A sequence symmetry analysis of the interrelationships between statins, diabetes and skin infections.

AIMS: To determine statins' impact on skin infection risk in view of conflicting literature: that statins may reduce infection risk, but are also associated with diabetes mellitus, a risk factor for skin and soft tissue infections (SSTIs). METHODS: Sequence symmetry analysis was performed on prescription claims (2001-2011) from the Australian Department of Veterans' Affairs to determine the interrelationships between: (i) statins and SSTIs; (ii) statins and diabetes; and (iii) diabetes and SSTIs; as well as whether statins increased the risk of SSTIs, independent of diabetes status. Chi-square tests were performed to detect differences in Index of Relative Socio-economic Advantage and Disadvantage scores of patients within each interrelationship. Prescriptions for statins, antidiabetic medication, and antistaphylococcal antibiotics were evaluated using nonidentifiable client numbers, prescription dates filled, residential electorates, and pharmaceutical codes. Adjusted sequence ratio and confidence interval were calculated at intervals of 91, 182 and 365 days for sequence symmetry analysis studies. RESULTS: Statins were associated with: (i) significant SSTI risk (adjusted sequence ratio > 1; confidence interval >1), (ii) significant diabetes risk, and (iii) diabetic patients had increased risk of SSTIs. Diabetic and nondiabetic statin users had significantly increased risks of SSTIs, while the influence from socioeconomic status was not significant for each of the 3 relationships. CONCLUSIONS: Statins are associated with increased risk of SSTIs via direct and indirect mechanisms, probably independent of diabetes or socioeconomic statuses. We believe that clinicians should be aware of the association between statins and SSTIs, and, where appropriate, monitor blood glucose levels of statin users.

In vitro antibacterial effects of statins against bacterial pathogens causing skin infections.

With financial considerations impeding research and development of new antibiotics, drug repurposing (finding new indications for old drugs) emerges as a feasible alternative. Statins are extensively prescribed around the world to lower cholesterol, but they also possess inherent antimicrobial properties. This study identifies statins with the greatest potential to be repurposed as topical antibiotics and postulates a mechanism of action for statins' antibacterial activity. Using broth microdilution, the direct antibacterial effects of all seven parent statins currently registered for human use and three selected statin metabolites were tested against bacterial skin pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens. Simvastatin and pitavastatin lactone exerted the greatest antibacterial effects (minimum inhibitory concentrations of 64 and 128 µg/mL, respectively) against S. aureus. None of the statins tested were effective against E. coli, P. aeruginosa, or S. marcescens, but simvastatin hydroxy acid acid might be active against S. aureus, E. coli, and S. marcescens at drug concentrations > 256 µg/mL. It was found that S. aureus may exhibit a paradoxical growth effect when exposed to simvastatin; thus, treatment failure at high drug concentrations is theoretically probable. Through structure-activity relationship analysis, we postulate that statins' antibacterial action may involve disrupting the teichoic acid structures or decreasing the number of alanine residues present on Gram-positive bacterial cell surfaces, which could reduce biofilm formation, diminish bacterial adhesion to environmental surfaces, or impede S. aureus cell division.

Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation.

LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.

Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect.

The excluded volume effect (EVE) rules all life processes. It is created by macromolecules that occupy a given volume thereby confining other molecules to the remaining space with large consequences on reaction kinetics and molecular assembly. Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by procollagen C-proteinase (PCP/BMP-1) and proteolytic modification of its allosteric regulator, PCOLCE1. This led to a 20-30- and 3-6-fold increased collagen deposition in two- and three-dimensional cultures, respectively, and creation of crosslinked collagen footprints beneath cells. Important parameters correlating with accelerated deposition were hydrodynamic radius of macromolecules and their negative charge density.

In vitro enhancement of collagen matrix formation and crosslinking for applications in tissue engineering: a preliminary study.

The construction of stable engineered tissue depends on the formation of a functional connective tissue produced by cells locally. A major component of connective tissue is collagen. Its deposition into a stable matrix depends on the enzymatic extracellular conversion of procollagen to collagen. This step is very slow in vitro and we hypothesized that this is due to a lack of crowdedness and insufficient excluded volume effect (EVE) in culture media. We used neutral (670 kDa) and negatively charged dextran sulfate (DxS, 500 kDa) to create EVE in cell cultures and to enhance in vitro matrix formation by accelerating procollagen conversion. Biochemical analyses in 2 human fibroblast lines revealed mostly unprocessed procollagen in uncrowded culture medium, whereas in the presence of DxS, procollagen conversion occurred and most of the collagen was associated with the cell layer. Immunocytochemistry confirmed DxS-related collagen deposition that colocalized with fibronectin. The large neutral dextran showed, in identical concentration ranges, no effects that correlated well with its smaller hydrodynamic radius as determined by dynamic light scattering. This predicted a 10 times bigger crowding power of DxS and benchmarks it as a potentially promising crowding agent facilitating the formation of extracellular matrix in vitro.

