Honoring the gift: The transformative potential of transplant-declined human organs.

For decades, transplantation has been a life-saving treatment for those fortunate enough to gain access. Nevertheless, many patients die waiting for an organ and countless more never make it onto the waitlist because of a shortage of donor organs. Concurrently, thousands of donated organs are declined for transplant each year because of concerns about poor outcomes post-transplant. The decline of any donated organ-even if medically justified-is tragic for both the donor family and potential recipients. In this Personal Viewpoint, we discuss the need for a new mindset in how we honor the gift of organ donation. We believe that the use of transplant-declined human organs in translational research has the potential to hasten breakthrough discoveries in a multitude of scientific and medical areas. More importantly, such breakthroughs will allow us to properly value every donated organ. We further discuss the many practical challenges that such research presents and offer some possible solutions based on experiences in our own research laboratories. Finally, we share our perspective on what we believe are the necessary next steps to ensure a future where every donated organ realizes its full potential to impact the lives of current and future patients.

Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris.

Hydrophobins are small highly surface-active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression host Pichia pastoris. One-, 2-, and 3-copy-HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction. Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression of PDI1 or ERO1 caused insignificant changes in HFBI secretion in 1- and 2-copy strains, but a statistically significant HFBI secretion increase in 3-copy strain. KAR2 overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3-copy-HFBI secreting 22±1.6 fold more than the 1-copy-HFBI/no chaperone strain. The highest increase was seen in 3-copy-HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1-copy-HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small-scale format used in this study.

Foreign body response to synthetic polymer biomaterials and the role of adaptive immunity.

Implanted biomaterials elicit a series of distinct immune and repair-like responses that are collectively known as the foreign body reaction (FBR). These include processes involving innate immune inflammatory cells and wound repair cells that contribute to the encapsulation of biomaterials with a dense collagenous and largely avascular capsule. Numerous studies have shown that the early phase is dominated by macrophages that fuse to form foreign body giant cells that are considered a hallmark of the FBR. With the advent of more precise cell characterization techniques, specific macrophage subsets have been identified and linked to more or less favorable outcomes. Moreover, studies comparing synthetic- and natural-based polymer biomaterials have allowed the identification of macrophage subtypes that distinguish between fibrotic and regenerative responses. More recently, cells associated with adaptive immunity have been shown to participate in the FBR to synthetic polymers. This suggests the existence of cross-talk between innate and adaptive immune cells that depends on the nature of the implants. However, the exact participation of adaptive immune cells, such as T and B cells, remains unclear. In fact, contradictory studies suggest either the independence or dependence of the FBR on these cells. Here, we review the evidence for the involvement of adaptive immunity in the FBR to synthetic polymers with a focus on cellular and molecular components. In addition, we examine the possibility that such biomaterials induce specific antibody responses resulting in the engagement of adaptive immune cells.

Overexpression of Spry1 in chondrocytes causes attenuated FGFR ubiquitination and sustained ERK activation resulting in chondrodysplasia.

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.

Meat Bars: A Survey To Assess Consumer Familiarity and Preparation Parameters and a Challenge Study To Quantify Viability of Shiga Toxin-Producing Escherichia coli Cells during Processing and Storage.

