Burial of nonpolar surface area and thermodynamic stabilization of globins as a function of chain elongation

Proteins are biosynthesized from N to C terminus before they depart from the ribosome and reach their bioactive state in the cell. At present, very little is known about the evolution of conformation and the free energy of the nascent protein with chain elongation. These parameters critically affect the extent of folding during ribosome‐assisted biosynthesis. Here, we address the impact of vectorial amino acid addition on the burial of nonpolar surface area and on the free energy of native‐like structure formation in the absence of the ribosomal machinery. We focus on computational predictions on proteins bearing the globin fold, which is known to encompass the 3/3, 2/2, and archaeal subclasses. We find that the burial of nonpolar surface increases progressively with chain elongation, leading to native‐like conformations upon addition of the last C‐terminal residues, corresponding to incorporation of the last two helices. Additionally, the predicted folding entropy for generating native‐like structures becomes less unfavorable at nearly complete chain lengths, suggesting a link between the late burial of nonpolar surface and water release. Finally, the predicted folding free energy takes a progressive favorable dip toward more negative values, as the chain gets longer. These results suggest that thermodynamic stabilization of the native structure of newly synthesized globins during translation in the cell is significantly enhanced as the chain elongates. This is especially true upon departure of the last C‐terminal residues from the ribosomal tunnel, which hosts ca., 30–40 amino acids. Hence, we propose that release from the ribosome is a crucial step in the life of single‐domain proteins in the cell. Proteins 2014; 82:2318–2331. © 2014 Wiley Periodicals, Inc.     

Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe,Protective Vaccine Antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.     

Neutrophil Gelatinase-Associated Lipocalin (NGAL) Predicts Response to Neoadjuvant Chemotherapy and Clinical Outcome in Primary Human Breast Cancer

In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC.     

Acute colitis induced by dextran sulfate sodium progresses to chronicity in C57BL/6 but not in BALB/c mice: correlation between symptoms and inflammation

Exposure to dextran sulfate sodium (DSS) induces acute colitis, which is normally resolved after DSS removal. To study chronicity, mice are typically subjected to three to five cycles of weekly DSS exposures, each followed by a 1- to 2-wk rest period. Here, we describe a novel and convenient way of inducing chronic, progressive colitis by a single exposure to DSS. C57BL/6 mice exposed to DSS for 5 days developed acute colitis that progressed to severe chronic inflammation. The plasma haptoglobin levels remained high during the chronic phase, showing that the inflammation was active. Surprisingly, the mice regained their original weight along with the progression of colitis, and the only apparent symptom was loose feces. Histopathological changes 4 wk after DSS removal were dense infiltrates of mononuclear cells, irregular epithelial structure, and persistent deposits of collagen. A progressive production of the cytokines IL-1beta, IL-12 p70, and IL-17 correlated with the extensive cellular infiltration, whereas high IFN-gamma production was mainly found late in the chronic phase. Similar to C57BL/6 mice, BALB/c mice exposed to 5 days of DSS developed acute colitis as previously described. The acute colitis was accompanied by elevated plasma levels of haptoglobin and increased colonic levels of IL-1alpha/beta, IL-6, IL-18, and granulocyte colony-stimulating factor. However, soon after DSS removal, BALB/c mice recovered and were symptom free within 2 wk and completely recovered 4 wk after DSS removal in terms of histopathology, haptoglobin levels, and local cytokine production. In summary, these data stress the effect of genetic background on the outcome of DSS provocation. We believe that the present protocol to induce chronic colitis in C57BL/6 mice offers a robust model for validating future therapies for treatment of inflammatory bowel disease.     

Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus–Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the β-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this β-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.     

Four distinct chondrocyte populations in the fetal bovine growth plate: highest expression levels of PTH/PTHrP receptor,Indian hedgehog,and MMP-13 in hypertrophic chondrocytes and their suppression by PTH (1-34) and PTHrP (1-40)

Differentiation and growth of chondrocytes in fetal growth plates of vertebrate long bones and ribs appear to occur in a gradual, continuous manner between the resting zone through the proliferation zone, maturation zone, and upper and lower hypertrophic zones, with a continuous increase in cell size up to 10-fold of the volume of a resting chondrocyte. Here we provide evidence, however, that after centrifugation through a continuous Percoll gradient growth plate chondrocytes separate into four distinct cell populations (B1 to B4) which differ markedly in density, size, and gene expression. These populations collect in the absence of any phase borders in the gradient which might serve as concentration barriers. Fractions B1 and B2 contained the largest cells with the lowest buoyant density and showed the highest expression levels for type X collagen (Col X), but only the B1 population expressed high levels of matrix metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly smaller and expressed little Col X, while cells in fraction B4 were of similar size to cells in the resting zone without significant Col X expression. The highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR-1), and Indian hedgehog (Ihh) expression were also found in the hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as expected from in situ hybridization data on PTHR-1 expression in fetal rodent or chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and PTHR-1 by more than 50% and the expression of Ihh nearly completely. In contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These findings confirm the existence of distinct differentiation stages within chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP fragments controlling the differentiation of proliferating to prehypertrophic chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic chondrocytes.     

In vivo motion of the rectus femoris muscle after tendon transfer surgery

Rectus femoris transfer surgery is performed to convert the rectus femoris muscle from a knee extensor to a knee flexor. In this surgery, the distal tendon of the rectus femoris is detached from the patella and reattached to one of the knee flexor tendons. The outcomes of this procedure are variable, and it is not known if the surgery successfully converts the muscle to a knee flexor. We measured the motion of muscle tissue within the rectus femoris and vastus intermedius during knee extension in 10 unimpaired control subjects (10 limbs) and 6 subjects (10 limbs) after rectus femoris transfer using cine phase-contrast magnetic resonance imaging. Displacements of the vastus intermedius during knee extension were similar between control and tendon transfer subjects. In the control subjects, the rectus femoris muscle consistently moved in the direction of the knee extensors and displaced more than the vastus intermedius. The rectus femoris also moved in the direction of the knee extensors in the tendon transfer subjects; however, the transferred rectus femoris displaced less than the vastus intermedius. These results suggest that the rectus femoris is not converted to a knee flexor after its distal tendon is transferred to the posterior side of the knee, but its capacity for knee extension is diminished by the surgery.