Statins: antimicrobial resistance breakers or makers?

INTRODUCTION: The repurposing of non-antibiotic drugs as adjuvant antibiotics may help break antimicrobial resistance (AMR). Statins are commonly prescribed worldwide to lower cholesterol. They also possess qualities of AMR "breakers", namely direct antibacterial activity, synergism with antibiotics, and ability to stimulate the host immune system. However, statins' role as AMR breakers may be limited. Their current extensive use for cardiovascular protection might result in selective pressures for resistance, ironically causing statins to be AMR "makers" instead. This review examines statins' potential as AMR breakers, probable AMR makers, and identifies knowledge gaps in a statin-bacteria-human-environment continuum. The most suitable statin for repurposing is identified, and a mechanism of antibacterial action is postulated based on structure-activity relationship analysis. METHODS: A literature search using keywords "statin" or "statins" combined with "minimum inhibitory concentration" (MIC) was performed in six databases on 7th April 2017. After screening 793 abstracts, 16 relevant studies were identified. Unrelated studies on drug interactions; antifungal or antiviral properties of statins; and antibacterial properties of mevastatin, cerivastatin, antibiotics, or natural products were excluded. Studies involving only statins currently registered for human use were included. RESULTS: Against Gram-positive bacteria, simvastatin generally exerted the greatest antibacterial activity (lowest MIC) compared to atorvastatin, rosuvastatin, and fluvastatin. Against Gram-negative bacteria, atorvastatin generally exhibited similar or slightly better activity compared to simvastatin, but both were more potent than rosuvastatin and fluvastatin. DISCUSSION: Statins may serve as AMR breakers by working synergistically with existing topical antibiotics, attenuating virulence factors, boosting human immunity, or aiding in wound healing. It is probable that statins' mechanism of antibacterial activity involves interference of bacterial cell regulatory functions via binding and disrupting cell surface structures such as wall teichoic acids, lipoteichoic acids, lipopolysaccharides, and/or surface proteins. The widespread use of statins for cardiovascular protection may favor selective pressures or co-selection for resistance, including dysbiosis of the human gut microbiota, sublethal plasma concentrations in bacteremic patients, and statin persistence in the environment, all possibly culminating in AMR. CONCLUSION: Simvastatin appears to be the most suitable statin for repurposing as a novel adjuvant antibiotic. Current evidence better supports statins as potential AMR breakers, but their role as plausible AMR makers cannot be excluded. Elucidating the mechanism of statins' antibacterial activity is perhaps the most important knowledge gap to address as this will likely clarify statins' role as AMR breakers or makers.

Statin therapy causes gut dysbiosis in mice through a PXR-dependent mechanism.

BACKGROUND: Statins are a class of therapeutics used to regulate serum cholesterol and reduce the risk of heart disease. Although statins are highly effective in removing cholesterol from the blood, their consumption has been linked to potential adverse effects in some individuals. The most common events associated with statin intolerance are myopathy and increased risk of developing type 2 diabetes mellitus. However, the pathological mechanism through which statins cause these adverse effects is not well understood. RESULTS: Using a murine model, we describe for the first time profound changes in the microbial composition of the gut following statin treatment. This remodelling affected the diversity and metabolic profile of the gut microbiota and was associated with reduced production of butyrate. Statins altered both the size and composition of the bile acid pool in the intestine, tentatively explaining the observed gut dysbiosis. As also observed in patients, statin-treated mice trended towards increased fasting blood glucose levels and weight gain compared to controls. Statin treatment affected the hepatic expression of genes involved in lipid and glucose metabolism. Using gene knockout mice, we demonstrated that the observed effects were mediated through pregnane X receptor (PXR). CONCLUSION: This study demonstrates that statin therapy drives a profound remodelling of the gut microbiota, hepatic gene deregulation and metabolic alterations in mice through a PXR-dependent mechanism. Since the demonstrated importance of the intestinal microbial community in host health, this work provides new perspectives to help prevent the statin-associated unintended metabolic effects.

Cloning and characterization of a novel zinc finger protein (rZFP96) in the rat corpus luteum.

