A computational simulated control system for a high-force pneumatic muscle actuator: system definition and application as an augmented orthosis

High-force pneumatic muscle actuators (PMAs) are used for force assistance with minimal displacement applications. However, poor control due to dynamic nonlinearities has limited PMA applications. A simulated control system is developed consisting of: (1) a controller relating an input position angle to an output proportional pressure regulator voltage, (2) a phenomenological model of the PMA with an internal dynamic force loop (system time constant information), (3) a physical model of a human sit-to-stand task and (4) an external position angle feed-back loop. The results indicate that PMA assistance regarding the human sit-to-stand task is feasible within a specified PMA operational pressure range.     

Image-derived modeling of nucleus strain amplification associated with chromatin heterogeneity

Beyond the critical role of cell nuclei in gene expression and DNA replication, they also have a significant influence on cell mechanosensation and migration. Nuclear stiffness can impact force transmission and, furthermore, act as a physical barrier to translocation across tight spaces. As such, it is of wide interest to accurately characterize nucleus mechanical behavior. In this study, we present a computational investigation of the in situ deformation of a heterogeneous chondrocyte nucleus. A methodology is developed to accurately reconstruct a three-dimensional finite-element model of a cell nucleus from confocal microscopy. By incorporating the reconstructed nucleus into a chondrocyte model embedded in pericellular and extracellular matrix, we explore the relationship between spatially heterogeneous nuclear DNA content, shear stiffness, and resultant shear strain. We simulate an externally applied extracellular matrix shear deformation and compute intranuclear strain distributions, which are directly compared with corresponding experimentally measured distributions. Simulations suggest that the mechanical behavior of the nucleus is highly heterogeneous, with a nonlinear relationship between experimentally measured grayscale values and corresponding local shear moduli (μ_n). Three distinct phases are identified within the nucleus: a low-stiffness mRNA-rich interchromatin phase (0.17 kPa ≤ μ_n ≤ 0.63 kPa), an intermediate-stiffness euchromatin phase (1.48 kPa ≤ μ_n ≤ 2.7 kPa), and a high-stiffness heterochromatin phase (3.58 kPa ≤ μ_n ≤ 4.0 kPa). Our simulations also indicate that disruption of the nuclear envelope associated with lamin A/C depletion significantly increases nuclear strain in regions of low DNA concentration. We further investigate a phenotypic shift of chondrocytes to fibroblast-like cells, a signature for osteoarthritic cartilage, by increasing the contractility of the actin cytoskeleton to a level associated with fibroblasts. Peak nucleus strains increase by 35% compared to control, with the nucleus becoming more ellipsoidal. Our findings may have broad implications for current understanding of how local DNA concentrations and associated strain amplification can impact cell mechanotransduction and drive cell behavior in development, migration, and tumorigenesis.     

Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay

Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, the authors designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96-well plate to fully characterize T-cell viability, function, growth and differentiation. Peripheral blood mononuclear cells (PBMCs) from six consenting donors were seeded at 100 000 cells per well with pools of cytomegalovirus peptides from IE1 and pp65 and combinations of IL-15, IL-6, IL-21, interferon alpha, IL-12, IL-18, IL-4 and IL-7. Ten-day cultures were tested by 13-color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers and interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) expression. Combinations of IL-15/IL-6 and IL-4/IL-7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold, respectively, compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL-15/IL-6 and 9-fold in IL-4/IL-7 cultures (P < 0.0001). CD4+ T cells expanded 27-fold in IL-4/IL-7 and 15-fold in IL-15/IL-6 (P < 0.0001). CD45RO+ CCR7– effector memory (CD45RO+ CCR7– CD3+), central memory (CD45RO+ CCR7+ CD3+), terminal effector (CD45RO– CCR7– CD3+), and naive (CD45RO– CCR7+ CD3+). T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL-6 and IL-15/IL-7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL-15/IL-6 producing IFNγ (7.6% producing both TNFα and IFNγ) and 18.5% of CD3+ cells grown in IL-4/IL-7 producing IFNγ (9% producing both TNFα and IFNγ). This study shows the utility of this single-plate assay to rapidly identify optimal growth conditions for VST manufacture using only 10⁷ PBMCs.     

Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions

Foxp3(+) regulatory T (T(reg)) cells suppress different types of immune responses to help maintain homeostasis in the body. How T(reg) cells regulate humoral immunity, including germinal center reactions, is unclear. Here we identify a subset of T(reg) cells expressing CXCR5 and Bcl-6 that localize to the germinal centers in mice and humans. The expression of CXCR5 on T(reg) cells depends on Bcl-6. These CXCR5(+)Bcl-6(+) T(reg) cells are absent in the thymus but can be generated de novo from CXCR5(-)Foxp3(+) natural T(reg) precursors. A lack of CXCR5(+) T(reg) cells leads to greater germinal center reactions including germinal center B cells, affinity maturation of antibodies and the differentiation of plasma cells. These results unveil a Bcl-6-CXCR5 axis in T(reg) cells that drives the development of follicular regulatory T (T(FR)) cells that function to inhibit the germinal center reactions.     

Insect immune responses to nematode parasites

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.     

PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells

Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(ΔB) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(ΔB) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing.     

Determination of free desmosine and isodesmosine as urinary biomarkers of lung disorder using ultra performance liquid chromatography-ion mobility-mass spectrometry

The elastin degradation products, desmosine (DES) and isodesmosine (IDES) are highly stable, cross-linking amino-acids that are unique to mature elastin. The excretion of DES/IDES in urine, in the free form and with associated peptide fragments, provides an indicator of lung damage in chronic obstructive pulmonary disease (COPD). A quantitative ion mobility-mass spectrometry (IM-MS) method has been developed for the analysis of free DES/IDES in urine with deuterated IDES as an internal standard. Resolution of DES/IDES isomers was achieved in less than five minutes using ultra performance liquid chromatography (UPLC) combined with ion pairing. The optimized UPLC-IM-MS method provided a linear dynamic range of 10-300 ng/mL and a limit of quantitation of 0.028 ng/mL for IDES and 0.03 ng/mL for DES (0.55 ng and 0.61 ng on column respectively). The method reproducibility (%RSD) was <4% for DES and IDES. The UPLC-IM-MS method was applied to the analysis of urine samples obtained from healthy volunteers and COPD patients. The DES/IDES concentrations in healthy and COPD urine showed an increase in DES (79%) and IDES (74%) in the COPD samples, relative to healthy controls. The incorporation of an IM separation prior to m/z measurement by MS was shown to reduce non-target ion responses from the bio-fluid matrix.     

A connection between colony biomass and death in Caribbean reef-building corals

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994-2007), eleven years in the Exuma Cays, Bahamas (1995-2006), and four years in Puerto Morelos, Mexico (2003-2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1-4 m) compared to deeper-dwelling conspecifics (12-15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels.     

Abrogation of the twin arginine transport system in Salmonella enterica serovar Typhimurium leads to colonization defects during infection

TatC (STM3975) is a highly conserved component of the Twin Arginine Transport (Tat) systems that is required for transport of folded proteins across the inner membrane in gram-negative bacteria. We previously identified a ΔtatC mutant as defective in competitive infections with wild type ATCC14028 during systemic infection of Salmonella-susceptible BALB/c mice. Here we confirm these results and show that the ΔtatC mutant is internalized poorly by cultured J774-A.1 mouse macrophages a phenotype that may be related to the systemic infection defect. This mutant is also defective for short-term intestinal and systemic colonization after oral infection of BALB/c mice and is shed in reduced numbers in feces from orally infected Salmonella-resistant (CBA/J) mice. We show that the ΔtatC mutant is highly sensitive to bile acids perhaps resulting in the defect in intestinal infection that we observe. Finally, the ΔtatC mutant has an unusual combination of motility phenotypes in Salmonella; it is severely defective for swimming motility but is able to swarm well. The ΔtatC mutant has a lower amount of flagellin on the bacterial surface during swimming motility but normal levels under swarming conditions.