Matrix Mechanotransduction via Yes-Associated Protein in Human Lamina Cribrosa Cells in Glaucoma.

Purpose: Extracellular matrix stiffening is characteristic of both aging and glaucoma, and acts as a promoter and perpetuator of pathological fibrotic remodeling. Here, we investigate the role of a mechanosensitive transcriptional coactivator, Yes-associated protein (YAP), a downstream effector of multiple signaling pathways, in lamina cribrosa (LC) cell activation to a profibrotic, glaucomatous state. Methods: LC cells isolated from glaucomatous human donor eyes (GLC; n = 3) were compared to LC cells from age-matched nonglaucomatous controls (NLC; n = 3) to determine differential YAP expression, protein levels, and proliferation rates. NLC cells were then cultured on soft (4 kPa), and stiff (100 kPa), collagen-1 coated polyacrylamide hydrogel substrates. Quantitative real-time RT-PCR, immunoblotting, and immunofluorescence microscopy were used to measure the expression, activity, and subcellular location of YAP and its downstream targets, respectively. Proliferation rates were examined in NLC and GLC cells by methyl thiazolyl tetrazolium salt assays, across a range of incrementally increased substrate stiffness. Endpoints were examined in the presence or absence of a YAP inhibitor, verteporfin (2 µM). Results: GLC cells show significantly (P < 0.05) increased YAP gene expression and total-YAP protein compared to NLC cells, with significantly increased proliferation. YAP regulation is mechanosensitive, because NLC cells cultured on pathomimetic, stiff substrates (100 kPa) show significantly upregulated YAP gene and protein expression, increased YAP phosphorylation at tyrosine 357, reduced YAP phosphorylation at serine 127, increased nuclear pooling, and increased transcriptional target, connective tissue growth factor. Accordingly, myofibroblastic markers, α-smooth muscle actin (α-SMA) and collagen type I, alpha 1 (Col1A1) are increased. Proliferation rates are elevated on 50 kPa substrates and tissue culture plastic. Verteporfin treatment significantly inhibits YAP-mediated cellular activation and proliferation despite a stiffened microenvironment. Conclusions: These data demonstrate how YAP plays a pivotal role in LC cells adopting a profibrotic and proliferative phenotype in response to the stiffened LC present in aging and glaucoma. YAP provides an attractive and novel therapeutic target, and its inhibition via verteporfin warrants further clinical investigation.

The Admont Grassland Experiment: 70 years of fertilizer application and its effects on soil and vegetation properties in an alluvial meadow managed under a three-cut regime.

Fertilizer application is a widely used management technique for increasing forage production from agricultural grassland. Fertilization is also a key driver of changes in soil nutrient status and plant species composition of grassland as shown in many short-term studies. Results from long-term experiments can further improve understanding of plant-soil relationships and help with management recommendations for agricultural and environmental outcomes. We collected data from a long-term experiment on alluvial meadow (Admont Grassland Experiment, Austria; established 1946) with 24 fertilization treatments managed under a three-cut regime. Soil sampling in autumn 2015 and vegetation sampling in spring 2016 were conducted in seven selected treatments. Combinations of N (nitrogen 80 kg ha-1), P (phosphorus 35 kg ha-1) and K (potassium 100 kg ha-1) were applied annually and compared with a non-fertilized control. Treatments were: Control, N, P, K, NP, NK, PK and NPK fertilization. Long-term different fertilization affected soil pH and nutrient concentrations in the soil and plant species composition, but no significant effects on species richness were found. Short species (<0.5 m height) prevailed in all treatments regardless of nutrient application, probably as a result of the three-cut defoliation. The dry matter biomass (DMB) yield in the Control was limited by N and P and synergisticly co-limited by N, P and K, and DMB yields of more than 5 t ha-1 per year were achieved under nutrient combinations containing P (NP, PK, NPK) without loss of species richness. Results from the Admont Grassland Experiment show that the tested nutrient combinations significantly increased DMB yield and changed the species composition, but without significant effects on species richness. Long-term biomass yields of more than 5 t ha-1 DMB per year can be achieved with any nutrient combination containing P without loss species richness in an alluvial meadow managed under a three-cut regime.

