Longitudinal progression of blood biomarkers reveals a key role of astrocyte reactivity in preclinical Alzheimer's disease.

Defining the progression of blood biomarkers of Alzheimer's disease (AD) is essential for targeting treatments in patients most likely to benefit from early intervention. We delineated the temporal ordering of blood biomarkers a decade prior to the onset of AD symptoms in participants in the Baltimore Longitudinal Study of Aging. We show that increased astrocyte reactivity, assessed by elevated glial fibrillary acidic protein (GFAP) levels is an early event in the progression of blood biomarker changes in preclinical AD. In AD-converters who are initially cognitively unimpaired (N=158, 377 serial plasma samples), higher plasma GFAP levels are observed as early as 10-years prior to the onset of cognitive impairment due to incident AD compared to individuals who remain cognitively unimpaired (CU, N=160, 379 serial plasma samples). Plasma GFAP levels in AD-converters remain elevated 5-years prior to and coincident with the onset of cognitive impairment due to AD. In participants with neuropathologically confirmed AD, plasma GFAP levels are elevated relative to cognitively normal individuals and intermediate in those who remain cognitively unimpaired despite significant AD pathology (asymptomatic AD). Higher plasma GFAP levels at death are associated with greater severity of both neuritic plaques and neurofibrillary tangles. In the 5XFAD transgenic model of AD, we observed greater GFAP levels in the cortex and hippocampus of transgenic mice relative to wild-type prior to the development of cognitive impairment. Reactive astrocytosis, an established biological response to neuronal injury, may be an early initiator of AD pathogenesis and a promising therapeutic target.

Neurogranin expression regulates mitochondrial function and redox balance in endothelial cells.

Endothelial dysfunction and endothelial activation are common early events in vascular diseases and can arise from mitochondrial dysfunction. Neurogranin (Ng) is a 17kD protein well known to regulate intracellular Ca2+-calmodulin (CaM) complex signaling, and its dysfunction is significantly implicated in brain aging and neurodegenerative diseases. We found that Ng is also expressed in human aortic endothelial cells (HAECs), and depleting Ng promotes Ca2+-CaM complex-dependent endothelial activation and redox imbalances. Endothelial-specific Ng knockout (Cre-CDH5-Ngf/f) mice demonstrate a significant delay in the flow-mediated dilation (FMD) response. Therefore, it is critical to characterize how endothelial Ng expression regulates reactive oxygen species (ROS) generation and affects cardiovascular disease. Label-free quantification proteomics identified that mitochondrial dysfunction and the oxidative phosphorylation pathway are significantly changed in the aorta of Cre-CDH5-Ngf/f mice. We found that a significant amount of Ng is expressed in the mitochondrial fraction of HAECs using western blotting and colocalized with the mitochondrial marker, COX IV, using immunofluorescence staining. Seahorse assay demonstrated that a lack of Ng decreases mitochondrial respiration. Treatment with MitoEbselen significantly restores the oxygen consumption rate in Ng knockdown cells. With the RoGFP-Orp1 approach, we identified that Ng knockdown increases mitochondrial-specific hydrogen peroxide (H2O2) production, and MitoEbselen treatment significantly reduced mitochondrial ROS (mtROS) levels in Ng knockdown cells. These results suggest that Ng plays a significant role in mtROS production. We discovered that MitoEbselen treatment also rescues decreased eNOS expression and nitric oxide (NO) levels in Ng knockdown cells, which implicates the critical role of Ng in mtROS-NO balance in the endothelial cells.

Shake It Off! Acoustic Manipulation of Lipid Vesicles for Mass Spectrometric Analysis.

Analyzing lipid assemblies, including liposomes and extracellular vesicles (EVs), is challenging due to their size, diverse composition, and tendency to aggregate. Such vesicles form with a simple phospholipid bilayer membrane, and they play important roles in drug discovery and delivery. The use of mass spectrometry (MS) allows for broad analysis of lipids from different classes; however, their release from the higher order structural aggregates is typically achieved by chemical means. Mechanical disruption by high frequency surface acoustic waves (SAW) is presented as an appealing alternative to preparing lipid vesicles for MS sampling. In this work, SAWs used to disrupt liposomes allow for the direct analysis of their constituent lipids by employing SAW nebulization with corona discharge (CD) ionization. We explore the effects of duration, frequency, and incorporation of nonpolar lipids, including cholesterol, on the SAW's ability to disrupt the liposome. We also report on the successful MS analysis of liposome-derived lipids along with cytochrome C in solution, thus demonstrating applications to aqueous samples and native MS conditions.

