Whole-genome sequences of Campylobacter coli and Campylobacter jejuni isolates from rhesus macaques (Macaca mulatta) with and without intestinal disease.

Campylobacter jejuni or Campylobacter coli infection can lead to post-infectious irritable bowel syndrome in humans and may produce a similar syndrome in rhesus macaques (Macaca mulatta). We report the complete genomes of 8 C. jejuni isolates and 103 C. coli isolates obtained from rhesus macaques with and without intestinal disease.

Analysis of cell populations in the normal rhesus macaque (Macaca mulatta) lower intestinal tract and diagnostic thresholds for chronic enterocolitis.

Rhesus macaques (Macaca mulatta) are used extensively in biomedical research, often with a focus on the gastrointestinal tract, and yet a full characterization of their normal resident intestinal cell populations has not been published. In addition, chronic enterocolitis (CE), also known as idiopathic chronic diarrhea, affects up to 25% of colony-housed rhesus macaques, often requiring euthanasia for welfare concerns and severely limiting their value as a breeding animal or research subject. We aimed to characterize subjective and objective variables in sections of the ileum, cecum, colon, and rectum in 16 healthy rhesus macaques and compare these results with a cohort of 37 animals euthanized for CE to produce relevant diagnostic thresholds and to improve case definitions for future studies. We found neutrophils to be an infrequent but expected component of the large intestinal leukocyte population. Animals with CE had significantly increased total leukocyte populations between crypts in the cecum, colon, and rectum; variable increases in specific cell populations across all levels of the distal intestinal tract; and significantly increased intraepithelial CD3+ T cells in the colon and rectum. Concentrations of enteroendocrine cells, enterochromaffin cells, and intestinal mast cells were not significantly different between healthy and affected individuals. This study characterizes individual leukocyte populations in the rhesus macaque lower intestinal tract, is the first to evaluate rhesus macaque intestinal mast cells, and provides key diagnostic thresholds for evaluating animals with potential CE.

Transformation and regeneration of DNA polymerase Θ mutant rice plants.

Agrobacterium T-DNA integration into the plant genome is essential for the process of transgenesis and is widely used for genome engineering. The importance of the non-homologous end-joining (NHEJ) protein DNA polymerase Θ, encoded by the PolQ gene, for T-DNA integration is controversial, with some groups claiming it is essential whereas others claim T-DNA integration in Arabidopsis and rice polQ mutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenic polQ mutant plants. We describe a protocol for regenerating transgenic polQ mutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species.

Characterization of T-Circles and Their Formation Reveal Similarities to Agrobacterium T-DNA Integration Patterns.

Agrobacterium transfers T-DNA to plants where it may integrate into the genome. Non-homologous end-joining (NHEJ) has been invoked as the mechanism of T-DNA integration, but the role of various NHEJ proteins remains controversial. Genetic evidence for the role of NHEJ in T-DNA integration has yielded conflicting results. We propose to investigate the formation of T-circles as a proxy for understanding T-DNA integration. T-circles are circular double-strand T-DNA molecules, joined at their left (LB) and right (RB) border regions, formed in plants. We characterized LB-RB junction regions from hundreds of T-circles formed in Nicotiana benthamiana or Arabidopsis thaliana. These junctions resembled T-DNA/plant DNA junctions found in integrated T-DNA: Among complex T-circles composed of multiple T-DNA molecules, RB-RB/LB-LB junctions predominated over RB-LB junctions; deletions at the LB were more frequent and extensive than those at the RB; microhomology was frequently used at junction sites; and filler DNA, from the plant genome or various Agrobacterium replicons, was often present between the borders. Ku80 was not required for efficient T-circle formation, and a VirD2 ω mutation affected T-circle formation and T-DNA integration similarly. We suggest that investigating the formation of T-circles may serve as a surrogate for understanding T-DNA integration.

Comparative molecular genomic analyses of a spontaneous rhesus macaque model of mismatch repair-deficient colorectal cancer.

Colorectal cancer (CRC) remains the third most common cancer in the US with 15% of cases displaying Microsatellite Instability (MSI) secondary to Lynch Syndrome (LS) or somatic hypermethylation of the MLH1 promoter. A cohort of rhesus macaques from our institution developed spontaneous mismatch repair deficient (MMRd) CRC with a notable fraction harboring a pathogenic germline mutation in MLH1 (c.1029C

Vasocutaneous fistula formation and repair following inguinal hernia repair in a rhesus monkey (Macaca mulatta).