The role of lamina cribrosa tissue stiffness and fibrosis as fundamental biomechanical drivers of pathological glaucoma cupping.

The primary biomechanical driver of pathological glaucomatous cupping remains unknown. Finite element modeling indicates that stress and strain play key roles. In this article, primarily a review, we utilize known biomechanical data and currently unpublished results from our lab to propose a three-stage, tissue stiffness-based model to explain glaucomatous cupping occurring at variable levels of translaminar pressure (TLP). In stage 1, a short-term increase in TLP gradient induces a transient increase in lamina cribrosa (LC) strain. Beyond a critical level of strain, the tissue stiffness rises steeply provoking cellular responses via integrin-mediated mechanotransduction. This early mechanoprotective cellular contraction reduces strain, which reduces tissue stiffness by return of the posteriorly deflected LC to baseline. In stage 2 a prolonged period of TLP increase elicits extracellular matrix (ECM) production leading to fibrosis, increasing baseline tissue stiffness and strain and diminishing the contractile ability/ability to return to the baseline LC position. This is supported by our three-dimensional collagen contraction assays, which show significantly reduced capacity to contract in glaucoma compared with normal LC cells. Second, 15% cyclic strain in LC cells over 24 h elicits a typical increase in ECM profibrotic genes in normal LC cells but a highly blunted response in glaucoma LC cells. Stage 3 is characterized by persistent fibrosis causing further stiffening and inducing a feed-forward ECM production cycle. Repeated cycles of increased strain and stiffness with profibrotic ECM deposition prevent optic nerve head (ONH) recoil from the new deflected position. This incremental maladaptive modeling leads to pathological ONH cupping.

Dysregulated claudin-5 cycling in the inner retina causes retinal pigment epithelial cell atrophy.

Age-related macular degeneration (AMD) is the leading cause of central retinal vision loss worldwide, with an estimated 1 in 10 people over the age of 55 showing early signs of the condition. There are currently no forms of therapy available for the end stage of dry AMD, geographic atrophy (GA). Here, we show that the inner blood-retina barrier (iBRB) is highly dynamic and may play a contributory role in GA development. We have discovered that the gene CLDN5, which encodes claudin-5, a tight junction protein abundantly expressed at the iBRB, is regulated by BMAL1 and the circadian clock. Persistent suppression of claudin-5 expression in mice exposed to a cholesterol-enriched diet induced striking retinal pigment epithelium (RPE) cell atrophy, and persistent targeted suppression of claudin-5 in the macular region of nonhuman primates induced RPE cell atrophy. Moreover, fundus fluorescein angiography in human and nonhuman primate subjects showed increased retinal vascular permeability in the evening compared with the morning. These findings implicate an inner retina-derived component in the early pathophysiological changes observed in AMD, and we suggest that restoring the integrity of the iBRB may represent a novel therapeutic target for the prevention and treatment of GA secondary to dry AMD.

Tight Junctions of the Outer Blood Retina Barrier.

The outer blood retina barrier (oBRB) formed by the retinal pigment epithelium (RPE) is critical for maintaining retinal homeostasis. Critical to this modified neuro-epithelial barrier is the presence of the tight junction structure that is formed at the apical periphery of contacting cells. This tight junction complex mediates size-selective passive diffusion of solutes to and from the outer segments of the retina. Unlike other epithelial cells, the apical surface of the RPE is in direct contact with neural tissue and it is centrally involved in the daily phagocytosis of the effete tips of photoreceptor cells. While much is known about the intracellular trafficking of material within the RPE, less is known about the role of the tight junction complexes in health and diseased states. Here, we provide a succinct overview of the molecular composition of the RPE tight junction complex in addition to highlighting some of the most common retinopathies that involve a dysregulation of RPE integrity.

