Rapid, Phase-free Detection of Long Identity-by-Descent Segments Enables Effective Relationship Classification.

Identity-by-descent (IBD) segments are a useful tool for applications ranging from demographic inference to relationship classification, but most detection methods rely on phasing information and therefore require substantial computation time. As genetic datasets grow, methods for inferring IBD segments that scale well will be critical. We developed IBIS, an IBD detector that locates long regions of allele sharing between unphased individuals, and benchmarked it with Refined IBD, GERMLINE, and TRUFFLE on 3,000 simulated individuals. Phasing these with Beagle 5 takes 4.3 CPU days, followed by either Refined IBD or GERMLINE segment detection in 2.9 or 1.1 h, respectively. By comparison, IBIS finishes in 6.8 min or 7.8 min with IBD2 functionality enabled: speedups of 805-946× including phasing time. TRUFFLE takes 2.6 h, corresponding to IBIS speedups of 20.2-23.3×. IBIS is also accurate, inferring ≥7 cM IBD segments at quality comparable to Refined IBD and GERMLINE. With these segments, IBIS classifies first through third degree relatives in real Mexican American samples at rates meeting or exceeding other methods tested and identifies fourth through sixth degree pairs at rates within 0.0%-2.0% of the top method. While allele frequency-based approaches that do not detect segments can infer relationship degrees faster than IBIS, the fastest are biased in admixed samples, with KING inferring 30.8% fewer fifth degree Mexican American relatives correctly compared with IBIS. Finally, we ran IBIS on chromosome 2 of the UK Biobank dataset and estimate its runtime on the autosomes to be 3.3 days parallelized across 128 cores.

Transcriptional regulation of proteoglycan 4 by 17ß-estradiol in immortalized baboon temporomandibular joint disc cells.

Temporomandibular joint disorders (TMDs) affect a significant portion of the population of the USA, with the majority of those seeking treatment being women of childbearing age. Owing to this striking sexual dimorphism it has been postulated that sex hormones play a role in the maintenance of normal temporomandibular joint (TMJ) function. Proteoglycan 4 (PRG4) is a secreted lubricating molecule required for maintaining low frictional levels within articular joints; however, its role in the TMJ is not well characterized. In this study we describe the development of immortalized baboon cells isolated from specific regions of the TMJ disc and their use in the investigation of PRG4 expression and localization patterns in the TMJ. We identified conserved estrogen response elements within the 5' flanking region of the PRG4 gene of several species, and found that treatment of baboon TMJ disc cells with estrogen led to reduced PRG4 promoter activity and reduced expression of PRG4 mRNA in vitro. The observed negative regulation of PRG4 by estrogen could lead to increased friction and degradation of joint components over time. This study, for the first time, provides evidence of the regulatory potential of estrogen on PRG4 gene expression and suggests a novel etiology for the gender disparity observed among TMD patients.

3D-Printed Multidrug-Eluting Stent from Graphene-Nanoplatelet-Doped Biodegradable Polymer Composite.

Patients with percutaneous coronary intervention generally receive either bare metal stents or drug-eluting stents to restore the normal blood flow. However, due to the lack of stent production with an individual patient in mind, the same level of effectiveness may not be possible in treating two different clinical scenarios. This study introduces for the first time the feasibility of a patient-specific stenting process constructed from direct 3D segmentation of medical images using direct 3D printing of biodegradable polymer-graphene composite with dual drug incorporation. A biodegradable polymer-carbon composite is prepared doped with graphene nanoplatelets to achieve controlled release of combinatorics as anticoagulation and antirestenosis agents. This study develops a technology prototyped for personalized stenting. An in silico analysis is performed to optimize the stent design for printing and its prediction of sustainability under force exerted by coronary artery or blood flow. A holistic approach covering in silico to in situ-in vivo establishes the structural integrity of the polymer composite, its mechanical properties, drug loading and release control, prototyping, functional activity, safety, and feasibility of placement in coronary artery of swine.

Transcriptional organization of pneumococcal psrP-secY2A2 and impact of GtfA and GtfB deletion on PsrP-associated virulence properties.

Pneumococcal serine-rich repeat protein (PsrP) is a glycoprotein that mediates Streptococcus pneumoniae attachment to lung cells and promotes biofilm formation. Herein, we investigated the transcriptional organization of psrP-secY2A2, the 37-kbp pathogenicity island encoding PsrP and its accessory genes. PCR amplification of cDNA and RNA-seq analysis found psrP-secY2A2 to be minimally composed of three operons: psrP-glyA, glyB, and glyC-asp5. Transcription of all three operons was greatest during biofilm growth and immunoblot analyses confirmed increased PsrP production by biofilm pneumococci. Using gas chromatography-mass spectrometry we identified monomeric N-acetylglucosamine as the primary glycoconjugate present on a recombinant intracellular version of PsrP, i.e. PsrP1-734. This finding was validated by immunoblot using lectins with known carbohydrate specificities. We subsequently deleted gtfA and gtfB, the GTFs thought to be responsible for addition of O-linked N-acetylglucosamine, and tested for PsrP and its associated virulence properties. These deletions negatively affected our ability to detect PsrP1-734 in bacterial whole cell lysates. Moreover, S. pneumoniae mutants lacking these genes pheno-copied the psrP mutant and were attenuated for: biofilm formation, adhesion to lung epithelial cells, and pneumonia in mice. Our studies identify the transcriptional organization of psrP-secY2A2 and show the indispensable role of GtfA and GtfB on PsrP-mediated pneumococcal virulence.