The corpus luteum (CL) is a temporary organ involved in the maintenance of pregnancy. In the course of its life-cycle, the CL undergoes two distinct and consecutive processes for its inevitable removal through apoptosis: functional and structural luteolysis. We isolated a gene encoding for a novel rat zinc finger protein (ZFP), named rat ZFP96 (rZFP96) from an ovarian lambda cDNA library. Sequence analysis revealed close sequence and structural similarity to mouse ZFP96 and human zinc finger protein 305 (ZNF305). Quantitative reverse transcription-polymerase chain reaction analysis revealed a positive correlation with the end of pregnancy, that is, the onset of structural luteolysis of the CL. Messenger RNA levels increased 3-fold (P < 0.01) between days 13 and 22 of pregnancy and 8-fold (P < 0.01) between day 13 of pregnancy and day 1 post-partum. In addition, we detected rZFP96 expression in mammary, placenta, heart, kidney and skeletal muscle. Sequence analysis predicted that rZFP96 has a high probability of localizing to the nuclear compartment. The presence of both a perfect consensus TGEKP linker sequence between zinc fingers 2 and 3 as well as several similar sequences between the other zinc fingers suggests physical interaction with DNA. Speculatively, rZFP96 may therefore function as a transcription factor, switching-off pro-survival genes and/or upregulating pro-apoptotic genes and thereby contributing to the demise of the CL.

Defensins HNP1 and HBD2 stimulation of wound-associated responses in human conjunctival fibroblasts.

PURPOSE: To study the responses of human conjunctival fibroblasts (HCFs) to stimulation by human neutrophil defensin 1 (HNP1) and beta defensin2 (HBD2). METHODS: Defensin-stimulated gene expression in primary cultures of HCFs was analyzed by real-time PCR after exposure to various concentrations of HNP1 or HBD2. Gene and protein expression for selected collagens, matrix metalloproteinases, and tissue inhibitors of metalloproteinases were determined by real-time PCR and ELISA analysis. Activation of p42/44 mitogen-activated protein (MAP) and Akt was analyzed by Western blot. RESULTS: HCFs did not express significant levels of the genes for HNP1 or HBD1-3. However, HNP1 and HBD2 stimulated HCF proliferation, the activation of p42/44 MAP kinase, and Akt kinase in a dose-dependent manner. HNP1 and HBD2 were not found to be chemotoxic for HCFs. It was demonstrated with the use of U0126 and wortmannin that the activation of p42/44 MAP kinase and Akt was responsible for the increased HCF proliferation observed under HNP1 and HBD2 stimulation. HNP1 stimulated the expression of the genes for collagen I, III, VI, and VIII. In addition, it reduced the secretion of collagen I protein but increased its intracellular retention. HNP1 and HBD2 upregulated the transcription and translation of MMP1. Small increases were observed in MMP14 gene expression after HNP1 stimulation and MMP2 gene expression after HBD2 stimulation. CONCLUSIONS: The results of this study suggest that HNP1 and HBD2 have a potential role in the biosynthetic and tissue remodeling responses of conjunctival fibroblasts.

A histological and micro-CT investigation in to the effect of NGF and EGF on the periodontal, alveolar bone, root and pulpal healing of replanted molars in a rat model - a pilot study.

BACKGROUND: This study aims to investigate, utilising micro-computed tomography (micro-CT) and histology, whether the topical application of nerve growth factor (NGF) and/or epidermal growth factor (EGF) can enhance periodontal, alveolar bone, root and pulpal tissue regeneration while minimising the risk of pulpal necrosis, root resorption and ankylosis of replanted molars in a rat model. METHODS: Twelve four-week-old male Sprague-Dawley rats were divided into four groups: sham, collagen, EGF and NGF. The maxillary right first molar was elevated and replanted with or without a collagen membrane impregnated with either the growth factors EGF or NGF, or a saline solution. Four weeks after replantation, the animals were sacrificed and the posterior maxilla was assessed using histological and micro-CT analysis. The maxillary left first molar served as the control for the corresponding right first molar. RESULTS: Micro-CT analysis revealed a tendency for all replanted molars to have reduced root length, root volume, alveolar bone height and inter-radicular alveolar bone volume. It appears that the use of the collagen membrane had a negative effect while no positive effect was noted with the incorporation of EGF or NGF. Histologically, the incorporation of the collagen membrane was found to negatively affect pulpal, root, periodontal and alveolar bone healing with pulpal inflammation and hard tissue formation, extensive root resorption and alveolar bone fragmentation. The incorporation of EGF and NGF did not improve root, periodontal or alveolar bone healing. However, EGF was found to improve pulp vascularisation while NGF-improved pulpal architecture and cell organisation, although not to the level of the control group. CONCLUSIONS: Results indicate a possible benefit on pulpal vascularisation and pulpal cell organisation following the incorporation of EGF and NGF, respectively, into the alveolar socket of replanted molars in the rat model. No potential benefit of EGF and NGF was detected in periodontal or root healing, while the use of a collagen membrane carrier was found to have a negative effect on the healing response.