Single-ion adsorption and switching in carbon nanotubes.

Single-ion detection has, for many years, been the domain of large devices such as the Geiger counter, and studies on interactions of ionized gasses with materials have been limited to large systems. To date, there have been no reports on single gaseous ion interaction with microelectronic devices, and single neutral atom detection techniques have shown only small, barely detectable responses. Here we report the observation of single gaseous ion adsorption on individual carbon nanotubes (CNTs), which, because of the severely restricted one-dimensional current path, experience discrete, quantized resistance increases of over two orders of magnitude. Only positive ions cause changes, by the mechanism of ion potential-induced carrier depletion, which is supported by density functional and Landauer transport theory. Our observations reveal a new single-ion/CNT heterostructure with novel electronic properties, and demonstrate that as electronics are ultimately scaled towards the one-dimensional limit, atomic-scale effects become increasingly important.

The effect of a thermal renal denervation cycle on the mechanical properties of the arterial wall.

The aim of this study was to determine the effect that a thermal renal denervation cycle has on the mechanical properties of the arterial wall. Porcine arterial tissue specimens were tested in three groups: native tissue, decellularized tissue, decellularized with collagen digestion (e.g. elastin only). One arterial specimen was used as an unheated control specimen while another paired specimen was subjected to a thermal cycle of 70°C for 120s (n=10). The specimens were subjected to tensile loading and a shrinkage analysis. We observed two key results: The mechanical properties associated with the elastin extracellular matrix (ECM) were not affected by the thermal cycle. The effect of the thermal cycle on the collagen (ECM) was significant, in both the native and decellularized groups the thermal cycle caused a statistically significant decrease in stiffness, and failure strength, moreover the native tissue demonstrated a 27% reduction in lumen area post exposure to the thermal cycle. We have demonstrated that a renal denervation thermal cycle can significantly affect the mechanical properties of an arterial wall, and these changes in stiffness and failure strength were associated with alterations to the collagen rather than the elastin extracellular matrix component.

Telavancin for hospital-acquired pneumonia: clinical response and 28-day survival.

U.S. Food and Drug Administration draft guidance for future antibiotic clinical trials of bacterial nosocomial pneumonia recommends the use of diagnostic criteria according to American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) guidelines and the use of a primary endpoint of 28-day all-cause mortality. The effect of applying these guidelines on outcomes of phase III nosocomial pneumonia studies of telavancin was evaluated in a post hoc analysis. ATS/IDSA criteria were applied in a blind fashion to the original all-treated (AT) group. Clinical cure rates at final follow-up were determined in the refined AT and clinically evaluable (CE) groups (ATS/IDSA-AT and ATS/IDSA-CE, respectively). The exploratory endpoint of 28-day survival was evaluated for the ATS/IDSA-AT group. Noninferiority of telavancin versus vancomycin was demonstrated, with similar cure rates in the ATS/IDSA-AT (59% versus 59%) and ATS/IDSA-CE (83% versus 80%) groups. Cure rates favored telavancin in ATS/IDSA-CE patients where Staphylococcus aureus was the sole pathogen (86% versus 75%). Overall, 28-day survival rates were similar in the telavancin (76%) and vancomycin (77%) groups but lower in telavancin-treated patients with preexisting moderate-to-severe renal impairment (creatinine clearance [CLCR] of <50 ml/min). Telavancin should be administered to patients with moderate-to-severe renal impairment only if treatment benefit outweighs the risk or if no suitable alternatives are available.

Integration of biostatistics and pharmacometrics computing platforms for efficient and reproducible PK/PD analysis: a case study.