Genomics of Tenacibaculum Species in British Columbia, Canada.

Tenacibaculum is a genus of Gram-negative filamentous bacteria with a cosmopolitan distribution. The research describing Tenacibaculum genomes stems primarily from Norway and Chile due to their impacts on salmon aquaculture. Canadian salmon aquaculture also experiences mortality events related to the presence of Tenacibaculum spp., yet no Canadian Tenacibaculum genomes are publicly available. Ribosomal DNA sequencing of 16S and four species-specific 16S quantitative-PCR assays were used to select isolates cultured from Atlantic salmon with mouthrot in British Columbia (BC), Canada. Ten isolates representing four known and two unknown species of Tenacibaculum were selected for shotgun whole genome sequencing using the Oxford Nanopore's MinION platform. The genome assemblies achieved closed circular chromosomes for seven isolates and long contigs for the remaining three isolates. Average nucleotide identity analysis identified T. ovolyticum, T. maritimum, T. dicentrarchi, two genomovars of T. finnmarkense, and two proposed novel species T. pacificus sp. nov. type strain 18-2881-AT and T. retecalamus sp. nov. type strain 18-3228-7BT. Annotation in most of the isolates predicted putative virulence and antimicrobial resistance genes, most-notably toxins (i.e., hemolysins), type-IX secretion systems, and oxytetracycline resistance. Comparative analysis with the T. maritimum type-strain predicted additional toxins and numerous C-terminal secretion proteins, including an M12B family metalloprotease in the T. maritimum isolates from BC. The genomic prediction of virulence-associated genes provides important targets for studies of mouthrot disease, and the annotation of the antimicrobial resistance genes provides targets for surveillance and diagnosis in veterinary medicine.

Targeted genomic sequencing with probe capture for discovery and surveillance of coronaviruses in bats.

Public health emergencies like SARS, MERS, and COVID-19 have prioritized surveillance of zoonotic coronaviruses, resulting in extensive genomic characterization of coronavirus diversity in bats. Sequencing viral genomes directly from animal specimens remains a laboratory challenge, however, and most bat coronaviruses have been characterized solely by PCR amplification of small regions from the best-conserved gene. This has resulted in limited phylogenetic resolution and left viral genetic factors relevant to threat assessment undescribed. In this study, we evaluated whether a technique called hybridization probe capture can achieve more extensive genome recovery from surveillance specimens. Using a custom panel of 20,000 probes, we captured and sequenced coronavirus genomic material in 21 swab specimens collected from bats in the Democratic Republic of the Congo. For 15 of these specimens, probe capture recovered more genome sequence than had been previously generated with standard amplicon sequencing protocols, providing a median 6.1-fold improvement (ranging up to 69.1-fold). Probe capture data also identified five novel alpha- and betacoronaviruses in these specimens, and their full genomes were recovered with additional deep sequencing. Based on these experiences, we discuss how probe capture could be effectively operationalized alongside other sequencing technologies for high-throughput, genomics-based discovery and surveillance of bat coronaviruses.

Neurogranin regulates calcium-dependent cardiac hypertrophy.

Intracellular Ca2+-calmodulin (CaM) signaling plays an important role in Ca2+-CaM-dependent kinase (CaMKII) and calcineurin (CaN)-mediated cardiac biology. While neurogranin (Ng) is known as a major Ca2+-CaM modulator in the brain, its pathophysiological role in cardiac hypertrophy has never been studied before. In the present study, we report that Ng is expressed in the heart and depletion of Ng dysregulates Ca2+ homeostasis and promotes cardiac failure in mice. 10-month-old Ng null mice demonstrate significantly increased heart-to-body weight ratios compared to wild-type. Using histological approaches, we identified that depletion of Ng increases cardiac hypertrophy, fibrosis, and collagen deposition near perivascular areas in the heart tissue of Ng null mice. Ca2+ spark experiments revealed that cardiac myocytes isolated from Ng null mice have decreased spark frequency and width, while the duration of sparks is significantly increased. We also identified that a lack of Ng increases CaMKIIδ signaling and periostin protein expression in these mouse hearts. Overall, we are the first study to explore how Ng expression in the heart plays an important role in Ca2+ homeostasis in cardiac myocytes as well as the pathophysiology of cardiac hypertrophy and fibrosis.

