Xanthomonas euvesicatoria pv. rosa strain Xer07 causes a leaf spot on a Rosa sp. and is closely related to X. euvesicatoria pv. euvesicatoria (Xee) and X. perforans (Xp), causal agents of bacterial spot of tomato. However, Xer07 is not pathogenic on tomato and elicits a hypersensitive reaction (HR). We compared the genomes of the three bacterial species to identify the factors that limit Xer07 on tomato. Comparison of pathogenicity associated factors including the type III secretion systems identified two genes, xopA and xer3856, in Xer07 that have lower sequence homology in tomato pathogens. xer3856 is a homolog of genes in X. citri (xac3856) and X. fuscans pv. aurantifolii, both of which have been reported to elicit HRs in tomato. When xer3856 was expressed in X. perforans and infiltrated in tomato leaflets, the transconjugant elicited an HR and significantly reduced bacterial populations compared to the wildtype X. perforans strain. When xer3856 was mutated in Xer07, the mutant strain still triggered an HR in tomato leaflets. The second gene identified codes for type III secreted effector XopA, which contains a harpin domain that is distinct from the xopA homologs in Xee and Xp. The Xer07-xopA, when expressed in X. perforans, did not elicit an HR in tomato leaflets, but significantly reduced bacterial populations. This indicates that xopA and xer3856 genes in combination with an additional factor(s) limit Xer07 in tomato.

Internal dynamics of proteins can play a critical role in the biological function of some proteins. Several well documented instances have been reported such as MBP, DHFR, hTS, DGCR8, and NSP1 of the SARS-CoV family of viruses. Despite the importance of internal dynamics of proteins, there currently are very few approaches that allow for meaningful separation of internal dynamics from structural aspects using experimental data. Here we present a computational approach named REDCRAFT that allows for concurrent characterization of protein structure and dynamics. Here, we have subjected DHFR (PDB-ID 1RX2), a 159-residue protein, to a fictitious, mixed mode model of internal dynamics. In this simulation, DHFR was segmented into 7 regions where 4 of the fragments were fixed with respect to each other, two regions underwent rigid-body dynamics, and one region experienced uncorrelated and melting event. The two dynamical and rigid-body segments experienced an average orientational modification of 7 degrees and 12 degrees respectively. Observable RDC data for backbone C '-N, N-H-N, and C '-H-N were generated from 102 uniformly sampled frames that described the molecular trajectory. The structure calculation of DHFR with REDCRAFT by using traditional Ramachandran restraint produced a structure with 29 angstrom of structural difference measured over the backbone atoms (bb-rmsd) over the entire length of the protein and an average bb-rmsd of more than 4.7 angstrom over each of the dynamical fragments. The same exercise repeated with context-specific dihedral restraints generated by PDBMine produced a structure with bb-rmsd of 21 angstrom over the entire length of the protein but with bb-rmsd of less than 3 angstrom over each of the fragments. Finally, utilization of the Dynamic Profile generated by REDCRAFT allowed for the identification of different dynamical regions of the protein and the recovery of individual fragments with bb-rmsd of less than 1 angstrom. Following the recovery of the fragments, our assembly procedure of domains (larger segments consisting of multiple fragments with a common dynamical profile) correctly assembled the four fragments that are rigid with respect to each other, categorized the two domains that underwent rigid-body dynamics, and identified one dynamical region for which no conserved structure could be defined. In conclusion, our approach was successful in identifying the dynamical domains, recovery of structure where it is meaningful, and relative assembly of the domains when possible.