Results of pharmacometric analyses influence high-level decisions such as clinical trial design, drug approval, and labeling. Key challenges for timely delivery of pharmacometric analyses are the data assembly process and tracking and documenting the modeling process and results. Since clinical efficacy and safety data typically reside in the biostatistics computing area, an integrated computing platform for pharmacometric and biostatistical analyses would be ideal. A case study is presented integrating a pharmacometric modeling platform into an existing statistical computing environment (SCE). The feasibility and specific configurations of running common PK/PD programs such as NONMEM and R inside of the SCE are provided. The case study provides an example of an integrated repository that facilitates efficient data assembly for pharmacometrics analyses. The proposed platform encourages a good pharmacometrics working practice to maintain transparency, traceability, and reproducibility of PK/PD models and associated data in supporting drug development and regulatory decisions.

Efficacy of telavancin in patients with specific types of complicated skin and skin structure infections.

BACKGROUND: Telavancin is approved in the USA and Canada for the treatment of Gram-positive complicated skin and skin structure infections (cSSSIs) based on the results of the Phase 3 Assessment of TeLAvancin in complicated Skin and skin structure infections (ATLAS) trials, which demonstrated non-inferiority of telavancin to vancomycin. METHODS: We conducted a post hoc analysis of the ATLAS studies (ClinicalTrials.gov identifiers NCT00091819 and NCT00107978) to explore the efficacy of telavancin in patients with various types of cSSSIs. RESULTS: A total of 1794 patients were included in this analysis; 1434 patients were clinically evaluable (CE) and 563 of these had methicillin-resistant Staphylococcus aureus (MRSA). Among CE patients with major abscesses (n = 619), cure rates were 91% for telavancin and 90% for vancomycin (95% CI for the difference -3.6 to 5.7). In patients with infective cellulitis (n = 519), cure was achieved in 87% and 88% of telavancin- and vancomycin-treated patients, respectively (95% CI for the difference -6.2 to 5.2). Cure rates in patients with wound infections were 85% in the telavancin group and 86% in the vancomycin group (95% CI for the difference -10.5 to 9.0). Cure rates for each type of cSSSI in patients infected with MRSA were also similar between the two treatment arms. Among CE patients infected with Panton-Valentine leucocidin (PVL)-positive MRSA (n = 447), cure rates were 93% for telavancin and 90% for vancomycin (95% CI for the difference -2.2 to 8.2). CONCLUSIONS: Cure rates were similar for telavancin and vancomycin in patients with different types of cSSSIs, including infections caused by MRSA and PVL-positive strains of MRSA.

The inability of tegaserod to affect platelet aggregation and coronary artery tone at supratherapeutic concentrations.

In 2007, the results from a meta analysis of 29 clinical studies indicated that tegaserod (Zelnorm®), a 5-hydroxytryptamine(4) (5-HT(4)) receptor agonist with gastrointestinal prokinetic activity, was associated with an increased incidence of cardiovascular ischemic events, resulting in its withdrawal from many markets around the world. Stimulation of platelet aggregation has been proposed to explain the phenomenon. However, data from recent epidemiological studies have suggested that there is no correlation between tegaserod use and the incidence of cardiovascular ischemia. In this study, the influence of tegaserod, at concentrations up to tenfold higher than the total plasma C (max) for the 6 mg clinical dose, has been investigated on platelet aggregation under standard conditions with platelet-rich plasma (PRP) obtained from healthy human subjects. Additionally, the influence of tegaserod on coronary artery tone was evaluated as an alternative pro-ischemic mechanism. The positive control, thrombopoietin, but not tegaserod, demonstrated a statistically significant increase in platelet aggregation using the same PRP samples with either adenosine diphosphate (ADP) or ADP plus 5-HT as an aggregation agonist. Tegaserod had no contractile activity in either porcine or human isolated coronary artery preparations, and only a small and variable response in canine coronary arteries at concentrations higher than those achieved clinically. Taken together, these studies do not identify a mechanism for the ischemic events that have been attributed to tegaserod in humans.

Telavancin versus vancomycin for the treatment of complicated skin and skin-structure infections associated with surgical procedures.