Longitudinal assessment of colonoscopy adverse events in the prospective Cooperative Studies Program no. 380 colorectal cancer screening and surveillance cohort.

BACKGROUND AND AIMS: Data are limited regarding colonoscopy risk during long-term, programmatic colorectal cancer screening and follow-up. We aimed to describe adverse events during follow-up in a colonoscopy screening program after the baseline examination and examine factors associated with increased risk. METHODS: Cooperative Studies Program no. 380 includes 3121 asymptomatic veterans aged 50 to 75 years who underwent screening colonoscopy between 1994 and 1997. Periprocedure adverse events requiring significant intervention were defined as major events (other events were minor) and were tracked during follow-up for at least 10 years. Multivariable odds ratios (ORs) were calculated for factors associated with risk of follow-up adverse events. RESULTS: Of 3727 follow-up examinations in 1983 participants, adverse events occurred in 105 examinations (2.8%) in 93 individuals, including 22 major and 87 minor events (examinations may have had >1 event). Incidence of major events (per 1000 examinations) remained relatively stable over time, with 6.1 events at examination 2, 4.8 at examination 3, and 7.2 at examination 4. Examinations with major events included 1 perforation, 3 GI bleeds requiring intervention, and 17 cardiopulmonary events. History of prior colonoscopic adverse events was associated with increased risk of events (major or minor) during follow-up (OR, 2.7; 95% confidence interval, 1.6-4.6). CONCLUSIONS: Long-term programmatic screening and surveillance was safe, as major events were rare during follow-up. However, serious cardiopulmonary events were the most common major events. These results highlight the need for detailed assessments of comorbid conditions during routine clinical practice, which could help inform individual decisions regarding the utility of ongoing colonoscopy follow-up.

Targeted genomic sequencing with probe capture for discovery and surveillance of coronaviruses in bats

Public health emergencies like SARS, MERS, and COVID-19 have prioritized surveillance of zoonotic coronaviruses, resulting in extensive genomic characterization of coronavirus diversity in bats. Sequencing viral genomes directly from animal specimens remains a laboratory challenge, however, and most bat coronaviruses have been characterized solely by PCR amplification of small regions from the best-conserved gene. This has resulted in limited phylogenetic resolution and left viral genetic factors relevant to threat assessment undescribed. In this study, we evaluated whether a technique called hybridization probe capture can achieve more extensive genome recovery from surveillance specimens. Using a custom panel of 20,000 probes, we captured and sequenced coronavirus genomic material in 21 swab specimens collected from bats in the Democratic Republic of the Congo. For 15 of these specimens, probe capture recovered more genome sequence than had been previously generated with standard amplicon sequencing protocols, providing a median 6.1-fold improvement (ranging up to 69.1-fold). Probe capture data also identified five novel alpha- and betacoronaviruses in these specimens, and their full genomes were recovered with additional deep sequencing. Based on these experiences, we discuss how probe capture could be effectively operationalized alongside other sequencing technologies for high-throughput, genomics-based discovery and surveillance of bat coronaviruses.

Screening Colonoscopy Findings Are Associated With Noncolorectal Cancer Mortality.