Essential modification of the Sircol Collagen Assay for the accurate quantification of collagen content in complex protein solutions.

Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophysiological tissues. Radiolabelling, chromatographic and calorimetric assays have been developed over the years for the accurate determination of collagen content through HyPro estimation. Recently, the Sircol Collagen Assay (SCA) has been almost exclusively adopted as the fastest and simplest colorimetric method for the determination of collagen concentration in complex protein solutions. We show here that the colorimetric SCA, which is based on the binding of Sirius red (SR) to collagen, is flawed by interference of non-collagenous proteins (e.g. serum). In fact, we demonstrate that SCA in cell culture systems and tissue hydrolysates results in a dramatic overestimation of collagen content ranging from 3- to 24-fold. In order to rescue this otherwise very practical assay, we introduce a simple purification procedure that allows the removal of interfering non-collagenous proteins from culture media and tissue samples so that accurate measurements with SCA are now possible.

Early adhesive behavior of bone-marrow-derived mesenchymal stem cells on collagen electrospun fibers.

A bioabsorbable nanofibrous scaffold was developed for early adhesion of mesenchymal stem cells (MSCs). Collagen nanofibers with diameters of 430 +/- 170 nm were fabricated by electrospinning. Over 45% of the MSC population adhered to this collagen nanofiber after 30 min at room temperature. Remarkably, collagen-coated P(LLA-CL) electrospun nanofibers were almost as efficient as collagen nanofibers whereas collagen cast film did not enhance early capture when it was applied on cover slips. The adhesive efficiency could be further increased to over 20% at 20 min and over 55% at 30 min when collagen nanofibers were grafted with monoclonal antibodies recognizing CD29 or CD49a. These data demonstrate that the early adhesive behavior is highly dependent on both the surface texture and the surface chemistry of the substrate. These findings have potential applications for early capture of MSCs in an ex vivo setting under time constraints such as in a surgical setting.

Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance.

The polymerase chain reaction's (PCR) phenomenal success in advancing fields as diverse as Medicine, Agriculture, Conservation, or Paleontology is based on the ability of using isolated prokaryotic thermostable DNA polymerases in vitro to copy DNA irrespective of origin. This process occurs intracellularly and has evolved to function efficiently under crowded conditions, namely in an environment packed with macromolecules. However, current in vitro practice ignores this important biophysical parameter of life. In order to more closely emulate conditions of intracellular biochemistry in vitro we added inert macromolecules into reverse transcription (RT) and PCR. We show dramatic improvements in all parameters of RT-PCR including 8- to 10-fold greater sensitivity, enhanced polymerase processivity, higher specific amplicon yield, greater primer annealing and specificity, and enhanced DNA polymerase thermal stability. The faster and more efficient reaction kinetics was a consequence of the cumulative molecular and thermodynamic effects of the excluded volume effect created by macromolecular crowding.

Regulated expression of inhibitor of apoptosis protein 3 in the rat corpus luteum.

We sought to investigate the role inhibitor of apoptosis proteins (IAPs) play in the life cycle of the corpus luteum (CL) of the rat. We isolated two clones with amino acid homology to rat IAP2 (BIRC 3) and three to rat IAP3 (rIAP3; BIRC 4). The expression of rIAP3 mRNA was examined in the rat CL during and after pregnancy, in Day 8 pregnant rats after 24-h treatment of gonadotropin-releasing hormone-agonist (GnRH-Ag), and in a CL organ culture model of spontaneous apoptosis in the absence of tropic support with and without superoxide dismutase. We used real-time RT-PCR to quantitate rIAP3 mRNA expression. Interestingly, a significant reduction in rIAP3 levels was seen at the time of CL regression in the course of natural pregnancy and the GnRH-Ag model. Surprisingly, rIAP3 mRNA levels in the CL organ culture model of spontaneous apoptosis failed to show significant changes, although TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) reaction showed 30%-40% of the cells undergoing DNA fragmentation after 2 h in culture. In situ hybridization revealed that rIAP3 expression was localized to the cytoplasm of luteal and granulosa cells. These data clearly demonstrate both the presence of IAPs in the rat CL and the regulation of rIAP3 during in vivo apoptotic cell death, indicating a role for IAPs in the maintenance of CL function and demise.