Autoantigens PLA2R and THSD7A in membranous nephropathy share a common epitope motif in the N-terminal domain.

Patients with membranous nephropathy have autoantibodies against PLA2R (up to 80%), or THSD7A (up to 2%). We previously described the immunodominant epitope within PLA2R but epitopes in THSD7A are still unknown. To find anti-THSD7A sera for this study, we screened 1843 sera from biopsy-proven MN patients by ELISA and identified 22 sera as anti-THSD7A positive representing 1.2% of MN cases. Anti-THSD7A positive sera were further characterized by western blotting and slot blotting on THSD7A protein fragments and peptides. Real time interaction analyses and antibodies off-rate could be reliably determined using bio-layer interferometry. A signature motif in the N-terminal domain of THSD7A (T28mer) with sequence homology to the major PLA2R epitope (P28mer) was identified. B-cell epitope prediction analysis and homology modelling revealed this sequence to be antigenic and surface available suggesting it is accessible for the antibody to bind. All ten selected sera bound to the T28mer confirming this sequence as a dominant epitope in THSD7A. Reactivity to this sequence was lost following kallikrein protease cleavage within the predicted epitope. Importantly, cross-reactivity of both PLA2R and THSD7A autoantibodies was observed at the peptide but not the protein level. We propose that this common motif shared by both autoantigens could be an epitope involved in the initial B-cell triggering event in MN.

Correction to: Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis.

We thank D Nicholson for initial advice on caspase activity purification and B Turk for advice on recombinant cathepsin B. We thank N Atanasova for cell death assays. The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund. Special thanks go to D Knight in the Faculty Biomolecular Analysis facility. We thank P Birch and M Kim for improving the manuscript. The project was partially funded by BBSRC Grants 34/P14516, BB/K009478/1 and China National High-Tech Research and Development Programme(863 programme)NO. 2015AA020903.

Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis.

Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation.

A Demonstration Project in New York and Virginia: Retrofitting Cost-Effective Roll-over Protective Structures (CROPS) on Tractors.

The NIOSH cost-effective roll-over protective structure (CROPS) demonstration project sought to determine whether three prototype roll-over protective structures (ROPS) designed to be retrofitted on Ford 8N, Ford 3000, Ford 4000, and Massey Ferguson 135 tractors could be installed in the field and whether they would be acceptable by the intended end users (farmers). There were a total of 50 CROPS. demonstrators (25 in New York and 25 in Virginia), with 45 observers attending the New York CROPS demonstrations and 36 observers attending the Virginia CROPS demonstrations, for a total of 70 participants in New York and 61 in Virginia. The oldest retrofitted tractors were 77 to 62 years old, while the newest retrofitted tractors were 40 to 37 years old. The most frequently retrofitted tractor in the CROPS demonstration project was a Ford 3000 series tractor (n = 19; 38%), followed by Ford 4000 (n = 11; 22%), Massey Ferguson 135 (n = 11; 22%), and Ford 8N (n = 9; 18%). A major issue of CROPS retrofitting was the rear wheel fenders. The effort involved in disassembling the fenders (removing the old bolts was often faster by cutting them with a torch), modifying the fender mounting brackets, and then reinstalling the fenders with the CROPS generally required the most time. In addition, various other semi-permanent equipment attachments, such as front-end loaders, required additional time and effort to fit with the CROPS. Demonstrators were asked to rank the reasons why they had not retrofitted their tractors with ROPS until they had enrolled in the CROPS demonstration program. ROPS "cost too much" was ranked as the primary reason for participants in both states (80% for New York and 88% for Virginia). The second highest ranked reasons were "ROPS wasn't available" for Virginia (80%) and "hassle to find ROPS" for New York (69%). The third highest ranked reasons were "not enough time to find ROPS" for New York (67%) and "hassle to find ROPS" for Virginia (79%). All demonstrators and observers indicated that they were glad to have participated in the CROPS project.

Techniques for Building an OSHA Compliant Guardrail Structure.

In the construction industry, workers falling to a lower level has been the primary cause of fatalities according to the Bureau of Labor Statistics Census of Fatal Occupational Injuries database. From 2006 to 2010, an average of 353 construction workers died annually as a result of falling to a lower level. An average of 126 workers (36%) died when falling from unguarded roof edges, and through roof and floor holes or skylights. The National Institute for Occupational Safety and Health evaluated the strength of job-built guardrail structures around an opening. The study focused on a 2'×4' opening typical of residential skylights. Nine full-time residential carpenters built guardrails for strength testing.