INTRODUCTION: Controversy exists regarding the impact of various risk factors on noncolorectal cancer (CRC) mortality in healthy screening populations. We examined the impact of known CRC risk factors, including baseline colonoscopy findings, on non-CRC mortality in a screening population. METHODS: Cooperative Studies Program (CSP) #380 is comprised of 3,121 veterans aged 50-75 years who underwent screening colonoscopy from 1994 to 97 and were then followed for at least 10 years or until death. Hazard ratios (HRs) for risk factors on non-CRC mortality were estimated by multivariate Cox proportional hazards. RESULTS: Current smoking (HR 2.12, 95% confidence interval [CI] 1.78-2.52, compared with nonsmokers) and physical activity (HR 0.89, 95% CI 0.84-0.93) were the modifiable factors most associated with non-CRC mortality in CSP#380. In addition, compared with no neoplasia at baseline colonoscopy, non-CRC mortality was higher in participants with ≥3 small adenomas (HR 1.43, 95% CI 1.06-1.94), advanced adenomas (HR 1.32, 95% CI 0.99-1.75), and CRC (HR 2.95, 95% CI 0.98-8.85). Those with 1-2 small adenomas were not at increased risk for non-CRC mortality (HR 1.15, 95% CI 0.94-1.4). DISCUSSION: In a CRC screening population, known modifiable risk factors were significantly associated with 10-year non-CRC mortality. Furthermore, those who died from non-CRC causes within 10 years were more likely to have had high-risk findings at baseline colonoscopy. These results suggest that advanced colonoscopy findings may be a risk marker of poor health outcomes. Integrated efforts are needed to motivate healthy lifestyle changes during CRC screening, particularly in those with high-risk colonoscopy findings and unaddressed risk factors.

The sacrificial adaptor protein Skp functions to remove stalled substrates from the ß-barrel assembly machine.

The biogenesis of integral ß-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the ß-barrel assembly machine (Bam) complex. We find that ß-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam-OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient ß-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

Ten or More Cumulative Lifetime Adenomas Are Associated with Increased Risk for Advanced Neoplasia and Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) screening guidelines recommend frequent colonoscopies and consideration of genetic testing in individuals with ≥10 cumulative adenomas. However, it is unclear how these guidelines apply to routine practice. AIMS: We estimated the proportion of participants found to have ≥10 cumulative adenomas in a screening population and described their outcomes of advanced neoplasia (AN), CRC, and extra-colonic malignancy. METHODS: We performed a secondary analysis of VA CSP#380, which includes 3121 veterans aged 50-75 who were followed up to 10 years after screening colonoscopy. We calculated the cumulative risk of ≥10 cumulative adenomas by Kaplan-Meier method. We compared baseline risk factors in those with and without ≥10 cumulative adenomas as well as the risk for AN (adenoma ≥1 cm, villous adenoma or high-grade dysplasia, or CRC) and extra-colonic malignancy by multivariate logistic regression. RESULTS: The cumulative risk of ≥10 cumulative adenomas over 10.5 years was 6.51% (95% CI 4.38%-9.62%). Age 60-69 or 70-75 at baseline colonoscopy was the only factors associated with the finding of ≥10 cumulative adenomas. Compared to those with 0-9 cumulative adenomas, participants with ≥10 cumulative adenomas were more likely to have had AN (OR 17.03; 95% CI 9.41-30.84), including CRC (OR 7.00; 95% CI 2.84-17.28), but not extra-colonic malignancies. CONCLUSIONS: Approximately 6.5% of participants in this screening population were found to have ≥10 cumulative adenomas over 10.5 years, which was uncommon before age 60. These participants were found to have AN and CRC significantly more often compared to those with lower cumulative adenomas.

Viscoelastic Testing and Coagulopathy of Traumatic Brain Injury.

A unique coagulopathy often manifests following traumatic brain injury, leading the clinician down a difficult decision path on appropriate prophylaxis and therapy. Conventional coagulation assays-such as prothrombin time, partial thromboplastin time, and international normalized ratio-have historically been utilized to assess hemostasis and guide treatment following traumatic brain injury. However, these plasma-based assays alone often lack the sensitivity to diagnose and adequately treat coagulopathy associated with traumatic brain injury. Here, we review the whole blood coagulation assays termed viscoelastic tests and their use in traumatic brain injury. Modified viscoelastic tests with platelet function assays have helped elucidate the underlying pathophysiology and guide clinical decisions in a goal-directed fashion. Platelet dysfunction appears to underlie most coagulopathies in this patient population, particularly at the adenosine diphosphate and/or arachidonic acid receptors. Future research will focus not only on the utility of viscoelastic tests in diagnosing coagulopathy in traumatic brain injury, but also on better defining the use of these tests as evidence-based and/or precision-based tools to improve patient outcomes.

Transitioning from cerebrospinal fluid to blood tests to facilitate diagnosis and disease monitoring in Alzheimer's disease.

