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This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.

Liquid handler systems can provide significant benefits to researchers by automating laboratory work, however, their unaffordable price provides a steep barrier to entry. Therefore, we provide the BioCloneBot, a versatile, low-cost, and open-source automated liquid handler. This system can be easily built with 3D-printed parts and readily available commercial components. The BioCloneBot is highly adaptive to user needs and facilitates various liquid handling tasks in research and diagnostics. Its user-friendly interface and programmable nature make it suitable for a wide range of applications, from small-scale experiments to larger laboratory setups. By utilizing BioCloneBot, researchers and scientists can streamline their liquid handling processes without the financial constraints posed by traditional systems. In this paper, we detail the design, construction, and validation of BioCloneBot, showcasing its precise control, accuracy, and repeatability in various liquid handling tasks. The open-source nature of the system encourages collaboration and customization, enabling researchers to contribute and adapt the technology to specific experimental requirements.

Various attempts to amplify an AQP11 cDNA from tissues of the spiny dogfish (Squalus acanthias) were made. Two pairs of deoxy-inosine-containing degenerate primers were designed based on conserved amino acid sequences from an AQP11 alignment. These primers yielded some faint bands from gill cDNA that were sequenced. Blast searches with the sequences showed they were not AQP11. An elasmobranch AQP11 nucleotide sequence alignment was produced to identify conserved regions to make further degenerate primers. One primer pair produced a short 148 bp fragment showing particularly strong amplification in gill and intestine. It was sequenced and represented a piece of the AQP11 gene. However, as the fragment may have resulted from contaminating genomic DNA (in total RNA used to make cDNA), 5′ and 3′ RACE were performed to amplify the two ends of the putative cDNA. Furthermore, 5′ and 3′ RACE amplifications depend on the presence of a 5′ cap nucleotide and a poly A tail, respectively on the putative AQP11 mRNA. Hence, successful amplification was only possible from cDNA and not genomic DNA. Nested RACE amplifications were performed using gill and intestinal RACE cDNA, but none of the DNA fragments sequenced were AQP11. Consequently, the spiny dogfish AQP11 gene may represent a pseudogene. [ABSTRACT FROM AUTHOR]

The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination of homology arms. Among them, Seamless Ligation Cloning Extract (SLiCE) is an affordable alternative solution that uses simple Escherichia coli lysates. However, the underlying molecular mechanisms remain unclear and the reconstitution of the extract by defined factors has not yet been reported. We herein show that the key factor in SLiCE is Exonuclease III (ExoIII), a double‐strand (ds) DNA‐dependent 3′‐5′ exonuclease, encoded by XthA. SLiCE prepared from the xthAΔ strain is devoid of recombination activity, whereas purified ExoIII alone is sufficient to assemble two blunt‐ended dsDNA fragments with homology arms. In contrast to SLiCE, ExoIII is unable to digest (or assemble) fragments with 3′ protruding ends; however, the addition of single‐strand DNA‐targeting Exonuclease T overcomes this issue. Through the combination of commercially available enzymes under optimized conditions, we achieved the efficient, reproducible, and affordable cocktail, "XE cocktail," for seamless DNA cloning. By reducing the cost and time required for DNA cloning, researchers will devote more resources to advanced studies and the careful validation of their own findings. [ABSTRACT FROM AUTHOR]

Positive selection vectors carry a lethal gene encoding a toxic product that is harmful to most laboratory E. coli strains. Previously, we reported a strategy for in-house production of a commercial positive selection vector, the pJET1.2/blunt cloning vector, using common laboratory E. coli strains. However, the strategy involves lengthy gel electrophoresis and extraction procedures to purify the linearized vector after digestion. Here, we streamlined the strategy to eliminate the gel-purification step. A uniquely designed short fragment called the Nawawi fragment was inserted into the coding sequence of the lethal gene of the pJET1.2 plasmid, resulting in the pJET1.2N plasmid that can be propagated in the E. coli strain DH5α. Digestion of the pJET1.2N plasmid with EcoRV released the Nawawi fragment, and the resulting blunt-ended pJET1.2/blunt cloning vector can be used directly for DNA cloning without prior purification. Cloning of a DNA fragment was not hindered by the Nawawi fragments carried over from the digestion step. After transformation, the pJET1.2N-derived pJET1.2/blunt cloning vector produced > 98% positive clones. The streamlined strategy accelerates the in-house production of the pJET1.2/blunt cloning vector and enables DNA cloning at a lower cost. [ABSTRACT FROM AUTHOR]

