Reversible Phospholipid Nanogels: An Alternative Matrix for Tunable High Resolution DNA Sieving with Enhanced Separation Efficiency and Accurate Extended Range Sizing by Capillary Gel Electrophoresis

In an aqueous solution the phospholipids dimyristoyl-sn-glycero-3- phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble to form thermo-responsive non-Newtonian fluids (i.e., pseudo-gels) in which small temperature changes of 5-6 °C decrease viscosity dramatically. This characteristic is useful for sieving-based electrophoretic separations (e.g., of DNA), as the high viscosity of linear sieving additives, such as linear polyacrylamide or polyethylene oxide, hinders the introduction and replacement of the sieving agent in microscale channels. While a practical advantage of utilizing phospholipid pseudo-gels for sieving is the ease with which they are introduced into the separation capillary, the separation performance of the material for DNA analyses is exceptional. Capillary electrophoresis separations of DNA are achieved with separation efficiencies ranging from 400,000 to 7,000,000 theoretical plates in a 25 micrometer inner diameter fused silica capillary. Assessment of the phospholipid nanogel with a Ferguson plot yields an apparent pore size of ~31 nm. Under isothermal conditions, Ogston sieving is achieved for DNA fragments smaller than 500 base pairs, whereas reptation-based transport occurs for DNA fragments larger than 500 base pairs. Single base resolution of short tandem repeats relevant to human identification is accomplished with 30 minute separations using traditional capillary electrophoresis instrumentation. Applications that do not require single base resolution are completed with faster separation times. This is demonstrated for a multiplex assay of biallelic single nucleotide polymorphisms relevant to warfarin sensitivity. The thermo-responsive pseudo-gel preparation described here provides a new innovation to sieving based capillary separations. Specific DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1,500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1,500 base pairs. This separation additive is used to evaluate size markers ranging between 200 and 1,500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these selfassembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency

A Thermally Responsive Phospholipid Pseudogel: Tunable DNA Sieving with Capillary Electrophoresis

In an aqueous solution the phospholipids dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-<i>sn</i>-glycero-3-phosphocholine (DHPC) self-assemble to form thermo-responsive non-Newtonian fluids (i.e., pseudogels) in which small temperature changes of 5–6 °C decrease viscosity dramatically. This characteristic is useful for sieving-based electrophoretic separations (e.g., of DNA), as the high viscosity of linear sieving additives, such as linear polyacrylamide or polyethylene oxide, hinders the introduction and replacement of the sieving agent in microscale channels. Advantages of utilizing phospholipid pseudogels for sieving are the ease with which they are introduced into the separation channel and the potential to implement gradient separations. Capillary electrophoresis separations of DNA are achieved with separation efficiencies ranging from 400,000 to 7,000,000 theoretical plates in a 25 μm i.d. fused silica capillary. Assessment of the phospholipid pseudogel with a Ferguson plot yields an apparent pore size of ∼31 nm. Under isothermal conditions, Ogston sieving is achieved for DNA fragments smaller than 500 base pairs, whereas reptation-based transport occurs for DNA fragments larger than 500 base pairs. Nearly single base resolution of short tandem repeats relevant to human identification is accomplished with 30 min separations using traditional capillary electrophoresis instrumentation. Applications that do not require single base resolution are completed with faster separation times. This is demonstrated for a multiplex assay of biallelic single nucleotide polymorphisms relevant to warfarin sensitivity. The thermo-responsive pseudogel preparation described here provides a new innovation to sieving-based capillary separations

A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection

Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer’s exact test and Cramer’s V2 values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated buclgenes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates

A Unique Set of the <i>Burkholderia</i> Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection

<div><p><i>Burkholderia pseudomallei</i> and <i>Burkholderia mallei</i>, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by <i>Burkholderia</i> are not fully understood, and correct diagnosis of <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>mallei</i> infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel <i>Burkholderia</i> collagen-like proteins (Bucl) that were identified among <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>mallei</i> select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that <i>bucl</i> genes are widespread in <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>mallei</i>; Fischer’s exact test and Cramer’s V² values indicate that the majority of <i>bucl</i> genes are highly associated with these pathogenic species versus nonpathogenic <i>B</i>. <i>thailandensis</i>. We designed a <i>bucl</i>-based quantitative PCR assay which was able to detect <i>B</i>. <i>pseudomallei</i> infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of <i>bucl</i> loci revealed considerable genomic plasticity and adaptation of <i>Burkholderia</i> spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated <i>bucl</i> genes commonly found in <i>Burkholderia</i> select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates.</p></div