Alzheimer's disease (AD) is increasingly prevalent worldwide, and disease-modifying treatments may soon be at hand; hence, now, more than ever, there is a need to develop techniques that allow earlier and more secure diagnosis. Current biomarker-based guidelines for AD diagnosis, which have replaced the historical symptom-based guidelines, rely heavily on neuroimaging and cerebrospinal fluid (CSF) sampling. While these have greatly improved the diagnostic accuracy of AD pathophysiology, they are less practical for application in primary care, population-based and epidemiological settings, or where resources are limited. In contrast, blood is a more accessible and cost-effective source of biomarkers in AD. In this review paper, using the recently proposed amyloid, tau and neurodegeneration [AT(N)] criteria as a framework towards a biological definition of AD, we discuss recent advances in biofluid-based biomarkers, with a particular emphasis on those with potential to be translated into blood-based biomarkers. We provide an overview of the research conducted both in CSF and in blood to draw conclusions on biomarkers that show promise. Given the evidence collated in this review, plasma neurofilament light chain (N) and phosphorylated tau (p-tau; T) show particular potential for translation into clinical practice. However, p-tau requires more comparisons to be conducted between its various epitopes before conclusions can be made as to which one most robustly differentiates AD from non-AD dementias. Plasma amyloid beta (A) would prove invaluable as an early screening modality, but it requires very precise tests and robust pre-analytical protocols.

Characterizing chronological accumulation of comorbidities in healthy veterans: a computational approach.

Understanding patient accumulation of comorbidities can facilitate healthcare strategy and personalized preventative care. We applied a directed network graph to electronic health record (EHR) data and characterized comorbidities in a cohort of healthy veterans undergoing screening colonoscopy. The Veterans Affairs Cooperative Studies Program #380 was a prospective longitudinal study of screening and surveillance colonoscopy. We identified initial instances of three-digit ICD-9 diagnoses for participants with at least 5 years of linked EHR history (October 1999 to December 2015). For diagnoses affecting at least 10% of patients, we calculated pairwise chronological relative risk (RR). iGraph was used to produce directed graphs of comorbidities with RR > 1, as well as summary statistics, key diseases, and communities. A directed graph based on 2210 patients visualized longitudinal development of comorbidities. Top hub (preceding) diseases included ischemic heart disease, inflammatory and toxic neuropathy, and diabetes. Top authority (subsequent) diagnoses were acute kidney failure and hypertensive chronic kidney failure. Four communities of correlated comorbidities were identified. Close analysis of top hub and authority diagnoses demonstrated known relationships, correlated sequelae, and novel hypotheses. Directed network graphs portray chronologic comorbidity relationships. We identified relationships between comorbid diagnoses in this aging veteran cohort. This may direct healthcare prioritization and personalized care.

The validation status of blood biomarkers of amyloid and phospho-tau assessed with the 5-phase development framework for AD biomarkers.

PURPOSE: The development of blood biomarkers that reflect Alzheimer's disease (AD) pathophysiology (phosphorylated tau and amyloid-ß) has offered potential as scalable tests for dementia differential diagnosis and early detection. In 2019, the Geneva AD Biomarker Roadmap Initiative included blood biomarkers in the systematic validation of AD biomarkers. METHODS: A panel of experts convened in November 2019 at a two-day workshop in Geneva. The level of maturity (fully achieved, partly achieved, preliminary evidence, not achieved, unsuccessful) of blood biomarkers was assessed based on the Biomarker Roadmap methodology and discussed fully during the workshop which also evaluated cerebrospinal fluid (CSF) and positron emission tomography (PET) biomarkers. RESULTS: Plasma p-tau has shown analytical validity (phase 2 primary aim 1) and first evidence of clinical validity (phase 3 primary aim 1), whereas the maturity level for Aß remains to be partially achieved. Full and partial achievement has been assigned to p-tau and Aß, respectively, in their associations to ante-mortem measures (phase 2 secondary aim 2). However, only preliminary evidence exists for the influence of covariates, assay comparison and cut-off criteria. CONCLUSIONS: Despite the relative infancy of blood biomarkers, in comparison to CSF biomarkers, much has already been achieved for phases 1 through 3 - with p-tau having greater success in detecting AD and predicting disease progression. However, sufficient data about the effect of covariates on the biomarker measurement is lacking. No phase 4 (real-world performance) or phase 5 (assessment of impact/cost) aim has been tested, thus not achieved.