Key message: In-house production of a positive selection cloning vector could be simple, efficient and low cost. DNA cloning technology requires a vector to harbour a gene of interest for multiplication of the gene in bacterial cells. Positive selection vector has become a popular type of cloning vector due to the simplicity and efficiency of the positive selection system. Due to the presence of a toxic gene, propagation of a commercial positive selection vector in common laboratory E. coli strains is infeasible. This study demonstrated a strategy for propagation and in-house production of a commercial positive selection vector, i.e., pJET1.2/blunt cloning vector, at low cost. This was done by insertion of a specially designed DNA fragment (MCS fragment), which can be easily removed later by EcoRV digestion, into the pJET1.2/blunt cloning vector to allow the propagation of the modified plasmid (termed pJET1.2M) in common E. coli strains. Removal of the MCS fragment from the pJET1.2M plasmid produces the pJET1.2/blunt cloning vector ready for gene cloning. The self-made pJET1.2/blunt cloning vector exhibited a cloning efficiency of 100%. [ABSTRACT FROM AUTHOR]

The discovery of the position effect variegation phenomenon and the subsequent comprehensive analysis of its molecular mechanisms led to understanding that the local chromatin composition has a dramatic effect on gene activity. To study this effect in a high-throughput mode and at the genome-wide level, the Thousands of Reporters Integrated in Parallel (TRIP) approach based on the usage of barcoded reporter gene constructs was recently developed. Here we describe the construction and quality checks of high-diversity barcoded plasmid libraries supposed to be used for high-throughput analysis of chromatin position effects in Drosophila cells. First, we highlight the critical parameters that should be considered in the generation of barcoded plasmid libraries and introduce a simple method to assess the diversity of random sequences (barcodes) of synthetic oligonucleotides using PCR amplification followed by Sanger sequencing. Second, we compare the conventional restriction-ligation method with the Gibson assembly approach for cloning barcodes into the same plasmid vector. Third, we provide optimized parameters for the construction of barcoded plasmid libraries, such as the vector : insert ratio in the Gibson assembly reaction and the voltage used for electroporation of bacterial cells with ligation products. We also compare different approaches to check the quality of barcoded plasmid libraries. Finally, we briefly describe alternative approaches that can be used for the generation of such libraries. Importantly, all improvements and modifications of the techniques described here can be applied to a wide range of experiments involving barcoded plasmid libraries.

Introduction: The relationship between the body and soul is a significant topic in various philosophical traditions, including ancient Greek, Islamic, and Western philosophy. These traditions have attempted to address the question of how the body and soul can coexist in a single entity, given their differing natures. The soul is often considered to be of a spiritual nature while the body is material. This raises the question of how these distinct entities can coexist and establish an existential relationship. Is the body like a container in which the soul is placed and, after death, it is separated from it, like a chicken freed from a cage? Or is the body like a tool that the soul needs for its actions, such as thinking and feeling, etc.? Which is no longer needed after death, or is the relationship between the body and the human soul a contract that is regulated by an external agent? Or is this relationship between the soul and the body a developmental thing? The process of material creation of the human body at the beginning in the mother's womb is such that this way of physical existence and its special structure can only be the origin of a unique spirit. In this article explores the relationship between the body and soul in Greek and Western divine philosophies. We also discussed the views of Sadr al-Maltahin Shirazi with the title "The physical theory of occurrence and the spiritual existence of the human soul", in which the soul, although given to the body by the active intellect or the Holy Spirit, is specifically formed in the mother's womb with the unique structures: an appropriate body must be prepared so that a suitable soul can be created in it. If the soul and body are not correlated, then it is impossible for a particular soul to be matched with a particular body. At the end of the article, we will try to compare this theory with the theory of Khwaja Nasir al-Din Tusi titled "The main component of the human body" and its adaptation to the theory of "simulation" in which the DNA of each human body is a unique thing that not only determines all the physical characteristics but also must determine a special soul that will refuse just any random soul. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the present in vivo study was to examine alterations of the microbial community structure in biofilms on different dental implant surfaces over the time. Materials and Methods: Zirconium nitride-coated glass (ZrN-glass) and ZrN-coated polished titanium (ZrN-Ti) disks were used as substrates and polished titanium (Ti-pol) was used as a control. The specimens were mounted on removable intraoral splints in one adult. After 24 hours and 14 days of intraoral exposure, the microbial biofilms were analyzed by generating 16S rRNA gene clone libraries. Results: ZrN coating of a Ti surface altered the microbial composition early on (24 hours), with a tendency to augment Lactobacillus-related phylotypes later. Long-term exposure (14 days) of dental implant surfaces to microbes resulted in a significantly different composition of the biofilm on all three tested surfaces. Conclusions: This preliminary study showed that a ZrN-Ti disk surface harbors a significantly different microbial composition from a polished Ti surface. Further improvement of ZrN physical vapor deposition coatings might help to influence the adhesion of bacteria that are less pathogenic, thereby reducing the risk of peri-implantitis. Int J Oral Maxillofac Implants 2012;27:811-819. [ABSTRACT FROM AUTHOR]

