Genetic Manipulation Techniques Research Articles

Genetic Manipulations of Schistosomiasis

Background: Genetic manipulation techniques can be beneficial to control schistosomiasis. These techniques can include such as adeno-associated virus (AAV) vectors, cluster regularly interspaced short palindromic repeats (CRISPR/Cas9) system, and microRNAs (miRNAs). Objectives: In this study, the author reports the progress in a study of schistosomiasis that focused on genetic manipulations for treating schistosomiasis as the objective of this study. Methodology: The author searched the PMC Database and PubMed Database for schistosomiasis articles. All were at National Center for Biotechnology Information (NCBI). The author also searched Google and ScienceDirect in this study. All articles were in English and were from unrestricted open access. Results: Genetic manipulation techniques can help for treating schistosomiasis infections. There several techniques are available: AAV, CRISPR/Cas9 system, microRNAs, RNA interference. In definite hosts, for example, rAAV8-mediated miR-203-3p shielded mice from Schistosoma japonicum schistosomiasis. In intermediate hosts, snail of the genus Biomphalaria, for example, CRISPR/Cas9 system can help to eliminate the parasite. Schistosoma U6 promoters may be beneficial for eliminating this parasite. Conclusions: Genetic manipulations can help to eliminate schistosomiasis in both intermediate hosts and definite hosts. These tools can include such as rAAV-mediated miR-203-3p, CRISPR/Cas9 system, and use of Schistosoma U6 promoters.

Efficient expressions of reporter genes in the industrial filamentous fungus Sclerotium rolfsii mediated by Agrobacterium tumefaciens

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters’ signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.

Integrative protocols for an in vitro generation of pancreatic progenitors from human dental pulp stem cells

Efficiency of the induction protocol is crucial for the generation of insulin-producing cells (IPCs) from human dental pulp stem cells (hDPSCs). Here, we established the integrative induction protocol by merging genetic manipulation technique with our previous published 3-step induction protocol aiming to enhance the pancreatic progenitor commitment and production yield. We found that the overexpression of PDX1 following with 3-step induction protocol were able to generate the 3-dimensional (3D) colony structure of pancreatic progenitors (PPs) with the beneficial trends of pancreatic endoderm commitment and production yield, while other protocols using the prolong maintenance of PDX1-overexpressed hDPSCs and the PDX1 overexpression after definitive endoderm induction were unable to generate and sustain the 3D structure of the colonies. Further Notch signaling manipulation by DAPT treatment showed lesser degree of positive effects on progenitor commitment and production yield. Although the generated PPs from the integrative protocol expressed pancreatic mRNA markers along with pro-insulin and insulin proteins, they still contained the defective glucose-responsive C-peptide secretion. Only basal secreted C-peptide level was observed. In summary, the integrative induction protocol potentially enhanced the PP generation with high colony production yield and could serve as an efficient platform for further hDPSC-derived IPC production and maturation.

Genetic Manipulation to Improve Athletes Performance - A Critical Review

Genetic manipulation as the name suggests is the modification of genes. It is the process of changing the structure of the genes of a living thing in order to make it healthier, stronger, or more useful. It is the direct manipulation of an organism’s genes using biotechnology used to change the genetic makeup of cells including transfer of genes. The need of genetic modification is to enhance the performance of particular genes, this method is implemented effectively to enhance the quality of food, fight various chronic diseases like cancer, cloning etc. However, this paper is limited to the use of genetic manipulation to enhance Athletics performance of the athletes and healing process of the cells. There are various contradictory thoughts over the use of genetic manipulation in Athletics over its use being ethical or unethical. There have been various normative arguments both in favor and against its use in Athletics. However, no competent authority has prohibited its use in Athletics. There are various methods involved in gene modification, few prominent are discussed in this paper. Genetic manipulation, the process of inducing changes in gene expression and the expression of novel genes, has proven to be an indispensable tool in recent genetic research. The implementation of increasingly powerful genetic tools to mouse embryonic stem (ES) cells has led to an explosion of data concerning the specific properties of an extremely large array of genes. Current available techniques allow for the specific elimination of target gene expression, tissue specific induction of reporter gene expression, the over expression of cellular genes, and more. It is not surprising, therefore, that a short time after the derivation of human ES cells, several studies describing genetic manipulation of these cells have been reported. This focuses on the recent advances in the genetic manipulation of human ES cells, the methods currently available, and their possible utilization. A brief discussion of advanced genetic manipulation techniques not yet demonstrated in these cells is also presented. Through this paper the author has tried to summarize various methods, aspects, approaches and arguments involved with genetic modification in Athletic persons.