2020 update on the clinical validity of cerebrospinal fluid amyloid, tau, and phospho-tau as biomarkers for Alzheimer's disease in the context of a structured 5-phase development framework.

PURPOSE: In the last decade, the research community has focused on defining reliable biomarkers for the early detection of Alzheimer's disease (AD) pathology. In 2017, the Geneva AD Biomarker Roadmap Initiative adapted a framework for the systematic validation of oncological biomarkers to cerebrospinal fluid (CSF) AD biomarkers-encompassing the 42 amino-acid isoform of amyloid-ß (Aß42), phosphorylated-tau (P-tau), and Total-tau (T-tau)-with the aim to accelerate their development and clinical implementation. The aim of this work is to update the current validation status of CSF AD biomarkers based on the Biomarker Roadmap methodology. METHODS: A panel of experts in AD biomarkers convened in November 2019 at a 2-day workshop in Geneva. The level of maturity (fully achieved, partly achieved, preliminary evidence, not achieved, unsuccessful) of CSF AD biomarkers was assessed based on the Biomarker Roadmap methodology before the meeting and presented and discussed during the workshop. RESULTS: By comparison to the previous 2017 Geneva Roadmap meeting, the primary advances in CSF AD biomarkers have been in the area of a unified protocol for CSF sampling, handling and storage, the introduction of certified reference methods and materials for Aß42, and the introduction of fully automated assays. Additional advances have occurred in the form of defining thresholds for biomarker positivity and assessing the impact of covariates on their discriminatory ability. CONCLUSIONS: Though much has been achieved for phases one through three, much work remains in phases four (real world performance) and five (assessment of impact/cost). To a large degree, this will depend on the availability of disease-modifying treatments for AD, given these will make accurate and generally available diagnostic tools key to initiate therapy.

Convalescent plasma-mediated resolution of COVID-19 in a patient with humoral immunodeficiency.

Convalescent plasma (CP) is widely used to treat COVID-19, but without formal evidence of efficacy. Here, we report the beneficial effects of CP in a severely ill COVID-19 patient with prolonged pneumonia and advanced chronic lymphocytic leukemia (CLL), who was unable to generate an antiviral antibody response of her own. On day 33 after becoming symptomatic, the patient received CP containing high-titer (ID50 > 5,000) neutralizing antibodies (NAbs), defervesced, and improved clinically within 48 h and was discharged on day 37. Hence, when present in sufficient quantities, NAbs to SARS-CoV-2 have clinical benefit even if administered relatively late in the disease course. However, analysis of additional CP units revealed widely varying NAb titers, with many recipients exhibiting endogenous NAb responses far exceeding those of the administered units. To obtain the full therapeutic benefits of CP immunotherapy, it will thus be important to determine the neutralizing activity in both CP units and transfusion candidates.

Multiple factors are involved in regulation of extracellular membrane vesicle biogenesis in Streptococcus mutans.

Streptococcus mutans, a major etiological agent of human dental caries, produces membrane vesicles (MVs) that contain protein and extracellular DNA. In this study, functional genomics, along with in vitro biofilm models, was used to identify factors that regulate MV biogenesis. Our results showed that when added to growth medium, MVs significantly enhanced biofilm formation by S. mutans, especially during growth in sucrose. This effect occurred in the presence and absence of added human saliva. Functional genomics revealed several genes, including sfp, which have a major effect on S. mutans MVs. In Bacillus sp. sfp encodes a 4'-phosphopantetheinyl transferase that contributes to surfactin biosynthesis and impacts vesiculogenesis. In S. mutans, sfp resides within the TnSmu2 Genomic Island that supports pigment production associated with oxidative stress tolerance. Compared to the UA159 parent, the Δsfp mutant, TW406, demonstrated a 1.74-fold (p < .05) higher MV yield as measured by BCA protein assay. This mutant also displayed increased susceptibility to low pH and oxidative stressors, as demonstrated by acid killing and hydrogen peroxide challenge assays. Deficiency of bacA, a putative surfactin synthetase homolog within TnSmu2, and especially dac and pdeA that encode a di-adenylyl cyclase and a phosphodiesterase, respectively, also significantly increased MV yield (p < .05). However, elimination of bacA2, a bacitracin synthetase homolog, resulted in a >1.5-fold (p < .05) reduction of MV yield. These results demonstrate that S. mutans MV properties are regulated by genes within and outside of the TnSmu2 island, and that as a major particulate component of the biofilm matrix, MVs significantly influence biofilm formation.