Elevated A20 contributes to age-dependent macrophage dysfunction in the lungs.

Advanced age is associated with chronic low-grade inflammation (i.e. inflamm-aging) and poor macrophage function that includes a weak pro-inflammatory cytokine response to bacteria and diminished phagocytosis (i.e. age-dependent macrophage dysfunction [ADMD]). One reason for this is that ADMD is associated with poor NFκB and MAPK activation following Toll-like receptor stimulation. Herein, we tested the hypothesis that inflamm-aging induces production of A20, a cytosolic and homeostatic suppressor of the NFκB and MAPK signaling cascades that deubiquitinates (i.e. inactivates) the common upstream signaling molecule TRAF6, and this is responsible for ADMD. Western blots and immunohistochemistry comparing tissues from young, mature, and aged C57BL/6 mice indicated that A20 was strongly elevated in the lungs of aged mice but not in other tissues. Elevated A20 was also detected in alveolar macrophages (AM) from aged mice. In contrast CYLD, a second deubiquitinase that also negatively regulates the NFκB pathway was decreased with aging. Following co-incubation of AM with the bacteria Streptococcus pneumoniae, TRAF6 polyubiquitination was diminished in AM isolated from aged versus young mice. A20 production was inducible in the J774A.1 macrophage cell line and C57BL/6AM by overnight incubation with TNFα but not IL-6. Retrovirus-induced expression of A20 in J774A.1 cells resulted in their diminished production of IL-6 following exposure to S. pneumoniae but had no effect on levels of phagocytosis. Overnight incubation of AM from young mice with TNFα also resulted in a dampened IL-6 response to S. pneumoniae. Finally, dietary supplementation of aged mice with anti-inflammatory n-3 polyunsaturated fatty acids in the form of fish oil lowered lung A20 levels and enhanced resistance, including a 100-fold reduction in bacterial titers in the lungs, to experimental challenge with S. pneumoniae. We conclude that elevated A20 due to TNFα partially explains the ADMD phenotype and that ADMD is potentially reversible.

Streptococcus pneumoniae biofilm formation is strain dependent, multifactorial, and associated with reduced invasiveness and immunoreactivity during colonization.

UNLABELLED: Biofilms are thought to play an important role during colonization of the nasopharynx by Streptococcus pneumoniae, yet how they form in vivo and the determinants responsible remain unknown. Using scanning electron microscopy, we show that biofilm aggregates of increasing complexity form on murine nasal septa following intranasal inoculation. These biofilms were highly distinct from in vitro biofilms, as they were discontiguous and appeared to incorporate nonbacterial components such as intact host cells. Biofilms initially formed on the surface of ciliated epithelial cells and, as cells were sloughed off, were found on the basement membrane. The size and number of biofilm aggregates within nasal lavage fluid were digitally quantitated and revealed strain-specific capabilities that loosely correlated with the ability to form robust in vitro biofilms. We tested the ability of isogenic mutants deficient in CbpA, pneumolysin, hydrogen peroxide, LytA, LuxS, CiaR/H, and PsrP to form biofilms within the nasopharynx. This analysis revealed that CiaR/H was absolutely required for colonization, that PsrP and SpxB strongly impacted aggregate formation, and that other determinants affected aggregate morphology in a modest fashion. We determined that mice colonized with ΔpsrP mutants had greater levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1ß, and KC in nasal lavage fluid than did mice colonized with wild-type controls. This phenotype correlated with a diminished capacity of biofilm pneumococci to invade host cells in vitro despite enhanced attachment. Our results show that biofilms form during colonization and suggest that they may contribute to persistence through a hyperadhesive, noninvasive state that elicits a dampened cytokine response. IMPORTANCE: This work demonstrates the first temporal characterization of Streptococcus pneumoniae biofilm formation in vivo. Our results show that the morphology of biofilms formed by both invasive and noninvasive clinical isolates in vivo is distinct from that of formed biofilms in vitro, yet propensity to form biofilms in vivo loosely correlates with the degree of in vitro biofilm formation on a microtiter plate. We show that host components, including intact host cells, influence the formation of in vivo structures. We also found that efficient biofilm formation in vivo requires multiple bacterial determinants. While some factors are essential for in vivo biofilm formation (CiaRH, PsrP, and SpxB), other factors are less critical (CbpA, LytA, LuxS, and pneumolysin). In comparison to their planktonic counterparts, biofilm pneumococci are hyperadhesive but less invasive and elicit a weaker proinflammatory cytokine response. These findings give insight into the requirements for and potential role of biofilms during prolonged asymptomatic colonization.