Extracellular Vesicles, also referred to as EVs, are spherical lipid membrane-bound vesicles produced by both Gram positive and Gram negative bacteria. These vesicles are secreted into the extracellular space and play important roles in cellular and host communication, elimination of competitors, virulence, detoxification of environmental stress, and nutrient sensing. They are often packed with proteins, enzymes, lipids, nucleic acids and other biomolecules. Streptococcus thermophilus is a lactic acid bacteria (LAB), inhabiting the human digestive tract, that has been shown to produce EVs. The bacterial florae are known to influence the host immune system, metabolism, and neurological processes, but little is known about the biochemical pathways involved. Since EVs are involved in host communication, they may play a key role in affecting the biochemical processes of the host. To study the content and potential effects of EVs, we have grown different LAB strains under anaerobic conditions, including S. thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus. Our goal is to isolate EVs from these LAB strains and identify small regulatory RNA (sRNA) molecules that mediate communication and biochemical effects on the host systems. As part of this study, we have begun to identify sRNA genes in LAB strains that may mediate communication with other bacteria or the host. In S. thermophilus, we have focused on the AsdS sRNA transcript, that is 152 base pairs in length, and is involved in quorum sensing. This gene is conserved among other streptococcal species and in the human pathogen, S. pyogenes, a homologous sRNA, known as MarS, is associated with the regulation of virulence factors. Since S. thermophilus is a non-pathogenic species, the AsdS gene cannot be involved in virulence, but based on functional bioinformatics predictions, it may be responsible for intraspecies communication, biofilm formation, and transport processes. Our current studies aim to identify additional sRNA transcripts that may be found in EVs and mediate effects on the host systems. © FASEB.

Streptococcus pyogenes is a human pathogen that is also known as group A Streptococcus (GAS), which is responsible for the development of sore throats and skin infections as well as the more severe condition, necrotizing fasciitis. The streptolysin associated gene A, sagA, codes for the protein streptolysin A (SagA), which is modified and exported as the cytotoxin streptolysin S (SLS). SLS is a virulence factor that inhibits the response of neutrophils during necrotizing fasciitis. The sagA gene is contained within the pleiotropic effect locus (pel). Previous studies have identified sagA as both the gene for the streptolysin S peptide, and the small regulatory RNA (sRNA), known as Pel, which influences expression of multiple virulence genes. The 5' UTR of sagA/Pel contains the regions necessary for both transcription initiation and ribosomal binding for translation. Focusing on the 5' UTR of sagA, we hypothesize that the RNA structure of this region affects the transcription of sagA and the initiation of translation. Our goal is to characterize the secondary and 3D structure of the sagA/Pel mRNA/sRNA, which will allow for a better understanding for the role it plays in streptococcal pathogenesis. In this study, RNA constructs containing predicted structured regions were produced by in vitro transcription. Secondary structures were predicted using the mfold and RNAfold webserver based programs. The predicted secondary structures were used to generate three-dimensional models with the program FARFAR through the ROSIE webserver. Differential scanning fluorimetry was also used to assess the structural stability of the RNA constructs and to screen a variety of conditions to be used in crystallization studies. These experiments provided evidence that the 5' UTR of sagA/Pel contains significant structured regions and optimal conditions were identified for future structural studies. Potential interactions between the sagA/Pel sRNA and regulated mRNA targets were predicted using the TargetRNA2 webserver. The potential sites for recognition were used to evaluate the functional significance of the structured regions within the 5' UTR of sagA/Pel. Identification of structural motifs necessary for regulating the expression of virulence genes will aid in the design of therapeutic strategies to inhibit the production of the virulence factors, such as SLS. In future studies, we plan to use structural techniques such as NMR spectroscopy and X-ray crystallography to help further understand the role that sagA/Pel plays in streptococcal diseases. © FASEB.

Adeno-associated virus vectors are the most used delivery method for liver-directed gene editing. Still, they are associated with significant disadvantages that can compromise the safety and efficacy of therapies. Here, we investigate the effects of electroporating CRISPR-Cas9 as mRNA and ribonucleoproteins (RNPs) into primary hepatocytes regarding on-target activity, specificity, and cell viability. We observed a transfection efficiency of >60% and on-target insertions/deletions (indels) of up to 95% in primary mouse hepatocytes electroporated with Cas9 RNPs targeting Hpd, the gene encoding hydroxyphenylpyruvate dioxygenase. In primary human hepatocytes, we observed on-target indels of 52.4% with Cas9 RNPs and >65% viability after electroporation. These results establish the impact of using electroporation to deliver Cas9 RNPs into primary hepatocytes as a highly efficient and potentially safe approach for therapeutic liver-directed gene editing and the production of liver disease models.