A 6-year-old adult male rhesus macaque (Macaca mulatta) developed a vasocutaneous fistula following an anatomic inguinal hernia repair years earlier. The vasocutaneous fistula was surgically repaired, the vas deferens was ligated, and the wound was closed in layers with non-overlapping suture lines with no further adverse sequalae of events.

Comprehensive diagnostic catheterization in children with major aortopulmonary collateral arteries: A review of catheterization technique and anatomic nomenclature.

Achieving an optimal surgical result in patients with major aortopulmonary collateral arteries (MAPCAs) requires a thorough preoperative evaluation of the anatomy and physiology of the pulmonary circulation. This review provides a detailed description of diagnostic catheterization in patients with MAPCAs, including a summary of catheterization techniques, an overview of commonly used terms, and a review of MAPCA and pulmonary artery angiographic anatomy.

Plant DNA Repair and Agrobacterium T-DNA Integration.

Agrobacterium species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T-DNA often contains, at its junctions with plant DNA, deletions of T-DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T-DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T-DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T-DNA integration into the plant genome.

Agrobacterium VirE2 Protein Modulates Plant Gene Expression and Mediates Transformation From Its Location Outside the Nucleus.

Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation.

Balloon Angioplasty for Pulmonary Artery Stenosis After Complete Unifocalization and Repair of Tetralogy of Fallot With Major Aortopulmonary Collaterals.

OBJECTIVES: The objective of this study was to assess procedural outcomes of balloon pulmonary artery (PA) angioplasty procedures after complete repair of tetralogy of Fallot with major aortopulmonary collateral arteries (TOF/MAPCAs). BACKGROUND: Our approach to patients with TOF/MAPCAs emphasizes early complete unifocalization and repair. Major PA reinterventions are relatively uncommon. Balloon PA angioplasty is often used, but the effectiveness of balloon PA angioplasty in this population is unknown. METHODS: The study cohort comprised patients who underwent complete unifocalization and repair of TOF/ MAPCAs at our center between 2002-2018 and underwent balloon PA angioplasty after repair. To assess immediate procedural outcomes, pre- and postintervention PA measurements were compared. RESULTS: We reviewed 134 vessels that were dilated a median of 1.1 years after repair in 60 patients (median 2 PA branches per patient). Treated vessels included 15 central, 64 lobar, and 55 segmental branches. The median PA diameter at the level of stenosis increased from 1.9 mm to 3.3 mm (P<.001), and the median diameter increase was 50%. All but 6 treated vessels were enlarged. The stenosis-distal diameter ratio increased from a median of 64% to 89% (P<.001). The median central PA to aortic systolic pressure ratio was 47% before and 39% after intervention (P<.001). CONCLUSIONS: Balloon PA angioplasty was acutely effective at treating most stenoses of reconstructed PA branches after repair of TOF/MAPCAs. Simple angioplasty can be a useful tool in treating isolated or modest stenoses after unifocalization/PA reconstruction surgery using our approach.

Bernard J. Miller: MD, ScD. (Hon), FACS: Lifelong Surgeon-Scientist and Critical Contributor to the Gibbon Heart-Lung Machine.

Bernard J. Miller, MD, ScD. (Hon), FACS, is known as a critical contributor for his work in the John H. Gibbon, MD, laboratory for his work on the heart-lung machine (HLM). In this setting, Dr. Miller developed the fluid control servo system, which was necessary to prevent malfunctioning of the HLM and prevent air emboli. Additionally, Dr. Miller assisted in conceiving and testing the left ventricular vent, the positive-negative pressure ventilator, and the HLM oxygenator; these inventions were all the product of extensive collaboration between the International Business Machines Corporation and the members of Dr. Gibbon's laboratory. Furthermore, Dr. Miller was a surgical assistant and perfusionist in the first successful open-heart surgery. Herein, we seek to describe Dr. Miller's story and his contributions to the HLM, as well as the contributions that were developed by the laboratory at that time. Additionally, we describe critical events leading up to the first successful use of the HLM on May 6, 1953, including a previously unreported use of the HLM for partial bypass of the right heart at Pennsylvania Hospital in 1952. Finally, we present the rest of Dr. Miller's professional and personal successes after his work on the HLM ended.

Agrobacterium T-DNA integration in somatic cells does not require the activity of DNA polymerase θ.

