Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.     

Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca²⁺. The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H₂O₂) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.     

Middle phalanx skeletal morphology in the hand: Can it predict flexor tendon size and attachments?

Specific sites on the palmar diaphysis of the manual middle phalanges provide attachment for the flexor digitorum superficialis (FDS) tendon. It has been assumed in the literature that lateral palmar fossae on these bones reflect locations for these attachments and offer evidence for relative size of the flexor tendon. This assumption has led to predictions about relative FDS muscle force potential from sizes of fossae on fossil hominin middle phalanges. Inferences about locomotor capabilities of fossil hominins in turn have been drawn from the predicted force potential of the flexor muscle. The study reported here provides a critical first step in evaluating hypotheses about behavioral implications of middle phalangeal morphology in fossil hominins, by testing the hypothesis that the lateral fossae reflect the size of the FDS tendon and the location of the terminal FDS tendon attachments on the middle phalanx. The middle phalangeal region was dissected in 43 individuals from 16 primate genera, including humans. Qualitative observations were made of tendon attachment locations relative to the lateral fossae. Length measurements of the fossae were tested as predictors of FDS tendon cross-sectional area and of FDS attachment tendon lengths. Our results lead to the conclusion that the hypothesis must be rejected, and that future attention should focus on functional implications of the palmar median bar associated with the lateral fossae.     

Male inclusion in randomized controlled trials of lifestyle weight loss interventions

The prevalence of obesity is similar for men (32.2%) and women (35.5%). It has been assumed that lifestyle weight loss interventions have been developed and tested in predominately female samples, but this has not been systematically investigated. The aim of this review was to investigate total and ethnic male inclusion in randomized controlled trials of lifestyle interventions. PUBMED, MEDLINE, and PSYCHINFO were searched for randomized controlled trials of lifestyle weight loss interventions (N = 244 studies with a total of 95,207 participants) published in the last 10 years (1999-2009). A trial must be in English, included weight loss as an outcome, and tested a dietary, exercise, and/or other behavioral intervention for weight loss. Results revealed samples were on average 27% male vs. 73% female (P < 0.001). Trials recruiting a diseased sample included a larger proportion of males than those not targeting a disease (35% vs. 21%; P < 0.001). About 32% of trials used exclusively female samples, whereas only 5% used exclusively male samples (P < 0.001). No studies in the past 10 years specifically targeted minority males. Ethnic males identified composed 1.8% of total participants in US studies. Only 24% of studies that underrepresented males provided a reason. Males, especially ethnic males, are underrepresented in lifestyle weight loss trials.     

WNT5A signaling affects pituitary gland shape

Wnt signaling is important in organogenesis, and aberrant signaling in mature cells is associated with tumorigenesis. Several members of the Wnt family of signaling molecules are expressed in the developing pituitary gland. Wnt5a is expressed in the neuroectoderm that induces pituitary gland development and has been proposed to influence pituitary cell specification. We discovered that mice deficient in Wnt5a display abnormal morphology in the dorsal part of the developing pituitary. The expression of downstream effectors of the canonical Wnt pathway is not altered, and expression of genes in other signaling pathways such as Shh, Fgf8, Fgf10 and Fgfr2b is intact. Prop1 and Hesx1 are also important for normal shape of the pituitary primordium, but their expression is unaltered in the Wnt5a mutants. Specification of the hormone-producing cell types of the mature anterior pituitary gland occurs appropriately. This study suggests that the primary role of Wnt5a in the developing pituitary gland is in establishment of the shape of the gland.     

Give What You Get: Capuchin Monkeys (Cebus apella) and 4-Year-Old Children Pay Forward Positive and Negative Outcomes to Conspecifics

The breadth of human generosity is unparalleled in the natural world, and much research has explored the mechanisms underlying and motivating human prosocial behavior. Recent work has focused on the spread of prosocial behavior within groups through paying-it-forward, a case of human prosociality in which a recipient of generosity pays a good deed forward to a third individual, rather than back to the original source of generosity. While research shows that human adults do indeed pay forward generosity, little is known about the origins of this behavior. Here, we show that both capuchin monkeys (Cebus apella) and 4-year-old children pay forward positive and negative outcomes in an identical testing paradigm. These results suggest that a cognitively simple mechanism present early in phylogeny and ontogeny leads to paying forward positive, as well as negative, outcomes.     

Energetic conditions promoting top-down control of prey by predators

Humans remove large amounts of biomass from natural ecosystems, and large bodied high trophic level animals are especially sensitive and vulnerable to exploitation. The effects of removing top-predators on food webs are often difficult to predict because of limited information on species interaction strengths. Here we used a three species predator-prey model to explore relationships between energetic properties of trophodynamic linkages and interaction strengths to provide heuristic rules that indicate observable energetic conditions that are most likely to lead to stable and strong top-down control of prey by predator species. We found that strong top-down interaction strengths resulted from low levels of energy flow from prey to predators. Strong interactions are more stable when they are a consequence of low per capita predation and when predators are subsidized by recruitment. Diet composition also affects stability, but the relationship depends on the form of the functional response. Our results imply that for generalist satiating predators, strong top-down control on prey is most likely for prey items that occupy a small portion of the diet and when density dependent recruitment is moderately high.     

Posttranslational modification of gluten shapes TCR usage in celiac disease

Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced enteropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational conversion of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide-MHC complex. In this study, we report that there is a biased usage of TCR Vβ6.7 chain among TCRs reactive to the immunodominant DQ2-α-II gliadin epitope. We isolated Vβ6.7 and DQ2-αII tetramer-positive CD4(+) T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semipublic response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3β loop. Functional testing of a prototype DQ2-α-II-reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-α-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags.     

A practical approach in bioreactor scale‐up and process transfer using a combination of constant P/V and vvm as the criterion

Bioreactor scale‐up is a critical step in the production of therapeutic proteins such as monoclonal antibodies (MAbs). With the scale‐up criterion such as similar power input per volume or O₂ volumetric mass transfer coefficient ( ), adequate oxygen supply and cell growth can be largely achieved. However, CO₂ stripping in the growth phase is often inadequate. This could cascade down to increased base addition and osmolality, as well as residual lactate increase and compromised production and product quality. Here we describe a practical approach in bioreactor scale‐up and process transfer, where bioreactor information may be limited. We evaluated the sparger and (CO₂ volumetric mass transfer coefficient) from a range of bioreactor scales (3–2,000 L) with different spargers. Results demonstrated that for oxygen is not an issue when scaling from small‐scale to large‐scale bioreactors at the same gas flow rate per reactor volume (vvm). Results also showed that sparging CO₂ stripping, , is dominated by the gas throughput. As a result, a combination of a minimum constant vvm air or N₂ flow with a similar specific power was used as the general scale‐up criterion. An equation was developed to determine the minimum vvm required for removing CO₂ produced from cell respiration. We demonstrated the effectiveness of using such scale‐up criterion with five MAb projects exhibiting different cell growth and metabolic characteristics, scaled from 3 to 2,000 L bioreactors across four sites. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1146–1159, 2017