Quantitative analysis of high-resolution, contrast-enhanced, cone-beam CT for the detection of intracranial in-stent hyperplasia

BACKGROUND: Intracranial in-stent hyperplasia is a stroke-associated complication that requires routine surveillance. OBJECTIVE: To compare the results of in vivo experiments to determine the accuracy and precision of in-stent hyperplasia measurements obtained with modified C-arm contrast-enhanced, cone-beam CT (CE-CBCT) imaging with those obtained by \u27gold standard\u27 histomorphometry. Additionally, to carry out clinical analyses comparing this CE-CBCT protocol with digital subtraction angiography (DSA). METHODS: A non-binned CE-CBCT protocol (VasoCT) was used that acquires x-ray images with a small field-of-view and applies a full-scale reconstruction algorithm providing high-resolution three-dimensional (3D) imaging with 100 microm isotropic voxels. In an vivo porcine model, VasoCT cross-sectional area measurements were compared with gold standard vessel histology. VasoCT and DSA were used to calculate in-stent stenosis in 23 imaging studies. RESULTS: Porcine VasoCT cross-sectional stent, lumen, and in-stent hyperplasia areas strongly correlated with histological measurements (r(2)=0.97, 0.93, 0.90; slope=1.14, 1.07, and 0.76, respectively; p\u3c0.0001). Clinical VasoCT percentage stenosis correlated well with DSA percentage stenosis (r(2)=0.84; slope=0.76), and the two techniques were free of consistent bias (Bland-Altman, bias=3.29%; 95% CI -14.75% to 21.33%). An illustrative clinical case demonstrated the advantages of VasoCT, including 3D capability and non-invasive IV contrast administration, for detection of in-stent hyperplasia. CONCLUSIONS: C-arm VasoCT is a high-resolution 3D capable imaging technique that has been validated in an animal model for measurement of in-stent tissue growth. Successful clinical implementation of the protocol was performed in a small case series. already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions

Intracellular bacillary burden reflects a burst size for Mycobacterium tuberculosis in vivo

We previously reported that Mycobacterium tuberculosis triggers macrophage necrosis in vitro at a threshold intracellular load of ~25 bacilli. This suggests a model for tuberculosis where bacilli invading lung macrophages at low multiplicity of infection proliferate to burst size and spread to naïve phagocytes for repeated cycles of replication and cytolysis. The current study evaluated that model in vivo, an environment significantly more complex than in vitro culture. In the lungs of mice infected with M. tuberculosis by aerosol we observed three distinct mononuclear leukocyte populations (CD11b(-) CD11c(+/hi), CD11b(+/lo) CD11c(lo/-), CD11b(+/hi) CD11c(+/hi)) and neutrophils hosting bacilli. Four weeks after aerosol challenge, CD11b(+/hi) CD11c(+/hi) mononuclear cells and neutrophils were the predominant hosts for M. tuberculosis while CD11b(+/lo) CD11c(lo/-) cells assumed that role by ten weeks. Alveolar macrophages (CD11b(-) CD11c(+/hi)) were a minority infected cell type at both time points. The burst size model predicts that individual lung phagocytes would harbor a range of bacillary loads with most containing few bacilli, a smaller proportion containing many bacilli, and few or none exceeding a burst size load. Bacterial load per cell was enumerated in lung monocytic cells and neutrophils at time points after aerosol challenge of wild type and interferon-γ null mice. The resulting data fulfilled those predictions, suggesting a median in vivo burst size in the range of 20 to 40 bacilli for monocytic cells. Most heavily burdened monocytic cells were nonviable, with morphological features similar to those observed after high multiplicity challenge in vitro: nuclear condensation without fragmentation and disintegration of cell membranes without apoptotic vesicle formation. Neutrophils had a narrow range and lower peak bacillary burden than monocytic cells and some exhibited cell death with release of extracellular neutrophil traps. Our studies suggest that burst size cytolysis is a major cause of infection-induced mononuclear cell death in tuberculosis

A recombinant herpesviral vector containing a near-full-length SIVmac239 genome produces SIV particles and elicits immune responses to all nine SIV gene products