Integration of Agrobacterium tumefaciens transferred DNA (T-DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T-DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end-joining (NHEJ) pathways. Recent evidence suggests that in Arabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T-DNA integration. We conducted quantitative transformation assays of wild-type and polQ mutant Arabidopsis and rice, analyzed T-DNA/plant DNA junction sequences, and (for Arabidopsis) measured the amount of integrated T-DNA in mutant and wild-type tissue. Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency of polQ mutants was c. 20% that of wild-type plants. T-DNA/plant DNA junctions from these transformed rice and Arabidopsis polQ mutants closely resembled those from wild-type plants, indicating that loss of PolQ activity does not alter the characteristics of T-DNA integration events. polQ mutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation. We suggest that either multiple redundant pathways function in T-DNA integration, and/or that integration requires some yet unknown pathway.

Sequence diversity analyses of an improved rhesus macaque genome enhance its biomedical utility.

The rhesus macaque (Macaca mulatta) is the most widely studied nonhuman primate (NHP) in biomedical research. We present an updated reference genome assembly (Mmul_10, contig N50 = 46 Mbp) that increases the sequence contiguity 120-fold and annotate it using 6.5 million full-length transcripts, thus improving our understanding of gene content, isoform diversity, and repeat organization. With the improved assembly of segmental duplications, we discovered new lineage-specific genes and expanded gene families that are potentially informative in studies of evolution and disease susceptibility. Whole-genome sequencing (WGS) data from 853 rhesus macaques identified 85.7 million single-nucleotide variants (SNVs) and 10.5 million indel variants, including potentially damaging variants in genes associated with human autism and developmental delay, providing a framework for developing noninvasive NHP models of human disease.

Angiographic Anatomy of Major Aortopulmonary Collateral Arteries and Association With Early Surgical Outcomes in Tetralogy of Fallot.

Background Due in part to the heterogeneity of the pulmonary circulation in patients with tetralogy of Fallot and major aortopulmonary collateral arteries (MAPCAs), research on this condition has focused on relatively basic anatomic characteristics. We aimed to detail pulmonary artery (PA) and MAPCA anatomy in a large group of infants, assess relationships between anatomy and early surgical outcomes, and consider systems for classifying MAPCAs. Methods and Results All infants ( <1 year of age) undergoing first cardiac surgery for tetralogy of Fallot/MAPCAs from 2001 to 2019 at Stanford University were identified. Preoperative angiograms delineating supply to all 18 pulmonary segments were reviewed for details of each MAPCA and the arborization and size of central PAs. We studied 276 patients with 1068 MAPCAs and the following PA patterns: 152 (55%) incompletely arborizing PAs, 48 (17%) normally arborizing PAs, 45 (16%) absent PAs, and 31 (11%) unilateral MAPCAs. There was extensive anatomic variability, but no difference in early outcomes according to PA arborization or the predominance of PAs or MAPCAs. Patients with low total MAPCA and/or PA cross-sectional area were less likely to undergo complete repair. Conclusions MAPCA anatomy is highly variable and essentially unique for each patient. Though each pulmonary segment can be supplied by a MAPCA, central PA, or both, all anatomic combinations are similarly conducive to a good repair. Total cross-sectional area of central PA and MAPCA material is an important driver of outcome. We elucidate a number of novel associations between anatomic features, but the extreme variability of the pulmonary circulation makes a granular tetralogy of Fallot/MAPCA classification system unrealistic.

Agrobacterium-delivered VirE2 interacts with host nucleoporin CG1 to facilitate the nuclear import of VirE2-coated T complex.

Agrobacterium tumefaciens is the causal agent of crown gall disease. The bacterium is capable of transferring a segment of single-stranded DNA (ssDNA) into recipient cells during the transformation process, and it has been widely used as a genetic modification tool for plants and nonplant organisms. Transferred DNA (T-DNA) has been proposed to be escorted by two virulence proteins, VirD2 and VirE2, as a nucleoprotein complex (T-complex) that targets the host nucleus. However, it is not clear how such a proposed large DNA-protein complex is delivered through the host nuclear pore in a natural setting. Here, we studied the natural nuclear import of the Agrobacterium-delivered ssDNA-binding protein VirE2 inside plant cells by using a split-GFP approach with a newly constructed T-DNA-free strain. Our results demonstrate that VirE2 is targeted into the host nucleus in a VirD2- and T-DNA-dependent manner. In contrast with VirD2 that binds to plant importin α for nuclear import, VirE2 directly interacts with the host nuclear pore complex component nucleoporin CG1 to facilitate its nuclear uptake and the transformation process. Our data suggest a cooperative nuclear import model in which T-DNA is guided to the host nuclear pore by VirD2 and passes through the pore with the assistance of interactions between VirE2 and host nucleoporin CG1. We hypothesize that this large linear nucleoprotein complex (T-complex) is targeted to the nucleus by a "head" guide from the VirD2-importin interaction and into the nucleus by a lateral assistance from the VirE2-nucleoporin interaction.