Defective Proventriculus Regulates Cell Specification in the Gastric Region of Drosophila Intestine.

The gastrointestinal tract in metazoans consists of diverse epithelial cells with distinct cell morphology and physiological functions. The development and homeostasis of gastrointestinal epithelia involve spatiotemporal regulation by many signaling pathways, essential to confer their region-specific function and identity. The adult Drosophila midgut and the mammalian intestine share a high degree of conservation between such signaling pathways. Due to availability of sophisticated techniques for genetic manipulation, Drosophila is an excellent model to study mechanisms of tissue homeostasis regulation in a regionally defined manner. The gastric region located in the Drosophila middle-midgut coincides with the region containing fewest number of stem cells. It is also known as the copper cell (CC) region since it is composed of specialized groups of acid-secreting CCs, along with interstitial cells and enteroendocrine cells. The generation and maintenance of these cell populations are determined by the bone morphogenic protein-like Decapentaplegic (Dpp) signaling pathway. The morphogenic gradient of the Dpp signaling activity induces differential expression of specific transcription factors labial (lab) and defective proventriculus (dve), which are required for the generation of various cell types specific to this region. In this study, we investigated the role of Dve in regulation of tissue homeostasis in the CC region. Our studies reveal that ectopic expression of dve in stem cells suppresses their self-renewal throughout the intestine. We further demonstrate that Dve is not required for generation of CCs. Higher levels of Dve can alter cell specification by inhibition of cut expression, which in turn prevents CC formation during homeostasis.

Chemoenzymatic Production and Engineering of Chitooligosaccharides and N-acetyl Glucosamine for Refining Biological Activities.

Chitooligosaccharides (COS) and N-acetyl glucosamine (GlcNAc) are currently of enormous relevance to pharmaceutical, nutraceutical, cosmetics, food, and agriculture industries due to their wide range of biological activities, which include antimicrobial, antitumor, antioxidant, anticoagulant, wound healing, immunoregulatory, and hypocholesterolemic effects. A range of methods have been developed for the synthesis of COS with a specific degree of polymerization along with high production titres. In this respect, chemical, enzymatic, and microbial means, along with modern genetic manipulation techniques, have been extensively explored; however no method has been able to competently produce defined COS and GlcNAc in a mono-system approach. Henceforth, the chitin research has turned toward increased exploration of chemoenzymatic processes for COS and GlcNAc generation. Recent developments in the area of green chemicals, mainly ionic liquids, proved vital for the specified COS and GlcNAc synthesis with better yield and purity. Moreover, engineering of COS and GlcNAc to generate novel derivatives viz. carboxylated, sulfated, phenolic acid conjugated, amino derived COS, etc., further improved their biological activities. Consequently, chemoenzymatic synthesis and engineering of COS and GlcNAc emerged as a useful approach to lead the biologically-active compound-based biomedical research to an advanced prospect in the forthcoming era.

MicroRNAs As Master Regulators of Atherosclerosis: From Pathogenesis to Novel Therapeutic Options.

Significance: Cardiovascular disease (CVD) remains the major cause of morbidity and mortality worldwide. Accumulating evidence indicates that atherosclerosis and its sequelae, coronary artery disease, contribute to the majority of cardiovascular deaths. Atherosclerosis is a chronic inflammatory disease of the arteries in which atherosclerotic plaques form within the vessel wall. Epidemiological studies have identified various risk factors for atherosclerosis, such as diabetes, hyperlipidemia, smoking, genetic predisposition, and sedentary lifestyle. Recent Advances: Through the advancement of genetic manipulation techniques and their use in cardiovascular biology, it was shown that small RNAs, especially microRNAs (miRNAs), are dynamic regulators of disease pathogenesis. They are considered to be central during the regulation of gene expression through numerous mechanisms and provide a means to develop biomarkers and therapeutic tools for the diagnosis and therapy of atherosclerosis. Circulating miRNAs encapsulated within membrane-surrounded vesicles, which originate from diverse subcellular compartments, are now emerging as novel regulators of intercellular communication. The miRNAs, in both freely circulating and vesicle-bound forms, represent a valuable tool for diagnosing and monitoring CVD, recently termed as "liquid biopsy." Critical Issues: However, despite the recent advancements in miRNA-based diagnostics and therapeutics, understanding how miRNAs can regulate atherosclerosis is still crucial to achieving an effective intervention and reducing the disease burden. Future Directions: We provide a landscape of the current developmental progression of RNA therapeutics as a holistic approach for treating CVD in different animal models and clinical trials. Future interrogations are warranted for the development of miRNA-based therapeutics to overcome challenges for the treatment of the disease.