Marine sponges have recently been recognized as the source ofenzymes, including members of hydrolases. Hydrolytic enzymes are extracellularly produced by sponge-associated bacteria to mediate the metabolism of complex organic matters, thereby assisting the sponge hosts in nutrition and metabolic processes. Among hydrolytic enzymes, amilaseshas attracted increasing attention due to their potential industrial applications. This research work was aimed atutilizing functional metagenomicsapproach for the discovery of amilases derived from the uncultured symbiotic bacteriaof the Indonesian marine sponge Theonella swinhoei. Weinitially constructed a small-insert metagenomiclibrary in Escherichia coliby cloning of metagenome in the size range of5-20 kb prepared from the sponge’s microbiome. Further functional screening of the resulting metagenomic library led to the isolation of two recombinant E. coli clones potentially harboring amilase genes, as indicated by the presence of clearing zones surroinding the selective medium containing 1% amilum.

Liquid handler systems can provide significant benefits to researchers by automating laboratory work, however, their unaffordable price provides a steep barrier to entry. Therefore, we provide the BioCloneBot, a versatile, low-cost, and open-source automated liquid handler. This system can be easily built with 3D-printed parts and readily available commercial components. The BioCloneBot is highly adaptive to user needs and facilitates various liquid handling tasks in research and diagnostics. Its user-friendly interface and programmable nature make it suitable for a wide range of applications, from small-scale experiments to larger laboratory setups. By utilizing BioCloneBot, researchers and scientists can streamline their liquid handling processes without the financial constraints posed by traditional systems. In this paper, we detail the design, construction, and validation of BioCloneBot, showcasing its precise control, accuracy, and repeatability in various liquid handling tasks. The open-source nature of the system encourages collaboration and customization, enabling researchers to contribute and adapt the technology to specific experimental requirements., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed. [ABSTRACT FROM AUTHOR]

Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manure-treated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment. [ABSTRACT FROM AUTHOR]

The human gut microbiota is well established as an important factor in health and disease. Fecal sample microbiota are often analyzed as a proxy for gut microbiota, and characterized with respect to their composition profiles. Modern approaches employ whole genome shotgun next-generation sequencing as the basis for these analyses. Sequencing depth as well as choice of next-generation sequencing data analysis method constitute two main interacting methodological factors for such an approach. In this study, we used 200 million sequence read pairs from one fecal sample for comparing different taxonomy classification methods, using default and custom-made reference databases, at different sequencing depths. A mock community data set with known composition was used for validating the classification methods. Results suggest that sequencing beyond 60 million read pairs does not seem to improve classification. The phylogeny prediction pattern, when using the default databases and the consensus database, appeared to be similar for all three methods. Moreover, these methods predicted rather different species. We conclude that the choice of sequencing depth and classification method has important implications for taxonomy composition prediction. A multi-method-consensus approach for robust gut microbiota NGS analysis is recommended. [ABSTRACT FROM AUTHOR]

Reptiles show varying degrees of facultative parthenogenesis. Here we use genetic methods to determine that an isolated, captive female Asian water dragon produced at least nine offspring via parthenogenesis. We identified microsatellites for the species from shotgun genomic sequences, selected and optimized primer sets, and tested all of the offspring for a set of seven microsatellites that were heterozygous in the mother. We verified that the seven loci showed high levels of polymorphism in four wild Asian water dragons from Vietnam. In all cases, the offspring (unhatched, but developed eggs, or hatched young) had only a single allele at each locus, and contained only alleles present in the mother’s genotype (i.e., were homozygous or hemizygous). The probability that our findings resulted from the female mating with one or more males is extremely small, indicating that the offspring were derived from a single female gamete (either alone or via duplication and/or fusion) and implicating parthenogenesis. This is the first documented case of parthenogenesis in the Squamate family Agamidae. [ABSTRACT FROM AUTHOR]

Population-based public health data on antibiotic resistance gene carriage is poorly surveyed. Research of the human microbiome as an antibiotic resistance reservoir has primarily focused on gut associated microbial communities, but data have shown more widespread microbial colonization across organs than originally believed, with organs previously considered as sterile being colonized. Our study demonstrates the utility of postmortem microbiome sampling during routine autopsy as a method to survey antibiotic resistance carriage in a general population. Postmortem microbial sampling detected pathogens of public health concern including genes for multidrug efflux pumps, carbapenem, methicillin, vancomycin, and polymixin resistances. Results suggest that postmortem assessments of host-associated microbial communities are useful in acquiring community specific data while reducing selective-participant biases. [ABSTRACT FROM AUTHOR]

