Objective Elevated inflammatory cytokine levels have been implicated in the pathogenesis of non3 healing chronic venous insufficiency (CVI) ulcers. The goal of this study was to determine the protein levels of a wide range of inflammatory cytokines in untreated CVI ulcer tissue before and after 4 weeks of high strength compression therapy. These levels were compared to cytokines present in healthy tissue. Methods Thirty limbs with untreated CVI and leg ulceration received therapy for 4 weeks with sustained high compression bandaging at an ambulatory wound center. Biopsies were obtained from healthy and ulcerated tissue before and after therapy. A multiplexed protein assay was used to measure multiple cytokines in a single sample. Patients were designated as rapid or delayed healers based on ulcer surface area change. Results The majority of pro-inflammatory cytokine protein levels were elevated in ulcer tissue compared to healthy tissue, and compression therapy significantly reduced these cytokines. TGF-β1 was up-regulated in ulcer tissue following compression therapy. Rapid healing ulcers had significantly higher levels of IL-1α, IL-1β, IFN-γ, IL-12p40 and GM-CSF before compression therapy, and IL-1 Ra after therapy. IFN-γ levels significantly decreased following therapy in the rapidly healing patients. Conclusion CVI ulcer healing is associated with a pro-inflammatory environment prior to treatment that reflects metabolically active peri-wound tissue that has the potential to heal. Treatment with compression therapy results in healing that is coupled with reduced pro-inflammatory cytokine levels and higher levels of the anti-inflammatory cytokine IL-1 Ra. Clinical Relevance This data suggests that cytokines may provide targets in which topical therapeutic inhibition or promotion at appropriate time points in the healing process may provide novel therapeutic approaches to the healing of CVI ulcers.

Elevated matrix metalloproteinases (MMP) levels have been implicated in the pathogenesis of chronic venous insufficiency ulcers. Quantitative measurements of a broad range of MMP proteins in human tissue treated with compression bandaging have not been reported. The goal of this study was to determine the expression of a wide range of proteases in untreated venous leg ulcer tissue and the changes in these levels after 4 weeks of high-strength compression therapy. Twenty-nine limbs with new or untreated chronic venous insufficiency and leg ulceration received therapy for 4 weeks with sustained high compression bandaging. Biopsies were obtained from healthy tissue and from ulcerated tissue before and after therapy. A novel multiplexed protein assay was used to measure multiple MMPs in a single sample. MMP protein activity, TIMP protein levels, and gene expression levels were also addressed. MMP1, 2, 3, 8, 9, 12, and 13 protein levels were elevated in ulcer tissue compared with healthy tissue. MMP8 and 9 were highly expressed in ulcer tissue. MMP3, 8, and 9 significantly decreased following treatment. Reduction in the levels of MMP1, 2, and 3 was associated with significantly higher rates of ulcer healing at 4 weeks. We conclude that compression therapy results in a reduction of the pro-inflammatory environment characterizing chronic venous ulcers, and ulcer healing is associated with resolution of specific elevated levels of protease expression.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser (FEL) sources has the potential to determine the structures of macromolecules beyond the limitation of radiation damage and without the need for crystals of sufficient size for conventional crystallography. In SFX, a liquid microjet is used to inject randomly oriented crystals suspended in their storage solution into the FEL beam. Settling of crystals in the reservoir prior to the injection has been found to complicate the data collection. This article details the development of an antisettling sample delivery instrument based on a rotating syringe pump, capable of producing flow rates and liquid pressures necessary for the operation of the injector. The device has been used successfully with crystals of different proteins, with crystal sizes smaller than 20 mm. Even after hours of continuous operation, no significant impairment of the experiments due to sample settling was observed. This article describes the working principle of the instrument and sets it in context with regard to the experimental conditions used for SFX. Hit rates for longer measuring periods are compared with and without the instrument operating. Two versions of the instrument have been developed, which both deliver sample at a constant flow rate but which differ in their minimum liquid flow rates and maximum pressures.

LAGLIDADG endonucleases bind across adjacent major grooves via a saddle-shaped surface and catalyze DNA cleavage. Some LAGLIDADG proteins, called maturases, facilitate splicing by group I introns, raising the issue of how a DNA-binding protein and an RNA have evolved to function together. In this report, crystallographic analysis shows that the global architecture of the bI3 maturase is unchanged from its DNA-binding homologs; in contrast, the endonuclease active site, dispensable for splicing facilitation, is efficiently compromised by a lysine residue replacing essential catalytic groups. Biochemical experiments show that the maturase binds a peripheral RNA domain 50 A from the splicing active site, exemplifying long-distance structural communication in a ribonucleoprotein complex. The bI3 maturase nucleic acid recognition saddle interacts at the RNA minor groove; thus, evolution from DNA to RNA function has been mediated by a switch from major to minor groove interaction.

Artificial SNARE analogues derived from SNARE proteins, which mediate synaptic membrane fusion, are of interest. They mimic the tetrameric α-helix bundle of the SNARE motif with various bio-oligomer recognition units. Interaction between complementary oligomers linked to the respective membrane by lipid or peptide anchors leads to proximity of vesicles and to fusion of lipid bilayers. β-Peptide nucleic acids were introduced as hybrid oligomers with the native SNARE protein transmembrane/linker sequence, in order to evaluate a fusion system that allows distance tuning of approaching membranes. Formation of a four-base pair β-PNA double strand with 20 Å length is sufficient for vesicle membrane fusion. Elongation of the recognition β-PNA duplex in the linker region yielded a 40 Å β-peptide duplex and provided a vesicle-vesicle distance that only supported hemifusion of vesicle membranes.