The properties of the human immunodeficiency virus (HIV) pose serious difficulties for the development of an effective prophylactic vaccine. Here we describe the construction and characterization of recombinant (r), replication-competent forms of rhesus monkey rhadinovirus (RRV), a gamma-2 herpesvirus, containing a near-full-length (nfl) genome of the simian immunodeficiency virus (SIV). A 306-nucleotide deletion in the pol gene rendered this nfl genome replication-incompetent as a consequence of deletion of the active site of the essential reverse transcriptase enzyme. Three variations were constructed to drive expression of the SIV proteins: one with SIV\u27s own promoter region, one with a cytomegalovirus (cmv) immediate-early promoter/enhancer region, and one with an RRV dual promoter (p26 plus PAN). Following infection of rhesus fibroblasts in culture with these rRRV vectors, synthesis of the early protein Nef and the late structural proteins Gag and Env could be demonstrated. Expression levels of the SIV proteins were highest with the rRRV-SIVcmv-nfl construct. Electron microscopic examination of rhesus fibroblasts infected with rRRV-SIVcmv-nfl revealed numerous budding and mature SIV particles and these infected cells released impressive levels of p27 Gag protein ( \u3e 150 ng/ml) into the cell-free supernatant. The released SIV particles were shown to be incompetent for replication. Monkeys inoculated with rRRV-SIVcmv-nfl became persistently infected, made readily-detectable antibodies against SIV, and developed T-cell responses against all nine SIV gene products. Thus, rRRV expressing a near-full-length SIV genome mimics live-attenuated strains of SIV in several important respects: the infection is persistent; \u3e 95% of the SIV proteome is naturally expressed; SIV particles are formed; and CD8+ T-cell responses are maintained indefinitely in an effector-differentiated state. Although the magnitude of anti-SIV immune responses in monkeys infected with rRRV-SIVcmv-nfl falls short of what is seen with live-attenuated SIV infection, further experimentation seems warranted

Extracellular Vesicles from Pseudomonas aeruginosa Suppress MHC-Related Molecules in Human Lung Macrophages

Pseudomonas aeruginosa, a Gram-negative bacterium, is one of the most common pathogens colonizing the lungs of cystic fibrosis patients. P. aeruginosa secrete extracellular vesicles (EVs) that contain LPS and other virulence factors that modulate the host\u27s innate immune response, leading to an increased local proinflammatory response and reduced pathogen clearance, resulting in chronic infection and ultimately poor patient outcomes. Lung macrophages are the first line of defense in the airway innate immune response to pathogens. Proper host response to bacterial infection requires communication between APC and T cells, ultimately leading to pathogen clearance. In this study, we investigate whether EVs secreted from P. aeruginosa alter MHC Ag expression in lung macrophages, thereby potentially contributing to decreased pathogen clearance. Primary lung macrophages from human subjects were collected via bronchoalveolar lavage and exposed to EVs isolated from P. aeruginosa in vitro. Gene expression was measured with the NanoString nCounter gene expression assay. DNA methylation was measured with the EPIC array platform to assess changes in methylation. P. aeruginosa EVs suppress the expression of 11 different MHC-associated molecules in lung macrophages. Additionally, we show reduced DNA methylation in a regulatory region of gene complement factor B (CFB) as the possible driving mechanism of widespread MHC gene suppression. Our results demonstrate MHC molecule downregulation by P. aeruginosa-derived EVs in lung macrophages, which is consistent with an immune evasion strategy employed by a prokaryote in a host-pathogen interaction, potentially leading to decreased pulmonary bacterial clearance

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases

CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice.

Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD

The Vaginal Microbiome: Disease, Genetics and the Environment

The vagina is an interactive interface between the host and the environment. Its surface is covered by a protective epithelium colonized by bacteria and other microorganisms. The ectocervix is nonsterile, whereas the endocervix and the upper genital tract are assumed to be sterile in healthy women. Therefore, the cervix serves a pivotal role as a gatekeeper to protect the upper genital tract from microbial invasion and subsequent reproductive pathology. Microorganisms that cross this barrier can cause preterm labor, pelvic inflammatory disease, and other gynecologic and reproductive disorders. Homeostasis of the microbiome in the vagina and ectocervix plays a paramount role in reproductive health. Depending on its composition, the microbiome may protect the vagina from infectious or non-infectious diseases, or it may enhance its susceptibility to them. Because of the nature of this organ, and the fact that it is continuously colonized by bacteria from birth to death, it is virtually certain that this rich environment evolved in concert with its microbial flora. Specific interactions dictated by the genetics of both the host and microbes are likely responsible for maintaining both the environment and the microbiome. However, the genetic basis of these interactions in both the host and the bacterial colonizers is currently unknown. _Lactobacillus_ species are associated with vaginal health, but the role of these species in the maintenance of health is not yet well defined. Similarly, other species, including those representing minor components of the overall flora, undoubtedly influence the ability of potential pathogens to thrive and cause disease. Gross alterations in the vaginal microbiome are frequently observed in women with bacterial vaginosis, but the exact etiology of this disorder is still unknown. There are also implications for vaginal flora in non-infectious conditions such as pregnancy, pre-term labor and birth, and possibly fertility and other aspects of women’s health. Conversely, the role of environmental factors in the maintenance of a healthy vaginal microbiome is largely unknown. To explore these issues, we have proposed to address the following questions:

*1.	Do the genes of the host contribute to the composition of the vaginal microbiome?* We hypothesize that genes of both host and bacteria have important impacts on the vaginal microbiome. We are addressing this question by examining the vaginal microbiomes of mono- and dizygotic twin pairs selected from the over 170,000 twin pairs in the Mid-Atlantic Twin Registry (MATR). Subsequent studies, beyond the scope of the current project, may investigate which host genes impact the microbial flora and how they do so.
*2.	What changes in the microbiome are associated with common non-infectious pathological states of the host?* We hypothesize that altered physiological (e.g., pregnancy) and pathologic (e.g., immune suppression) conditions, or environmental exposures (e.g., antibiotics) predictably alter the vaginal microbiome. Conversely, certain vaginal microbiome characteristics are thought to contribute to a woman’s risk for outcomes such as preterm delivery. We are addressing this question by recruiting study participants from the ~40,000 annual clinical visits to women’s clinics of the VCU Health System.
*3.	What changes in the vaginal microbiome are associated with relevant infectious diseases and conditions?* We hypothesize that susceptibility to infectious disease (e.g. HPV, _Chlamydia_ infection, vaginitis, vaginosis, etc.) is impacted by the vaginal microbiome. In turn, these infectious conditions clearly can affect the ability of other bacteria to colonize and cause pathology. Again, we are exploring these issues by recruiting participants from visitors to women’s clinics in the VCU Health System.

Three kinds of sequence data are generated in this project: i) rDNA sequences from vaginal microbes; ii) whole metagenome shotgun sequences from vaginal samples; and iii) whole genome shotgun sequences of bacterial clones selected from vaginal samples. The study includes samples from three vaginal sites: mid-vaginal, cervical, and introital. The data sets also include buccal and perianal samples from all twin participants. Samples from these additional sites are used to test the hypothesis of a per continuum spread of bacteria in relation to vaginal health. An extended set of clinical metadata associated with these sequences are deposited with dbGAP. We have currently collected over 4,400 samples from ~100 twins and over 450 clinical participants. We have analyzed and deposited data for 480 rDNA samples, eight whole metagenome shotgun samples, and over 50 complete bacterial genomes. These data are available to accredited investigators according to NIH and Human Microbiome Project (HMP) guidelines. The bacterial clones are deposited in the Biodefense and Emerging Infections Research Resources Repository ("http://www.beiresources.org/":http://www.beiresources.org/). 

In addition to the extensive sequence data obtained in this study, we are collecting metadata associated with each of the study participants. Thus, participants are asked to complete an extensive health history questionnaire at the time samples are collected. Selected clinical data associated with the visit are also obtained, and relevant information is collected from the medical records when available. This data is maintained securely in a HIPAA-compliant data system as required by VCU’s Institutional Review Board (IRB). The preponderance of these data (i.e., that judged appropriate by NIH staff and VCU’s IRB are deposited at dbGAP ("http://www.ncbi.nlm.nih.gov/gap":http://www.ncbi.nlm.nih.gov/gap). Selected fields of this data have been identified by NIH staff as ‘too sensitive’ and are not available in dbGAP. Individuals requiring access to these data fields are asked to contact the PI of this project or NIH Program Staff. 
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Heat stored in the Earth system 1960–2020: where does the energy go?

The Earth climate system is out of energy balance, and heat has accumulated continuously over the past decades, warming the ocean, the land, the cryosphere, and the atmosphere. According to the Sixth Assessment Report by Working Group I of the Intergovernmental Panel on Climate Change, this planetary warming over multiple decades is human-driven and results in unprecedented and committed changes to the Earth system, with adverse impacts for ecosystems and human systems. The Earth heat inventory provides a measure of the Earth energy imbalance (EEI) and allows for quantifying how much heat has accumulated in the Earth system, as well as where the heat is stored. Here we show that the Earth system has continued to accumulate heat, with 381±61 ZJ accumulated from 1971 to 2020. This is equivalent to a heating rate (i.e., the EEI) of 0.48±0.1 W m−2. The majority, about 89 %, of this heat is stored in the ocean, followed by about 6 % on land, 1 % in the atmosphere, and about 4 % available for melting the cryosphere. Over the most recent period (2006–2020), the EEI amounts to 0.76±0.2 W m−2. The Earth energy imbalance is the most fundamental global climate indicator that the scientific community and the public can use as the measure of how well the world is doing in the task of bringing anthropogenic climate change under control. Moreover, this indicator is highly complementary to other established ones like global mean surface temperature as it represents a robust measure of the rate of climate change and its future commitment. We call for an implementation of the Earth energy imbalance into the Paris Agreement's Global Stocktake based on best available science. The Earth heat inventory in this study, updated from von Schuckmann et al. (2020), is underpinned by worldwide multidisciplinary collaboration and demonstrates the critical importance of concerted international efforts for climate change monitoring and community-based recommendations and we also call for urgently needed actions for enabling continuity, archiving, rescuing, and calibrating efforts to assure improved and long-term monitoring capacity of the global climate observing system. The data for the Earth heat inventory are publicly available, and more details are provided in Table 4