Mats of the green alga Trentepohlia, a genus widely distributed in the tropics as well as temperate regions, have always been perceived as homogeneous (i.e., formed by only one species). As such, their general nature and specific feature play a supportive role in the species delimitation. However, the presence of morphologically dissimilar thalli was observed under the light microscope and when cultivating a piece of a single mat. To address this, we performed DNA cloning of the rbcL gene on mat fragments of T. abietina, T. annulata, T. jolithus and T. umbrina sampled in Europe to reveal if they are composed of one or more species. We revealed that more Trentepohlia haplotypes may coexist in a single mat. In consideration of this, we conclude that the use of material isolated in unialgal culture will be almost mandatory for a taxonomic reassessment of this complicated genus. Another direct implication of this problem is that herbarium specimens consisting of field‐collected material should not be used for direct sequencing. We further hypothesize the reasons why multiple haplotypes of Trentepohlia occur more frequently in the tuft‐like mats. Possibly, fragments and/or cells of other microalgae, including other species of Trentepohlia, might be retained in such mats more easily than in the crustose trentepohlialean mats. [ABSTRACT FROM AUTHOR]

Thoroughbred horses are finely-tuned athletes with a high aerobic capacity relative to skeletal muscle mass, attributable to centuries of genetic selection for speed and stamina. Polymorphisms in the myostatin gene (MSTN), a pronounced inhibitor of skeletal muscle growth, have been shown to almost singularly account for gene-based race distance aptitude in racehorses. In Thoroughbreds, two MSTN polymorphisms, a single nucleotide variation in the first intron (SNP g.66493737C>T) and a non-coding transposable element within the promoter region (a 227 bp SINE insertion) are of particular interest. Until now, it has not been clear which of these variants affect skeletal muscle phenotypes or whether both can impact racing performance. In a large cohort of Thoroughbreds, we observed a complete concordance between the SNP and the SINE insertion. By means of in vitro assays in C2C12 myoblasts, we isolated the SNP variant from the SINE polymorphism and showed the latter is exclusively responsible for adversely affecting transcription initiation and gene expression thereby limiting myostatin protein production. Mapping the MSTN transcription start site in horse skeletal muscle likewise revealed anomalous transcription initiation in the presence of the SINE insertion. Our data provides mechanistic evidence that the SINE insertion uniquely accounts for the MSTN “speed gene” effect on race distance aptitude in the Thoroughbred horse. [ABSTRACT FROM AUTHOR]

Honey bees are considered large-scale monitoring tools due to their environmental exploration and foraging activities. Traces of these activities can be recovered in the honey that also may reflect the hive ecological micro-conditions in which it has been produced. This study applied a next generation sequencing platform (Ion Torrent) for shotgun metagenomic analysis of honey environmental DNA (eDNA). The study tested a methodological framework to interpret DNA sequence information useful to describe the complex ecosystems of the honey bee colony superorganism, its pathosphere and the heterogeneity of the agroecological environments and environmental sources that left DNA marks in the honey. Analysis of two honeys reported sequence reads from five main organism groups (kingdoms or phyla): arthropods (that mainly included reads from Apis mellifera, several other members of the Hymenotpera, in addition to members of the Diptera, Coleoptera and Lepidoptera, as well as aphids and mites), plants (that clearly confirmed the botanical origin of the two honeys, i.e. orange tree blossom and eucalyptus tree blossom honeys), fungi and bacteria (including common hive and honey bee gut microorganisms, honey bee pathogens and plant pathogens), and viruses (which accounted for the largest number of reads in both honeys, mainly assigned to Apis mellifera filamentous virus). The shotgun metagenomic approach that was used in this study can be applied in large scale experiments that might have multiple objectives according to the multi-kingdom derived eDNA that is contained in the honey. [ABSTRACT FROM AUTHOR]

The article focuses on the construction of an infectious horsepox virus from synthesized DNA. It mentions that the study ease the development of smallpox virus (variola) using synthetic biology. It focuses on the benefits and risks of synthetic biology. It focuses on the construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments.

The article presents author's comments on the synthetic horsepox viruses with thedebate on dual use research. It focuses on the dual use research of concern (DURC), as the method that can be used to assemble horsepox virus can be used to develop variola which is the virus that causes smallpox. It focuses on recovering vaccinia virus (VACV) from a bacmid.