BACKGROUND: We compared telavancin with vancomycin for the treatment of complicated skin and skin-structure infections (cSSSI) caused by Gram-positive bacteria. METHODS: This was a retrospective analysis of clinical and microbiologic efficacy assessed at test-of-cure (7 to 14 days after completing therapy) in 194 patients from 2 randomized, double-blind clinical trials comparing telavancin (10 mg/kg intravenous [IV] every 24 hours; n = 101) with vancomycin (1 g IV every 12 hours; n = 93) for the treatment of cSSSI. RESULTS: Baseline characteristics were similar for both treatment groups. Clinical cure and microbiologic eradication rates demonstrated consistent trends favoring telavancin over vancomycin; however, the differences were not statistically significant. The incidence of adverse events was mostly similar between groups. CONCLUSIONS: The efficacy of telavancin was at least equivalent to that of vancomycin for the treatment of cSSSI. These data suggest that telavancin may be a useful alternative for treatment of cSSSI caused by S. aureus, particularly MRSA.

Telavancin versus vancomycin for the treatment of complicated skin and skin-structure infections caused by gram-positive organisms.

BACKGROUND: Telavancin is an investigational, rapidly bactericidal lipoglycopeptide with a multifunctional mechanism of action. METHODS: We conducted 2 parallel, randomized, double-blind, active-control, phase 3 studies with a prespecified pooled analysis design. Patients aged > or = 18 years who had complicated skin and skin-structure infections caused by suspected or confirmed gram-positive organisms were randomized to receive either telavancin (10 mg/kg intravenously every 24 h) or vancomycin (1 g intravenously every 12 h). RESULTS: A total of 1867 patients were randomized and received > or = 1 dose of study medication. In the clinically evaluable population, at 7-14 days after receipt of the last antibiotic dose, success was achieved in 88% and 87% of patients who received telavancin and vancomycin, respectively (95% confidence interval for the difference, -2.1 to 4.6). Methicillin-resistant Staphylococcus aureus was isolated at baseline from samples from 579 clinically evaluable patients. Among these patients with methicillin-resistant S. aureus infection, cure rates were 91% among patients who received telavancin and 86% among patients who received vancomycin (95% confidence interval for the difference, -1.1 to 9.3). Microbiologic eradication among patients infected with methicillin-resistant S. aureus was 90% in the telavancin treatment group and 85% in the vancomycin treatment group (95% confidence interval for the difference, -0.9 to 9.8). Therapy was discontinued because of adverse events in 8% and 6% of patients who received telavancin and vancomycin, respectively. Except for mild taste disturbance, nausea, vomiting, and serum creatinine concentration elevation in the telavancin treatment group and pruritus in the vancomycin treatment group, adverse events were similar between groups with regard to type and severity. CONCLUSIONS: Telavancin given once daily is at least as effective as vancomycin for the treatment of patients with complicated skin and skin-structure infections, including those infected with methicillin-resistant S. aureus.

Analyst Variation in Doing the Standard Plate Count as Described in Standard Methods for the Examination of Dairy Products 1, 2.

Five analysts participated in a study to evaluate the following aspects of the 13th edition of Standard Methods for the Examination of Dairy Products (SMEDP): (a) analyst variation in overall Standard Plate Counts (SPC), and (b) analyst duplication of bacterial colony counts on agar places. Each analyst prepared 24 samples of pasteurized, homogenized milk during a successive 8-day period (i.e., 3 samples/day), and then the analysts estimated the numbers of bacterial colonies for these, as well as other analysts' plates, initially after 48 h of incubation, and then 1 h later and 24 h later. Statistically significant differences in colony enumerations were found between analysts in preparation of agar plates on 3 days. Significant differences were also noted between analysts for bacterial counts of agar plates. Mean bacterial estimates of certain analysts ranged between 565 and 948, and fluctuated greatly between the initial, 1-h, and 24-h determinations. These results indicate that the "standards of accuracy" currently specified in SMEDP are not realistic, i.e., (a) among-analyst variation of 18.2% compared to 10%, and (b) within-analyst variation of 7.7% compared to 5% in SMEDP.