F4_plasmid pA or F4_plasmid pB could be recovered with an average conjugation frequencies of 3.42×10-4 or 4.19×10-4. F4_plasmid pA carried tmexCD1-toprJ1 and bla DHA-1 accompanied by genetic intermixing of TnAs1, Tn5393, TnAs3, and In641, while F4_plasmid pB, bearing bla CTX-M-174, had structural overlap of TnAs3 and In641. We suggested that plasmids carrying tmexCD1- toprJ1 might be strongly related to IS26-integrated loop intermediates. This study showed that due to the structural evolution of F4 and related strains, their resistances were so strong that effective antibiotics were virtually unavailable, therefore their spread and prevalence should be strictly controlled.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

Engineered Expression of Vip3A in Green Tissues as a Feasible Approach for the Control of Insect Pests in Maize.

Genetic engineering technology offers opportunities to improve many important agronomic traits in crops, including insect-resistance. However, genetically modified (GM) exogenous proteins in edible tissues of transgenic crops has become an issue of intense public concern. To advance the application of GM techniques in maize, a Cre/loxP-based strategy was developed for manipulating the transgenes in green tissues while locking them in non-green tissues. In the strategy, the site-specific excision can be used to switch on or off the expression of transgenes at specific tissues. In this work, two basic transgenic maize, named KEY, carrying the Cre gene, and LOCK, containing the Vip3A gene with a blocked element, were obtained based on their separate fusion gene cassettes. The expression level and concentration of Vip3A were observed with a high specific accumulation in the green tissues (leaf and stem), and only a small amount was observed in the root and kernel tissues in the KEY × LOCK hybrids. The insect resistance of transgenic maize against two common lepidopteran pests, Ostrinia furnacalis and Spodoptera frugiperda, was assessed in the laboratory and field. The results indicate that the hybrids possessed high resistance levels against the two pests, with mortality rates above 73.6% and damage scales below 2.4 compared with the control group. Our results suggest that the Cre/loxP-mediated genetic engineering approach has a competitive advantage in GM maize. Overall, the findings from this study are significant for providing a feasible strategy for transgenes avoiding expression in edible parts and exploring novel techniques toward the biosafety of GM plants.

High incidence of multiple intI1 genomic gene cassettes in Aeromonas strains

Antimicrobial resistance profiles of Aeromonas have been used as an effective indicator for monitoring resistances in aquatic environments. Integron gene cassettes are a major reservoir of antibiotic resistance genes in bacteria. To date, little has been reported on the antimicrobial resistance or integrons of Aeromonas in bullfrog aquaculture. In this study, 27 isolates belonging to four Aeromonas species (A. hydrophila, A. veronii, A. caviae, and A. jandaei) were isolated from farmed diseased American bullfrogs (Rana catesbeiana). all of them were multidrug-resistant, exhibiting resistance to at least seven drugs from five antibiotic classes. Class 1 integrons (intI1s), containing nine types of gene cassette arrays encoding resistance to trimethoprim (dfrA1, dfrA12, dfrA15, dfrA17), streptomycin/spectinomycin (aadA2), fluoroquinolones/aminoglycosides (aac(6′)-Ib-cr), rifampin (arr3), proline-rich antimicrobial peptides (ptrB), and multiple antimicrobial agents involved in biofilm development (dgs), were detected in 26 (26/27, 96.3%) of the isolates. Seven patterns were identified, and six of them contained two or three gene cassettes, in 27 isolates. This work highlights unusually high frequency (21/27, 77.8%) of multiple intI1 gene cassette arrays and three novel intI1 gene cassettes or arrays (dgc, ptrB, and aac(6′)-Ib-cr-arr3) in the isolates of Aeromonas spp. from American bullfrogs.

Application of Nanotechnology in Plant Genetic Engineering.

The ever-increasing food requirement with globally growing population demands advanced agricultural practices to improve grain yield, to gain crop resilience under unpredictable extreme weather, and to reduce production loss caused by insects and pathogens. To fulfill such requests, genome engineering technology has been applied to various plant species. To date, several generations of genome engineering methods have been developed. Among these methods, the new mainstream technology is clustered regularly interspaced short palindromic repeats (CRISPR) with nucleases. One of the most important processes in genome engineering is to deliver gene cassettes into plant cells. Conventionally used systems have several shortcomings, such as being labor- and time-consuming procedures, potential tissue damage, and low transformation efficiency. Taking advantage of nanotechnology, the nanoparticle-mediated gene delivery method presents technical superiority over conventional approaches due to its high efficiency and adaptability in different plant species. In this review, we summarize the evolution of plant biomolecular delivery methods and discussed their characteristics as well as limitations. We focused on the cutting-edge nanotechnology-based delivery system, and reviewed different types of nanoparticles, preparation of nanomaterials, mechanism of nanoparticle transport, and advanced application in plant genome engineering. On the basis of established methods, we concluded that the combination of genome editing, nanoparticle-mediated gene transformation and de novo regeneration technologies can accelerate crop improvement efficiently in the future.

Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.

Comparative Genomic Analysis Reveals the Emergence of ST-231 and ST-395 Klebsiella pneumoniae Strains Associated with the High Transmissibility of blaKPC Plasmids.

Conjugative transposons in Gram-negative bacteria have a significant role in the dissemination of antibiotic-resistance-conferring genes between bacteria. This study aims to genomically characterize plasmids and conjugative transposons carrying integrons in clinical isolates of Klebsiella pneumoniae. The genetic composition of conjugative transposons and phenotypic assessment of 50 multidrug-resistant K. pneumoniae isolates from a tertiary-care hospital (SQUH), Muscat, Oman, were investigated. Horizontal transferability was investigated by filter mating conjugation experiments. Whole-genome sequencing (WGS) was performed to determine the sequence type (ST), acquired resistome, and plasmidome of integron-carrying strains. Class 1 integrons were detected in 96% of isolates and, among integron-positive isolates, 18 stains contained variable regions. Horizontal transferability by conjugation confirmed the successful transfer of integrons between cells and WGS confirmed their presence in conjugative plasmids. Dihydrofolate reductase (dfrA14) was the most prevalent (34.8%) gene cassette in class 1 integrons. MLST analysis detected predominantly ST-231 and ST-395. BlaOXA-232 and blaCTX-M-15 were the most frequently detected carbapenemases and beta-lactamases in the sequenced isolates. This study highlighted the high transmissibility of MDR-conferring conjugative plasmids in clinical isolates of K. pneumoniae. Therefore, the wise use of antibiotics and the adherence to effective infection control measures are necessary to limit the further dissemination of multidrug-resistant bacteria.