Cancers must maintain their telomeres at lengths sufficient for cell survival. In several cancer subtypes, a recombination-like mechanism termed alternative lengthening of telomeres (ALT), is frequently used for telomere length maintenance. Cancers utilizing ALT often have lost functional ATRX, a chromatin remodeling protein, through mutation or deletion, thereby strongly implicating ATRX as an ALT suppressor. Herein, we have generated functional ATRX knockouts in four telomerase-positive, ALT-negative human glioma cell lines: MOG-G-UVW, SF188, U-251 and UW479. After loss of ATRX, two of the four cell lines (U-251 and UW479) show multiple characteristics of ALT-positive cells, including ultrabright telomeric DNA foci, ALT-associated PML bodies, and c-circles. However, telomerase activity and overall telomere length heterogeneity are unaffected after ATRX loss, regardless of cellular context. The two cell lines that showed ALT hallmarks after complete ATRX loss also did so upon ATRX depletion via shRNA-mediated knockdown. These results suggest that other genomic or epigenetic events, in addition to ATRX loss, are necessary for the induction of ALT in human cancer. [ABSTRACT FROM AUTHOR]

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens -mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for Hyg R or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community. [ABSTRACT FROM AUTHOR]

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies. [ABSTRACT FROM AUTHOR]

NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. As the roles of these genes are intimately involved with glioblastoma multiforme progression and exosomes are critical in intercellular communication, we conducted a detailed analysis of the association of the NANOG gene family with exosomes to identify diagnostic markers for cancer. Exosomes were precipitated from conditioned culture media from various cell lines, and NANOG gene fragments were directly amplified without DNA isolation using multiple primer sets. The use of the enzymes AlwNI and SmaI with restriction fragment length polymorphism analysis functioned to distinguish NANOGP8 from other NANOG family members. Collectively, results suggest that the NANOG DNA associated with exosomes is not full length and that mixed populations of the NANOG gene family exist. Furthermore, sequence analysis of exosomal DNA amplified with a NANOGP8 specific primer set frequently showed an insertion of a 22 bp sequence into the 3’ UTR. The occurrence rate of this insertion was significantly higher in exosomal DNA clones from cancer cells as compared to normal cells. We have detected mixed populations of NANOG DNA associated with exosomes and have identified preferential modulations in the sequences from cancer samples. Our findings, coupled with the properties of exosomes, may allow for the detection of traditionally inaccessible cancers (i.e. GBM) through minimally invasive techniques. Further analysis of exosomal DNA sequences of NANOG and other embryonic stemness genes (OCT3/4, SOX2, etc.) may establish a robust collection of exosome based diagnostic markers, and further elucidate the mechanisms of cancer formation, progression, and metastasis. [ABSTRACT FROM AUTHOR]

High-resolution insight into parasitic infections and diet of past populations in Northern Europe and the Middle East (500 BC- 1700 AD) was obtained by pre-concentration of parasite eggs from ancient latrines and deposits followed by shotgun sequencing of DNA. Complementary profiling of parasite, vertebrate and plant DNA proved highly informative in the study of ancient health, human-animal interactions as well as animal and plant dietary components. Most prominent were finding of soil-borne parasites transmitted directly between humans, but also meat-borne parasites that require consumption of raw or undercooked fish and pork. The detection of parasites for which sheep, horse, dog, pig, and rodents serves as definitive hosts are clear markers of domestic and synanthropic animals living in closer proximity of the respective sites. Finally, the reconstruction of full mitochondrial parasite genomes from whipworm (Ascaris lumbricoides) and roundworm species (Trichuris trichiura and Trichuris muris) and estimates of haplotype frequencies elucidates the genetic diversity and provides insights into epidemiology and parasite biology. [ABSTRACT FROM AUTHOR]

Archaeogenomic research has proven to be a valuable tool to trace migrations of historic and prehistoric individuals and groups, whereas relationships within a group or burial site have not been investigated to a large extent. Knowing the genetic kinship of historic and prehistoric individuals would give important insights into social structures of ancient and historic cultures. Most archaeogenetic research concerning kinship has been restricted to uniparental markers, while studies using genome-wide information were mainly focused on comparisons between populations. Applications which infer the degree of relationship based on modern-day DNA information typically require diploid genotype data. Low concentration of endogenous DNA, fragmentation and other post-mortem damage to ancient DNA (aDNA) makes the application of such tools unfeasible for most archaeological samples. To infer family relationships for degraded samples, we developed the software READ (Relationship Estimation from Ancient DNA). We show that our heuristic approach can successfully infer up to second degree relationships with as little as 0.1x shotgun coverage per genome for pairs of individuals. We uncover previously unknown relationships among prehistoric individuals by applying READ to published aDNA data from several human remains excavated from different cultural contexts. In particular, we find a group of five closely related males from the same Corded Ware culture site in modern-day Germany, suggesting patrilocality, which highlights the possibility to uncover social structures of ancient populations by applying READ to genome-wide aDNA data. READ is publicly available from . [ABSTRACT FROM AUTHOR]

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5’-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration. [ABSTRACT FROM AUTHOR]