Genetic Colorectal Cancer and Adenoma Risk Variants Are Associated with Increasing Cumulative Adenoma Counts.

BACKGROUND: The genetic basis for most individuals with high cumulative lifetime colonic adenomas is unknown. We investigated associations between known colorectal cancer-risk single-nucleotide polymorphisms (SNP) and increasing cumulative adenoma counts. METHODS: The Cooperative Studies Program #380 screening colonoscopy cohort includes 612 selected participants age 50 to 75 with genotyped blood samples and 10 years of clinical follow-up. We evaluated 41 published "colorectal cancer-risk SNPs" for associations with individual cumulative adenoma counts or having ≥10 cumulative adenomas. SNPs were analyzed singly or combined in a polygenic risk score (PRS). The PRS was constructed from eight published SNPs associated with multiple adenomas, termed "adenoma-risk SNPs." RESULTS: Four colorectal cancer-risk SNPs were associated with increasing cumulative adenoma counts (P < 0.05): rs12241008 (gene: VTI1A), rs2423279 (BMP2/HAO1), rs3184504 (SH2B3), and rs961253 (FERMT1/BMP2), with risk allele risk ratios of 1.31, 1.29, 1.24, and 1.23, respectively. Three colorectal cancer-risk SNPs were associated with ≥10 cumulative adenomas (P < 0.05), with risk allele odds ratios of 2.09 (rs3184504), 2.30 (rs961253), and 1.94 (rs3217901). A weighted PRS comprised of adenoma-risk SNPs was associated with higher cumulative adenomas (weighted rate ratio = 1.57; P = 0.03). CONCLUSIONS: In this mostly male veteran colorectal cancer screening cohort, several known colorectal cancer-risk SNPs were associated with increasing cumulative adenoma counts and the finding of ≥10 cumulative adenomas. In addition, an increasing burden of adenoma-risk SNPs, measured by a weighted PRS, was associated with higher cumulative adenomas. IMPACT: Future work will seek to validate these findings in different populations and then augment current colorectal cancer risk prediction tools with precancerous, adenoma genetic data.

High-Risk Adenomas at Screening Colonoscopy Remain Predictive of Future High-Risk Adenomas Despite an Intervening Negative Colonoscopy.

INTRODUCTION: Limited data inform the current postpolypectomy surveillance guidelines, which suggest a shortened interval to third colonoscopy after a negative second examination if high-risk adenomas (HRA) were present on the initial screening colonoscopy. Therefore, we examined the risk of HRA at third colonoscopy stratified by findings on 2 previous examinations in a prospective screening colonoscopy cohort of US veterans. METHODS: We identified participants who had 3 or more colonoscopies from CSP#380. We examined the risk of HRA on the third examination based on findings from the previous 2 examinations. Multivariate logistic regression was used to adjust for multiple covariates. RESULTS: HRA were found at the third examination in 114 (12.8%) of 891 participants. Those with HRA on both previous examinations had the greatest incidence of HRA at third examination (14/56, 25.0%). Compared with those with no adenomas on both previous examinations, participants with HRA on the first examination remained at significantly increased risk for HRA at the third examination at 3 years after a negative second examination (odds ratio [OR] 3.41, 95% confidence interval [CI] 1.28-9.08), 5 years (OR 3.14, 95% CI 1.49-6.61), and 7 years (OR 2.89, 95% CI 1.08-7.74). DISCUSSION: In a screening population, HRA on the first examination identified individuals who remained at increased risk for HRA at the third examination, even after a negative second examination. This finding supports current colorectal cancer surveillance guidelines, which suggest a shortened, 5-year time interval to third colonoscopy after a negative second examination if high-risk findings were present on the baseline examination.