Synthesis and application of a photoaffinity analog of dehydroepiandrosterone (DHEA)

We have synthesized an analog of dehydroepiandrosterone (DHEA, 1) containing both a benzophenone (BP) and a biotin (Bt) group (DHEA–BP–Bt, 8). Compound 8 was prepared by functionalization on C-17 of 1. Biocytin was reacted with 4-benzoylbenzoic acid and the product was condensed with 1 containing a diamine–hexane linker. We detected specific protein bands of approximately 55, 80, and 150 kDa by SDS–PAGE analysis of vascular endothelial cell plasma membranes which had been photoirradiated in the presence of 8.     

Conservation and Diversity of Influenza A H1N1 HLA-Restricted T Cell Epitope Candidates for Epitope-Based Vaccines

Background

The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated.

Methodology/Principal Findings

HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007–2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes.

Conclusions/Significance

Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.     

The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration

The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1-/-) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1-/- cells are unable to activate Rac upon alpha3beta1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.     

A flavanone from Baccharis retusa (Asteraceae) prevents elastase-induced emphysema in mice by regulating NF-κB,oxidative stress and metalloproteinases

BackgroundPulmonary emphysema is characterized by irreversible airflow obstruction, inflammation, oxidative stress imbalance and lung remodeling, resulting in reduced lung function and a lower quality of life. Flavonoids are plant compounds with potential anti-inflammatory and antioxidant effects that have been used in folk medicine. Our aim was to determine whether treatment with sakuranetin, a flavonoid extracted from the aerial parts of Baccharis retusa, interferes with the development of lung emphysema.MethodsIntranasal saline or elastase was administered to mice; the animals were then treated with sakuranetin or vehicle 2 h later and again on days 7, 14 and 28. We evaluated lung function and the inflammatory profile in bronchoalveolar lavage fluid (BALF). The lungs were removed to evaluate alveolar enlargement, extracellular matrix fibers and the expression of MMP-9, MMP-12, TIMP-1, 8-iso-PGF-2α and p65-NF-κB in the fixed tissues as well as to evaluate cytokine levels and p65-NF-κB protein expression.ResultsIn the elastase-treated animals, sakuranetin treatment reduced the alveolar enlargement, collagen and elastic fiber deposition and the number of MMP-9- and MMP-12-positive cells but increased TIMP-1 expression. In addition, sakuranetin treatment decreased the inflammation and the levels of TNF-α, IL-1β and M-CSF in the BALF as well as the levels of NF-κB and 8-iso-PGF-2α in the lungs of the elastase-treated animals. However, this treatment did not affect the changes in lung function.ConclusionThese data emphasize the importance of oxidative stress and metalloproteinase imbalance in the development of emphysema and suggest that sakuranetin is a potent candidate that should be further investigated as an emphysema treatment. This compound may be useful for counteracting lung remodeling and oxidative stress and thus attenuating the development of emphysema.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0233-3) contains supplementary material, which is available to authorized users.     

Long-term records of coral calcification across the central Great Barrier Reef: assessing the impacts of river runoff and climate change

Calcification rates are reported for 41 long-lived Porites corals from 7 reefs, in an inshore to offshore transect across the central Great Barrier Reef (GBR). Over multi-decadal timescales, corals in the mid-shelf (1947–2008) and outer reef (1952–2004) regions of the GBR exhibit a significant increase in calcification of 10.9 ± 1.1 % (1.4 ± 0.2 % per decade; ±1 SE) and 11.1 ± 3.9 % (2.1 ± 0.8 % per decade), respectively, while inner-shelf (1930–2008), reefs show a decline of 4.6 ± 1.3 % (0.6 ± 0.2 % per decade). This long-term decline in calcification for the inner GBR is attributed to the persistent ongoing effects of high sediment/nutrients loads from wet season river discharges, compounded by the effects of thermal stress, especially during the 1998 bleaching event. For the recent period (1990–2008), our data show recovery from the 1998 bleaching event, with no significant trend in the rates of calcification (1.1 ± 2.0 %) for the inner reefs, while corals from the mid-shelf central GBR show a decline of 3.3 ± 0.9 %. These results are in marked contrast to the extreme reef-wide declines of 14.2 % reported by De’ath et al. (2009) for the period of 1990–2005. The De’ath et al. (2009) results are, however, found to be compromised by the inclusion of incomplete final years, duplicated records, together with a bias toward inshore reefs strongly affected by the 1998 bleaching. Our new findings nevertheless continue to raise concerns, with the inner-shelf reefs continuing to show long-term declines in calcification consistent with increased disturbance from land-based effects. In contrast, the more ‘pristine’ mid- and outer-shelf reefs appear to be undergoing a transition from increasing to decreasing rates of calcification, possibly reflecting the effects of CO₂-driven climate change. Our study highlights the importance of properly undertaken, regular assessments of coral calcification that are representative of the distinctive cross-shelf environments and discriminate between local disturbances and the global impacts of climate change and ocean acidification.     

In vivo label‐free functional photoacoustic monitoring of ischemic reperfusion

Pressure ulcer formation is a common problem among patients confined to bed or restricted to wheelchairs. The ulcer forms when the affected skin and underlying tissues go through repeated cycles of ischemia and reperfusion, leading to inflammation. This theory is evident by intravital imaging studies performed in immune cell–specific, fluorescent reporter mouse skin with induced ischemia‐reperfusion (I‐R) injuries. However, traditional confocal or multiphoton microscopy cannot accurately monitor the progression of vascular reperfusion by contrast agents, which leaks into the interstitium under inflammatory conditions. Here, we develop a dual‐wavelength micro electro mechanical system (MEMS) scanning–based optical resolution photoacoustic microscopy (OR‐PAM) system for continuous label‐free functional imaging of vascular reperfusion in an IR mouse model. This MEMS‐OR‐PAM system provides fast scanning speed for concurrent dual‐wavelength imaging, which enables continuous monitoring of the reperfusion process. During reperfusion, the revascularization of blood vessels and the oxygen saturation (sO₂) changes in both arteries and veins are recorded, from which the local oxygen extraction ratios of the ischemic tissue and the unaffected tissue can be quantified. Our MEMS‐OR‐PAM system provides novel perspectives to understand the I‐R injuries. It solves the problem of dynamic label‐free functional monitoring of the vascular reperfusion at high spatial resolution.