Edward Jenner and his contemporaries believed that his variolae vaccinae originated in horses and molecular analyses show that modern vaccinia virus (VACV) strains share common ancestry with horsepox virus (HPXV). Given concerns relating to the toxicity of modern VACV vaccines, we asked whether an HPXV-based vaccine might provide a superior alternative. Since HPXV may be extinct and the only specimen of HPXV that has been identified is unavailable for investigation, we explored whether HPXV could be obtained by large-scale gene synthesis. Ten large (10–30 kb) fragments of DNA were synthesized based on the HPXV sequence along with two 157 nt VACV terminal sequences, and were recombined into a live synthetic chimeric HPXV (scHPXV) in cells infected with Shope fibroma virus (SFV). Sequencing of the 212 kbp scHPXV confirmed it encoded a faithful copy of the input DNA. We believe this is the first complete synthesis of a poxvirus using synthetic biology approaches. This scHPXV produced smaller plaques, produced less extracellular virus and exhibited less virulence in mice than VACV, but still provided vaccine protection against a lethal VACV challenge. Collectively, these findings support further development of scHPXV as a novel replication-proficient smallpox vaccine. [ABSTRACT FROM AUTHOR]

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science. [ABSTRACT FROM AUTHOR]

Due to the recent advances in high-throughput sequencing technologies, it becomes possible to directly analyze microbial communities in human body and environment. To understand how microbial communities adapt, develop, and interact with the human body and the surrounding environment, one of the fundamental challenges is to infer the interactions among different microbes. However, due to the compositional and high-dimensional nature of microbial data, statistical inference cannot offer reliable results. Consequently, new approaches that can accurately and robustly estimate the associations (putative interactions) among microbes are needed to analyze such compositional and high-dimensional data. We propose a novel framework called Microbial Prior Lasso (MPLasso) which integrates graph learning algorithm with microbial co-occurrences and associations obtained from scientific literature by using automated text mining. We show that MPLasso outperforms existing models in terms of accuracy, microbial network recovery rate, and reproducibility. Furthermore, the association networks we obtain from the Human Microbiome Project datasets show credible results when compared against laboratory data. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4–110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(−). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(−) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts. [ABSTRACT FROM AUTHOR]

Burkholderia spp. are genetically and physiologically diverse. Some strains are naturally transformable and capable of DNA catabolism. Burkholderia pseudomallei (Bp) strains 1026b and K96243 and B. thailandensis strain E264 are able to utilize DNA as a sole carbon source for growth, while only strains 1026b and E264 are naturally transformable. In this study, we constructed low-copy broad-host-range fosmid library, containing Bp strain 1026b chromosomal DNA fragments, and employed a novel positive selection approach to identify genes responsible for DNA uptake and DNA catabolism. The library was transferred to non-competent Bp K96243 and B. cenocepacia (Bc) K56-2, harboring chromosomally-inserted FRT-flanked sacB and pheS counter-selection markers. The library was incubated with DNA encoding Flp recombinase, followed by counter-selection on sucrose and chlorinated phenylalanine, to select for clones that took up flp-DNA, transiently expressed Flp, and excised the sacB-pheS cassette. Putative clones that survived the counter-selection were subsequently incubated with gfp-DNA and bacteria were visualized via fluorescent microscopy to confirm natural competency. Fosmid sequencing identified several 1026b genes implicated in DNA uptake, which were validated using chromosomal mutants. One of the naturally competent clones selected in Bc K56-2 enabled Bc, Bp and B. mallei to utilize DNA as a sole carbon source, and all fosmids were used to successfully create mutations in non-naturally-competent B. mallei and Bp strains. [ABSTRACT FROM AUTHOR]

The human gut has been continuously exposed to a broad spectrum of intestinal organisms, including viruses, bacteria, fungi, and parasites (protozoa and worms), over millions of years of coevolution, and plays a central role in human health. The modern lifestyles of Western countries, such as the adoption of highly hygienic habits, the extensive use of antimicrobial drugs, and increasing globalisation, have dramatically altered the composition of the gut milieu, especially in terms of its eukaryotic “citizens.” In the past few decades, numerous studies have highlighted the composition and role of human intestinal bacteria in physiological and pathological conditions, while few investigations exist on gut parasites and particularly on their coexistence and interaction with the intestinal microbiota. Studies of the gut “parasitome” through “omic” technologies, such as (meta)genomics, transcriptomics, proteomics, and metabolomics, are herein reviewed to better understand their role in the relationships between intestinal parasites, host, and resident prokaryotes, whether pathogens or commensals. Systems biology–based profiles of the gut “parasitome” under physiological and severe disease conditions can indeed contribute to the control of infectious diseases and offer a new perspective of omics-assisted tropical medicine. [ABSTRACT FROM AUTHOR]