Ectopic over expression of kiss1 may compensate for the loss of kiss2

Kisspeptin (KISS) is a neuropeptide which plays a central role in the regulation of the hypothalamic-pituitary–gonadal axis, and is essential for sexual maturation and fertility in mammals. Unlike mammals, which possess only one KISS gene, two paralogous genes, kiss1 and kiss2, have been identified in zebrafish and other non-mammalian vertebrates. Previous studies suggest that Kiss2, but not Kiss1, is the reproduction relevant form amongst the two. To better understand the role of each of these isoforms in reproduction, a loss of function approach was applied. Two genetic manipulation techniques—clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN)—were used to generate kiss1 and kiss2 knockout (KO) zebrafish lines, respectively. Examination of these KO lines showed that reproductive capability was not impaired, confirming earlier observations. Further analysis revealed that KO of kiss2 caused a significant increase in expression levels of kiss1, kiss2r and tac3a, while KO of kiss1 had no effect on the expression of any of the examined genes. In situ hybridization analysis revealed that kiss1 mRNA is expressed only in the habenula in wild type brains, while in kiss2 KO fish, kiss1 mRNA-expressing cells were identified also in the ventral telencephalon, the ventral part of the entopeduncular nucleus, and the dorsal and ventral hypothalamus. Interestingly, these regions are known to express kiss2r, and the ventral hypothalamus normally expresses kiss2. These results suggest that a compensatory mechanism, involving ectopic kiss1 expression, takes place in the kiss2 KO fish, which may substitute for Kiss2 activity.

A 96-well high-throughput, rapid-screening platform of extracellular electron transfer in microbial fuel cells

Microbial extracellular electron transfer (EET) stimulates a plethora of intellectual concepts leading to potential applications that offer environmentally sustainable advances in the fields of biofuels, wastewater treatment, bioremediation, desalination, and biosensing. Despite its vast potential and remarkable research efforts to date, bacterial electrogenicity is arguably the most underdeveloped technology used to confront the aforementioned challenges. Severe limitations are placed in the intrinsic energy and electron transfer processes of naturally occurring microorganisms. Significant boosts in this technology can be achieved with the growth of synthetic biology tools that manipulate microbial electron transfer pathways and improve their electrogenic potential. In particular, electrogenic Pseudomonas aeruginosa has been studied with the utility of its complete genome being sequenced coupled with well-established techniques for genetic manipulation. To optimize power density production, a high-throughput, rapid and highly sensitive test array for measuring the electrogenicity of hundreds of genetically engineered P. aeruginosa mutants is needed. This task is not trivial, as the accurate and parallel quantitative measurements of bacterial electrogenicity require long measurement times (~tens of days), continuous introduction of organic fuels (~tends of milliliters), architecturally complex and often inefficient devices, and labor-intensive operation. The overall objective of this work was to enable rapid (<30 min), sensitive (>100-fold improvement), and high-throughput (>96 wells) characterization of bacterial electrogenicity from a single 5 μL culture suspension. This project used paper as a substratum that inherently produces favorable conditions for easy, rapid, and sensitive control of an electrogenic microbial suspension. From 95 isogenic P. aeruginosa mutant, an hmgA mutant generated the highest power density (39 μW/cm2), which is higher than that of wild-type P. aeruginosa and even the strongly electrogenic organism, Shewanella oneidensis (25 μW/cm2). In summary, this work will serve as a springboard for the development of novel paradigms for genetic networks that will help develop mutations or over-expression and synthetic biology constructs to identify genes in P. aeruginosa and other organisms that enhance electrogenic performance in microbial fuel cells (MFCs).