Risk Factors of Recurrent Falls Among Older Adults Admitted to the Trauma Surgery Department.

Falls affect more than 29 million American adults ages ≥65 years annually. Many older adults experience recurrent falls requiring medical attention. These recurrent falls may be prevented through screening and intervention. In 2014 to 2015, records for 199 older adult patients admitted from a major urban teaching hospital's emergency department were queried. Open-ended variables from clinicians' notes were coded to supplement existing closed-ended variables. Of the 199 patients, 52 (26.1%) experienced one or more recurrent falls within 365 days after their initial fall. Half (50.0%) of all recurrent falls occurred within the first 90 days following discharge. A large proportion of recurrent falls among older adults appear to occur within a few months and are statistically related to identifiable risk factors. Prevention and intervention strategies, delivered either during treatment for an initial fall or upon discharge from an inpatient admission, may reduce the incidence of recurrent falls among this population.

Reproductive Outcomes in Rhesus Macaques (Macaca mulatta) with Naturally-acquired Trypanosoma cruzi Infection.

Chagas disease is a zoonotic vector-borne disease caused by infection with the protozoan parasite Trypanosoma cruzi. T. cruzi is found in Latin America and the Southern United States, where it infects many species, including humans and nonhuman primates (NHPs). NHPs are susceptible to natural infection and can develop clinical symptoms consistent with human disease, including Chagasic cardiomyopathy, gastrointestinal disease and transplacental transmission, leading to congenital infection. Due to evidence of Chagas transmission in Texas, this study hypothesized T. cruzi infection was present in a closed, outdoor-housed breeding colony of rhesus macaques (Macaca mulatta) located at a biomedical research facility in Central Texas. In addition, we questioned whether seropositive female rhesus macaques might experience reproductive complications consistent with maternal-fetal Chagas disease. The seroprevalence of T. cruzi infection in the colony was assessed using an Enzyme Linked Immunosorbant Assay (ELISA) to detect antibodies against Tc24 antigen as a screening assay, and a commercially available immunochromatographic test (Chagas Stat Pak) as a confirmatory assay. Retrospective serologic analysis was performed to confirm the status of all T. cruzi-infected animals between the years 2012 to 2016. The medical history of all seropositive and seronegative breeding females within the colony from 2012 to 2016 was reviewed to determine each animals' level of reproductive fitness. The percentage of T. cruzi-seropositive animals ranged from 6.7% to 9.7% in adult animals and 0% to 0.44% in juveniles or weanling animals, depending on the year. An overall 3.9% seroprevalence of T. cruzi infection was found in the total population. No significant differences in any measure of reproductive outcomes were identified between seropositive and seronegative females from 2012 to 2016. The lack of significant adverse reproductive outcomes reported here may help inform future management decisions regarding seropositive female rhesus macaques within breeding colonies.

Genome-Wide DNA Methylation and LncRNA-Associated DNA Methylation in Metformin-Treated and -Untreated Diabetes.

Metformin, which is used as a first line treatment for type 2 diabetes mellitus (T2DM), has been shown to affect epigenetic patterns. In this study, we investigated the DNA methylation and potential lncRNA modifications in metformin-treated and newly diagnosed adults with T2DM. Genome-wide DNA methylation and lncRNA analysis were performed from the peripheral blood of 12 screen-detected and 12 metformin-treated T2DM individuals followed by gene ontology (GO) and KEGG pathway analysis. Differentially methylated regions (DMRs) observed showed 22 hypermethylated and 11 hypomethylated DMRs between individuals on metformin compared to screen-detected subjects. Amongst the hypomethylated DMR regions were the SLC gene family, specifically, SLC25A35 and SLC28A1. Fifty-seven lncRNA-associated DNA methylation regions included the mitochondrial ATP synthase-coupling factor 6 (ATP5J). Functional gene mapping and pathway analysis identified regions in the axon initial segment (AIS), node of Ranvier, cell periphery, cleavage furrow, cell surface furrow, and stress fiber. In conclusion, our study has identified a number of DMRs and lncRNA-associated DNA methylation regions in metformin-treated T2DM that are potential targets for therapeutic monitoring in patients with diabetes.

Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.

KEY MESSAGE: Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.