Tissue engineered autologous cartilage-bone grafts for temporomandibular joint regeneration

Joint disorders can be detrimental to quality of life. There is an unmet need for precise functional reconstruction of native-like cartilage and bone tissues in the craniofacial space and particularly for the temporomandibular joint (TMJ). Current surgical methods suffer from lack of precision and comorbidities and frequently involve multiple operations. Studies have sought to improve craniofacial bone grafts without addressing the cartilage, which is essential to TMJ function. For the human-sized TMJ in the Yucatan minipig model, we engineered autologous, biologically, and anatomically matched cartilage-bone grafts for repairing the ramus-condyle unit (RCU), a geometrically intricate structure subjected to complex loading forces. Using image-guided micromilling, anatomically precise scaffolds were created from decellularized bone matrix and infused with autologous adipose-derived chondrogenic and osteogenic progenitor cells. The resulting constructs were cultured in a dual perfusion bioreactor for 5 weeks before implantation. Six months after implantation, the bioengineered RCUs maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a greater extent than either autologous bone-only engineered grafts or acellular scaffolds. Tracking of implanted cells and parallel bioreactor studies enabled additional insights into the progression of cartilage and bone regeneration. This study demonstrates the feasibility of TMJ regeneration using anatomically precise, autologous, living cartilage-bone grafts for functional, personalized total joint replacement. Inclusion of the adjacent tissues such as soft connective tissues and the TMJ disc could further extend the functional integration of engineered RCUs with the host

Phylogenetic analysis of <i>B</i>. <i>pseudomallei</i>, <i>B</i>. <i>mallei</i>, and <i>B</i>. <i>thailandensis</i> strains using individual <i>bucl3</i> and <i>bucl4</i> genes.

<p>Bayesian analysis was performed on nucleotide sequences of non-collagenous regions of a set of <i>Burkholderia</i> strains described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137578#pone.0137578.t003" target="_blank">Table 3</a>. Support values for each branch are shown as posterior probability from Bayesian analysis and bootstrap values from maximum parsimony analysis, respectively (PP/MP). Posterior probability values not supported by parsimony analysis are shown in red. Scale bar is representative of evolutionary distance in substitutions per nucleotide. Several clusters of strains corresponding to those observed in the concatenated analysis, C1-C7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137578#pone.0137578.g005" target="_blank">Fig 5</a>, were also observed in the individual trees.</p

Characterization of Bucl proteins in <i>Burkholderia</i>^a.

<p>^a Characteristics of Bucl proteins are shown based on analysis of completed genomes of 13 Bp, 11 Bm, and 9 Bt strains (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137578#pone.0137578.t003" target="_blank">Table 3</a>). The total protein length and length of protein sequences that are amino- and carboxyl-terminal to CL regions in each Bucl protein is shown as amino acid number, whereas the length of each CL region, which varies between strains, is expressed as the number of GXY repeats. Predominant GXY repeats are represented in bold text. Putative domains in the noncollagenous regions of each Bucl are shown: Talin-1 domain; Bac_export_1, bacterial export protein family 1; OEP, outer membrane efflux protein; and SBP_bac_3, bacterial extracellular solute-binding protein family 3.</p><p>^b SS; Signal sequence predictions are based on hidden Markov model predictions in the SignalP 3.0 server.</p><p>^c TM; Transmembrane domain predictions were made using TMPred.</p><p>Characterization of Bucl proteins in <i>Burkholderia</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137578#t002fn001" target="_blank">^a</a>.</p

Assessment of genomic plasticity of <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>mallei</i> using biomarkers.

<p>^a Organizational patterns (OP) of <i>bucl</i> genes were assigned to each chromosome, Ch1 and Ch2, according to position and orientation. OPs were labeled I-VI for chromosome 1, and I-III for chromosome 2. <i>bucl</i> position on the plus or minus strand is shown corresponding to each OP.</p><p>Assessment of genomic plasticity of <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>mallei</i> using biomarkers.</p