Background: Recognizing insufficient support for healthy eating and physical activity in early childhood education centers in Greenville, South Carolina, a group of stakeholders formed a workgroup as an organizing structure. Members developed and implemented a 2-year community-based participatory research initiative aimed at nutrition and physical activity policy, systems and environment change in 10 early childhood education centers.Objectives: This article 1) describes engagement efforts and partnerships leading to formation of the workgroup and initiative, 2) presents data on Workgroup members' knowledge and engagement, and 3) shares lessons learned.Methods: Workgroup member knowledge and engagement related to obesity prevention was measured at two time points during the ECE initiative using the "Stakeholder-driven Community Diffusion Survey."Lessons Learned: Knowledge and engagement scores increased over the measurement period. Scores for engagement were higher than scores for knowledge at both time points. There was a substantial increase in perceived leadership and stewardship, knowledge of intervention factors and how to intervene sustainably, and understanding of local resources and roles. An important strength was stakeholder buy-in and ownership of planning and implementation processes.

Objectives: Obesity is a major epidemic in our population and has emerged as a primary health concern. Consumption of a high fat, high sugar (HFHS) diet can specifically lead to gut dysbiosis, increased inflammation, and neuroinflammation. Interestingly, sex differences in the response to a HFHS diet are emerging. In this study, we investigated the effects of a HFHS diet compared to a low fat, low sugar (LFLS) diet in 8 week old male and female C57Bl/6 mice. Methods: The diet was administered for 14 weeks; body weights and food consumption were evaluated weekly. Results: Male and female mice fed the HFHS diet gained significantly more weight than LFLS-fed mice. However, in agreement with previous studies, males gained significantly more weight on the HFHS diet compared to females fed the same diet. Importantly, we determined significant sex and diet-induced differences to gut microbiome composition using next generation Illumina sequencing. We also observed significantly less astrocyte densitometry and no significant change to microglial morphology in the hypothalamus of Female HFHS compared to Female LFLS. On the other hand, Male HFHS revealed no change to hypothalamic astrogliosis, but increased microgliosis compared to Male LFLS. Discussion: In this study, we determined sex and diet-induced differences in both the gut and the brain, however, future studies will need to be performed in order to test the direct role of the gut microbiome to weight gain and neuroinflammation in male and female mice.

The auditory nerve (AN) of the inner ear is the primary conveyor of acoustic information from sensory hair cells to the brainstem. Approximately 95% of peripheral AN fibers are myelinated by glial cells. The integrity of myelin and the glial-associated paranodal structures at the node of Ranvier is critical for normal AN activity and axonal survival and function in the central auditory nervous system. However, little is known about the node of Ranvier's spatiotemporal development in the AN, how the aging process (or injury) affects the activity of myelinating glial cells, and how downstream alterations in myelin and paranodal structure contribute to AN degeneration and sensorineural hearing loss. Here, we characterized two types of Ranvier nodes-the axonal node and the ganglion node-in the mouse peripheral AN, and found that they are distinct in several features of postnatal myelination and age-related degeneration. Cellular, molecular, and structure-function correlations revealed that the two node types are each critical for different aspects of peripheral AN function. Neural processing speed and synchrony is associated with the length of the axonal node, while stimulus level-dependent amplitude growth and action potentials are associated with the ganglion node. Moreover, our data indicate that dysregulation of glial cells (e.g., satellite cells) and degeneration of the ganglion node structure are an important new mechanism of age-related hearing loss.

In this paper, we study the dynamics of HIV under gene therapy and latency reversing agents. While previous works modeled either the use of gene therapy or latency reversing agents, we consider the effects of a combination treatment strategy. For constant treatment controls, we establish global stability of the disease-free equilibrium and endemic equilibrium based on the value of R0. We then consider time-dependent controls and formulate an associated optimal control problem that emphasizes reduction of the latent reservoir. Characterizations for the optimal control profiles are found using Pontryagin's Maximum Principle. We perform numerical simulations of the optimal control model using the fourth-order Runge-Kutta forward-backward sweep method. We find that a combination treatment of gene therapy with latency reversing agents provides better remission times than gene therapy alone. We conclude with a discussion of our findings and future work.