Merging paired-end shotgun reads generated on high-throughput sequencing platforms can substantially improve various subsequent bioinformatics processes, including genome assembly, binning, mapping, annotation, and clustering for taxonomic analysis. With the inexorable growth of sequence data volume and CPU core counts, the speed and scalability of read-processing tools becomes ever-more important. The accuracy of shotgun read merging is crucial as well, as errors introduced by incorrect merging percolate through to reduce the quality of downstream analysis. Thus, we designed a new tool to maximize accuracy and minimize processing time, allowing the use of read merging on larger datasets, and in analyses highly sensitive to errors. We present BBMerge, a new merging tool for paired-end shotgun sequence data. We benchmark BBMerge by comparison with eight other widely used merging tools, assessing speed, accuracy and scalability. Evaluations of both synthetic and real-world datasets demonstrate that BBMerge produces merged shotgun reads with greater accuracy and at higher speed than any existing merging tool examined. BBMerge also provides the ability to merge non-overlapping shotgun read pairs by using k-mer frequency information to assemble the unsequenced gap between reads, achieving a significantly higher merge rate while maintaining or increasing accuracy. [ABSTRACT FROM AUTHOR]

The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent in vivo recombination of DNA fragments with overlapping ends using E. coli. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors–the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids–which can provide general guidance for selecting in vivo cloning parameters. The method may be used to accurately assemble up to 5 DNA fragments with 25 nt overlapping ends into relatively small plasmids, and 3 DNA fragments into plasmids up to 16 kb in size. The whole cloning procedure may be completed within 2 days by a researcher with little training in cloning. The combination of high accuracy and zero background eliminates the need for screening a large number of colonies. The method requires no enzymes other than Q5 DNA polymerase, has no sequence restriction, is highly reliable, and represents one of the simplest, fastest, and cheapest cloning techniques available. Our method is particularly suitable for common cloning tasks in the lab where the primary goal is to quickly generate a plasmid with a pre-defined sequence at low costs. [ABSTRACT FROM AUTHOR]

Immunocompromised individuals are at high risk of developing Clostridium difficile-associated disease (CDAD). Fecal microbiota transplantation (FMT) is a highly effective therapy for refractory or recurrent CDAD and, despite safety concerns, has recently been offered to immunocompromised patients. We investigated the genomics of bacterial composition following FMT in immunocompromised patients over a 1-year period. Metagenomic, strain and gene-level bacterial dynamics were characterized in two CDAD-affected hematopoietic stem cell (HCT) recipients following FMT. We found alterations in gene content, including loss of virulence and antibiotic resistance genes. These alterations were accompanied by long-term bacterial divergence at the species and strain levels. Our findings suggest limited durability of the specific bacterial consortium introduced with FMT and indicate that alterations of the functional potential of the microbiome are more complex than can be inferred by taxonomic information alone. Our observation that FMT alone cannot induce long-term donor-like alterations of the microbiota of HCT recipients suggests that FMT cannot indefinitely supersede environmental and/or host factors in shaping bacterial composition. [ABSTRACT FROM AUTHOR]

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose ‘Candidatus Maribrachyspira akoyae’ gen. nov, sp nov., for this bacterium. [ABSTRACT FROM AUTHOR]

Mycoplasma hyopneumoniae is the etiologic agent of swine enzootic pneumonia. However other mycoplasma species and secondary bacteria are found as inhabitants of the swine respiratory tract, which can be also related to disease. In the present study we have performed a total DNA metagenomic analysis from the lungs of pigs kept in a field condition, with suggestive signals of enzootic pneumonia and without any infection signals to evaluate the bacteria variability of the lungs microbiota. Libraries from metagenomic DNA were prepared and sequenced using total DNA shotgun metagenomic pyrosequencing. The metagenomic distribution showed a great abundance of bacteria. The most common microbial families identified from pneumonic swine’s lungs were Mycoplasmataceae, Flavobacteriaceae and Pasteurellaceae, whereas in the carrier swine’s lungs the most common families were Mycoplasmataceae, Bradyrhizobiaceae and Flavobacteriaceae. Analysis of community composition in both samples confirmed the high prevalence of M. hyopneumoniae. Moreover, the carrier lungs had more diverse family population, which should be related to the lungs normal flora. In summary, we provide a wide view of the bacterial population from lungs with signals of enzootic pneumonia and lungs without signals of enzootic pneumonia in a field situation. These bacteria patterns provide information that may be important for the establishment of disease control measures and to give insights for further studies. [ABSTRACT FROM AUTHOR]