A discrete subtype of neural progenitor crucial for cortical folding in the gyrencephalic mammalian brain.

An increase in the diversity of neural progenitor subtypes and folding of the cerebral cortex are characteristic features which appeared during the evolution of the mammalian brain. Here, we show that the expansion of a specific subtype of neural progenitor is crucial for cortical folding. We found that outer radial glial (oRG) cells can be subdivided by HOPX expression in the gyrencephalic cerebral cortex of ferrets. Compared with HOPX-negative oRG cells, HOPX-positive oRG cells had high self-renewal activity and were accumulated in prospective gyral regions. Using our in vivo genetic manipulation technique for ferrets, we found that the number of HOPX-positive oRG cells and their self-renewal activity were regulated by sonic hedgehog (Shh) signaling. Importantly, suppressing Shh signaling reduced HOPX-positive oRG cells and cortical folding, while enhancing it had opposing effects. Our results reveal a novel subtype of neural progenitor important for cortical folding in gyrencephalic mammalian cerebral cortex.

Production and Characterization of Human Macrophages from Pluripotent Stem Cells.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to differentmacrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

The origin and development of subcortical U-fibers in gyrencephalic ferrets

In the white matter of the human cerebrum, the majority of cortico-cortical fibers are of short range, connecting neighboring cortical areas. U-fibers represent connections between neighboring areas and are located in the white matter immediately deep to the cerebral cortex. Using gyrencephalic carnivore ferrets, here we investigated the neurochemical, anatomical and developmental features of U-fibers. We demonstrate that U-fibers were derived from neighboring cortical areas in ferrets. U-fiber regions in ferrets were intensely stained with Gallyas myelin staining and Turnbull blue iron staining. We further found that U-fibers were derived from neurons in both upper and lower layers in neighboring areas of the cerebral cortex and that U-fibers were formed later than axons in the deep white matter during development. Our findings shed light on the fundamental features of U-fibers in the gyrencephalic cerebral cortex. Because genetic manipulation techniques for ferrets are now available, ferrets should be an important option for investigating the development, functions and pathophysiological changes of U-fibers.

Transient Transfection of the Zoonotic Parasite Babesia microti.

The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5′-actin promoter, program FA100, and 50 μg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in &lt;em&gt;Chlamydia trachomatis&lt;/em&gt;

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

The CRISPR toolbox in medical mycology: State of the art and perspectives.

Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR–Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR–Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.

Phenylketonuria: Genes in phenylketonuria, diagnosis, and treatments

Introduction: Phenylketonuria (PKU) is a rare autosomal-recessive disorder inherited in accordance with the law of segregation. Detection tools for people with PKU can include Sanger Sequencing (SS) and Next Generation Sequencing (NGS). Diet therapy, Large Neutral Amino Acids (LNAA), and Specific Nutrient Combination (SNC) can help alleviate people with PKU. In the future, genetic manipulation techniques can help to eliminate PKU. Objectives: In this review, the author describes the progress in a study that focused on detection tools such as SS and NGS, the phenylalanine hydroxylase (PAH) gene and mutations in the PAH gene, use of drugs for PKU, and genetic manipulation techniques such as Adeno-associated virus (AAV) vectors and clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided FokI nuclease system (FokI-dCas9 system). AAV is abbreviation of AAV. CRISPR system is abbreviation of clustered regularly interspaced short palindromic repeats. Methods: The author searched the PubMed Database at National Center for Biotechnology Information (NCBI) for articles on PKU disorder. These articles were published between 2014 and 2019. Articles were open access and in English. Searches were also done at Google and ScienceDirect. Results: PKU derives from mutations in the PAH gene. Features of PKU may include ataxia, intellectual ability, and seizures. MassARRAY method, minisequencing method, SS and NGS can detect PKU on humans. Diet therapy, BH4, LNAA, SNC, enzyme therapy can help patients with PKU. However, these drugs cannot treat PKU permanently. In the future, genetic manipulation techniques can be used. AAV vectors and FokI-dCas9 system can be useful to eliminate PKU disorder. Conclusion: Guthrie method, MassARRAY, minisequencing, SS and NGS are tools for detecting PKU. Treatments with such as diet therapy, LNAA, SNC, and enzyme therapy are useful for PKU disorder. AAV vectors and FokI-dCas9 system are methods that can be useful for eliminating PKU in the future.

Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale.

Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. Here, we describe an optimized conjugation protocol enabling the transfer of autonomously replicating plasmids from an E. coli donor strain into Roseburia inulinivorans and Eubacterium rectale. The modular nature of the plasmids and their ability to be maintained in the recipient bacterium by autonomous replication makes them ideal for investigating heterologous gene expression, and as a platform for other genetic tools including antisense RNA silencing or mobile group II interon gene disruption strategies.

Establishment and application of a CRISPR\u2013Cas12a assisted genome-editing system in Zymomonas mobilis

BackgroundEfficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking.ResultsUsing Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed.ConclusionsThis study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum.

The heliobacteria are members of the bacterial order Clostridiales and form the only group of phototrophs in the phylum Firmicutes Several physiological and metabolic characteristics make them an interesting subject of investigation, including their minimalist photosynthetic system, nitrogen fixation abilities, and ability to reduce toxic metals. While the species Heliobacterium modesticaldum is an excellent candidate as a model system for the family Heliobacteriaceae, since an annotated genome and transcriptomes are available, studies in this organism have been hampered by the lack of genetic tools. We adapted techniques for genetic manipulation of related clostridial species for use with H. modesticaldum Five heliobacterial DNA methyltransferase genes were expressed in an Escherichia coli strain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation into H. modesticaldum is absolutely required for production of transconjugant colonies. The introduced shuttle vectors are maintained stably and can be recovered using a modified minipreparation procedure developed to inhibit endogenous DNase activity. Furthermore, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants.IMPORTANCE Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyll g as their main pigment and lacking a peripheral antenna system. Until now, research on this organism has been hampered by the lack of a genetic transformation system. Without such a system, gene knockouts, site-directed mutations, and gene expression studies cannot be performed to help us further understand or manipulate the organism. Here we report the genetic transformation of a heliobacterium, which should enable future genetic studies in this unique phototrophic organism.

Task-based fMRI of a free-viewing visuo-saccadic network in the marmoset monkey

Saccadic tasks are often used to index aberrations of cognitive function in patient populations, with several neuropsychiatric and neurologic disorders characterized by saccadic dysfunction. The common marmoset (Callithrix jacchus) has received recent attention as an additional primate model for studying the neural basis of these dysfunctions – marmosets are amenable to a host of genetic manipulation techniques and have a lissencephalic cortex, which is well suited for a variety of recording techniques (e.g., calcium imaging, laminar electrophysiology). Because the marmoset cortex is mostly lissencephalic, however, the locations of frontal saccade-related regions (e.g., frontal eye fields (FEF)) are less readily identified than in Old World macaque monkeys. Further, although high quality histology-based atlases do exist for marmosets, identifying these regions based on histology alone is not always accurate, with the cytoarchitectonic boundaries often inconsonant with functional boundaries. As such, there is a need to map the functional location of these regions directly. Task-based functional magnetic resonance imaging (fMRI) is of utility in this regard, allowing for detection of whole-brain signal changes in response to moving stimuli. Here, we conducted task-based fMRI in marmosets at ultra-high field (9.4 T) during a free-viewing visuo-saccadic task. We also conducted the same task in humans at ultra-high field (7 T) to validate that our simple task was indeed evoking the visuo-saccadic circuitry we expected (as defined by a meta-analysis of fMRI saccade studies). In the marmosets, we found that the task evoked a robust visuo-saccadic topology, with visual cortex (V1, V2, V3, V4) activation extending ventrally to MT, MST, FST and dorsally into V6, 19M, 23V. This topology also included putative cingulate eye field (area 32 and 24d), posterior parietal cortex (with strongest activation in lateral intraparietal area (LIP)), and a frontolateral peak in area 8 aV in marmosets, extending into 45, 46, 8aD, 6DR, 8c, 6 aV, 6DC. Overall, these results support the view that marmosets are a promising preclinical modelling species for studying saccadic dysfunction related to neuropsychiatric or neurodegenerative human brain diseases.