Meat bars are dried snacks containing a mixture of meat, berries, and nuts. To explore consumer awareness of meat bars, we conducted two online, nationally representative surveys and established that 70.8% (743 of 1,050) of U.S. citizens were unfamiliar with this product. When asked to check all answers that applied, most of the 545 respondents (who were recruited based on their familiarity with meat bars) preferred beef (n = 385) as the protein source, followed by chicken (n = 293), pork (n = 183), and turkey (n = 179). Most meat bars were purchased from grocery stores (n = 447), followed by online orders (n = 130) and outdoor stores (n = 120). When asked specifically whether they made their own meat bars, 17.8% of respondents (97 of 545) replied "yes," the majority (52 of 97, 54%) of which obtained recipes online. Some 69.1% (67 of 97) measured the internal temperature of the meat during dehydration, but only 10.3% (10 of 97) confirmed the internal temperature by using a thermometer. Given the paucity of information available on the fate of pathogenic or spoilage bacteria associated with meat bars, as another component of this study, batter was prepared with or without encapsulated citric acid (ECA; 0.74%) added to a formulation of ground beef (65%; 90% lean, 10% fat), chopped pecans (15%), golden flaxseed flour (9.7%), chopped cranberries (5.0%), chopped sunflower seeds (3.1%), sea salt (1.1%), black pepper (0.8%), and celery powder (0.35%). Batter was inoculated (ca. 6.5 log CFU/g) with Shiga toxin-producing Escherichia coli (STEC), portioned by hand (40 ± 0.1 g each), and then dried in a commercial dehydrator. Regardless of the drying treatment, inclusion of ECA in the batter resulted in a pH decrease from ca. 5.5 to ca. 4.7 to 5.0 in the finished product. Without ECA, when meat bars were dried at 62.8°C for 6 h, 71.1°C for 4 h, or 62.8°C for 2 h and then 71.1°C for 2 h, levels of STEC decreased by ca. 6.2, 6.3, or 5.2 log CFU/g, respectively. With ECA, STEC decreased by ca. 6.0, 6.6, or 6.0 log CFU/g in meat bars dried at 62.8°C for 6 h, 71.1°C for 4 h, or 62.8°C for 2 h and then 71.1°C for 2 h, respectively. Our results confirmed that a ≥5.0-log reduction in STEC could be achieved in meat bars formulated with or without ECA under all dehydration conditions tested.

Sef inhibits fibroblast growth factor signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent ERK activation.

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration as well as differentiation events such as angiogenesis, osteogenesis, and chondrogenesis. Recently, genetic screens in Drosophila and gene expression screens in zebrafish have resulted in the identification of several feedback inhibitors of FGF signaling. One of these, Sef (similar expression to fgf genes), encodes a transmembrane protein that belongs to the FGF synexpression group. Here we show that like zebrafish Sef (zSef), mouse Sef (mSef) interacts with FGFR1 and that the cytoplasmic domain of mSef mediates this interaction. Overexpression of mSef in NIH3T3 cells results in a decrease in FGF-induced cell proliferation associated with a decrease in Tyr phosphorylation of FGFR1 and FRS2. As a consequence, there is a reduction in the phosphorylation of Raf-1 at Ser(338), MEK1/2 at Ser(217) and Ser(221), and ERK1/2 at Thr(202) and Tyr(204). Furthermore, mSef inhibits ERK activation mediated by a constitutively activated FGFR1 but not by a constitutively active Ras and decreases FGF but not PDGF-mediated activation of Akt. These results indicate that Sef exerts its inhibitory effects at the level of FGFR and upstream of Ras providing an additional level of negative regulation of FGF signaling.

Sef interacts with TAK1 and mediates JNK activation and apoptosis.

Sef was recently identified as a negative regulator of fibroblast growth factor (FGF) signaling in a genetic screen of zebrafish and subsequently in mouse and humans. By inhibiting FGFR1 tyrosine phosphorylation and/or Ras downstream events, Sef inhibits FGF-mediated ERK activation and cell proliferation as well as PC12 cell differentiation. Here we show that Sef and a deletion mutant of Sef lacking the extracellular domain (SefIC) physically interact with TAK1 (transforming growth factor-beta-associated kinase) and activate JNK through a TAK1-MKK4-JNK pathway. Sef and SefIC overexpression also resulted in apoptotic cell death, while dominant negative forms of MKK4 and TAK1 blocked Sef-mediated JNK activation and attendant 293T cell apoptosis. These investigations reveal a novel activating function of Sef that is distinct from its inhibitory effect on FGF receptor signaling and ERK activation.