Cistanthe longiscapa is an endemic annual herb and characteristic element of the Chilean Atacama Desert. Principal threats are the destruction of its seed deposits by human activities and reduced germination rates due to the decreasing occurrence of precipitation events. To enable population genetic and phylogeographic analyses in this species we performed paired-end shotgun sequencing (2x100 bp) of genomic DNA on the Illumina HiSeq platform and identified microsatellite (SSR) loci in the resulting sequences. From 29 million quality-filtered read pairs we obtained 549,174 contigs (average length 614 bp; N50 = 904). Searching for SSRs revealed 10,336 loci with microsatellite motifs. Initially, we designed primers for 96 loci, which were tested for PCR amplification on three C. longiscapa individuals. Successfully amplifying loci were further tested on eight individuals to screen for length variation in the resulting amplicons, and the alleles were exemplarily sequenced to infer the basis for the observed length variation. Finally we arrived at 26 validated SSR loci for population studies in C. longiscapa, which resulted in 146 bi-allelic SSR markers in our test sample of eight individuals. The genomic sequences were also used to assemble the plastid genome of C. longiscapa, which provides an additional set of maternally inherited genetic markers. [ABSTRACT FROM AUTHOR]

The design and generation of an optimal expression construct is the first and essential step in in the characterization of a protein of interest. Besides evaluation and optimization of process parameters (e.g. selection of the best expression host or cell line and optimal induction conditions and time points), the design of the expression construct itself has a major impact. However, the path to this final expression construct is often not straight forward and includes multiple learning cycles accompanied by design variations and retesting of construct variants, since multiple, functional DNA sequences of the expression vector backbone, either coding or non-coding, can have a major impact on expression yields. To streamline the generation of defined expression constructs of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed. This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules with a minimal cloning burden. Combining these features with a standardized cloning strategy facilitates the identification of optimized DNA expression constructs in shorter time. The MoPET system currently consists of 53 defined DNA modules divided into eight functional classes and can be flexibly expanded. However, already with the initial set of modules, 792,000 different constructs can be rationally designed and assembled. Furthermore, this starting set was used to generate small and mid-sized combinatorial expression optimization libraries. Applying this screening approach, variants with up to 60-fold expression improvement have been identified by MoPET variant library screening. [ABSTRACT FROM AUTHOR]

Background: Paper currency by its very nature is frequently transferred from one person to another and represents an important medium for human contact with—and potential exchange of—microbes. In this pilot study, we swabbed circulating $1 bills obtained from a New York City bank in February (Winter) and June (Summer) 2013 and used shotgun metagenomic sequencing to profile the communities found on their surface. Using basic culture conditions, we also tested whether viable microbes could be recovered from bills. Results: Shotgun metagenomics identified eukaryotes as the most abundant sequences on money, followed by bacteria, viruses and archaea. Eukaryotic assemblages were dominated by human, other metazoan and fungal taxa. The currency investigated harbored a diverse microbial population that was dominated by human skin and oral commensals, including Propionibacterium acnes, Staphylococcus epidermidis and Micrococcus luteus. Other taxa detected not associated with humans included Lactococcus lactis and Streptococcus thermophilus, microbes typically associated with dairy production and fermentation. Culturing results indicated that viable microbes can be isolated from paper currency. Conclusions: We conducted the first metagenomic characterization of the surface of paper money in the United States, establishing a baseline for microbes found on $1 bills circulating in New York City. Our results suggest that money amalgamates DNA from sources inhabiting the human microbiome, food, and other environmental inputs, some of which can be recovered as viable organisms. These monetary communities may be maintained through contact with human skin, and DNA obtained from money may provide a record of human behavior and health. Understanding these microbial profiles is especially relevant to public health as money could potentially mediate interpersonal transfer of microbes. [ABSTRACT FROM AUTHOR]

Background/Aims: Studying the gut microbiota in unaffected relatives of people with Crohn’s disease (CD) may advance our understanding of the role of bacteria in disease aetiology. Methods: Faecal microbiota composition (16S rRNA gene sequencing), genetic functional capacity (shotgun metagenomics) and faecal short chain fatty acids (SCFA) were compared in unaffected adult relatives of CD children (CDR, n = 17) and adult healthy controls, unrelated to CD patients (HUC, n = 14). The microbiota characteristics of 19 CD children were used as a benchmark of CD ‘dysbiosis’. Results: The CDR microbiota was less diverse (p = 0.044) than that of the HUC group. Local contribution of β-diversity analysis showed no difference in community structure between the CDR and HUC groups. Twenty one of 1,243 (1.8%) operational taxonomic units discriminated CDR from HUC. The metagenomic functional capacity (p = 0.207) and SCFA concentration or pattern were similar between CDR and HUC (p>0.05 for all SCFA). None of the KEGG metabolic pathways were different between these two groups. Both of these groups (HUC and CDR) had a higher microbiota α-diversity (CDR, p = 0.026 and HUC, p<0.001) with a community structure (β-diversity) distinct from that of children with CD. Conclusions: While some alterations were observed, a distinct microbial ‘dysbiosis’, characteristic of CD patients, was not observed in their unaffected, genetically linked kindred. [ABSTRACT FROM AUTHOR]