Tractors and rollover protection in the United States.

There are approximately 4.2 million tractors on farms and ranches across the United States. The average age of tractors is over 25 years and some of the oldest models are the most popular. Older tractors are less safe than newer tractors, and many older tractors are operated by individuals with increased risk of being injured or killed on a tractor. A key tractor safety device, a rollover protective structure (ROPS), is missing from most tractors manufactured before 1985. Data from the US Department of Labor's Census of Fatal Occupational Injuries (CFOI) suggest that the production agriculture sector accounts for approximately 70.3% of the 3299 work deaths in the Agriculture, Forestry, and Fishing industry between 2003 and 2007. Nearly 900 of these incidents involve farm tractors and of these, approximately 43% were from tractor overturns. Efforts to reduce both the number of tractor overturn fatalities and injuries have been underway for years. These efforts primarily encompass worker education/training programs and activities, ROPS design and engineering applications, and research on more effective ways of encouraging tractor owners to retrofit their older tractors with ROPS. This paper reviews various approaches available to reduce the fatalities, serious injuries, and economic burden associated with tractor overturns. Past and current efforts to promote ROPS in the United States and in other countries, current safe tractor operations education and training programs, and ROPS-related safety engineering projects are discussed. Recommendations for advancing safe tractor operation and the number of tractors protected by ROPS are given. This review was prepared for the Agricultural Safety and Health Council of America/National Institute for Occupational Safety and Health conference, "Be Safe, Be Profitable: Protecting Workers in Agriculture," January 2010.

Evaluation of guardrail systems for preventing falls through roof and floor holes.

INTRODUCTION: Fall-related occupational injuries and fatalities are serious problems in the U.S. construction industry, especially incidents related to unguarded holes. The National Institute for Occupational Safety and Health, Division of Safety Research, Morgantown, WV conducted a project to evaluate the effectiveness of guardrail systems to prevent falls through roof and floor holes. METHODS: Two commercial edge-protection products were evaluated when used as perimeter guarding around a roof hole. Installations of the commercial products were compared to job-built guardrails constructed of 2('')x4('') construction-grade lumber. Occupational Safety and Health Administration (OSHA) regulations require that "a force of at least 200 pounds" must be supported by the top rail of a guardrail system "in any outward or downward direction at any point along the top edge." A laboratory testing system was developed to evaluate this requirement. A dynamic 200-lb force was generated against the top rail using a weighted manikin mounted on a hinged steel frame. Nine construction workers, who served as test subjects, each built five different guardrail configurations. RESULTS: All 45 configurations met the 200-lb OSHA requirement. Installation time for one commercial product was 32% quicker than the job-built configuration (25.6 min vs. 37.9 min). IMPACT ON INDUSTRY: This study: (a) indicates that the two edge-protection products can be used as perimeter guarding; (b) highlights the importance of using proper materials and fasteners to construct guardrails to protect workers from falling into unguarded roof and floor holes; and (c) discusses an overall-strength-testing methodology that can be used by fall-protection researchers.

ROPS performance during field upset and static testing.

Agriculture remains one of the most hazardous occupations in the U.S. By conservative estimates, tractor overturns alone claim 120 lives annually. A rollover protective structure (ROPS) and a seatbelt are a highly effective engineering safety control that can prevent many of these fatalities and reduce the severity of injuries associated with tractor overturn. SAE J2194 is a consensus performance standard established for agricultural ROPS. According to this standard, satisfactory ROPS performance can be demonstrated through static testing, field upset testing, or impact testing. A previous modeling study suggested that static testing may underpredict the strain induced in a ROPS during afield upset. In the current study, field upset testing and laboratory static testing results were compared. Field upset testing included six rear and six side upset tests performed according to SAE J2194 guidelines. Additionally, static testing was performed on a ROPS of the same model. The results support findings from the modeling study. Near the lowest sections of the ROPS, the plastic strain resulting from rear upset testing exceeded the plastic strain from static testing for 18 of 24 data points. Conversely, the ROPS plastic strain from side upset testing was typically less than plastic strain from laboratory static testing. However, data indicate that the side upset test may not be very repeatable. This study suggests that the longitudinal loading energy criterion for static testing might not be a conservative predictor of rear upset ROPS response.

Heparanase: a target for drug discovery in cancer and inflammation.

The remodelling of the extracellular matrix (ECM) has been shown to be highly upregulated in cancer and inflammation and is critically linked to the processes of invasion and metastasis. One of the key enzymes involved in specifically degrading the heparan sulphate (HS) component of the ECM is the endo-beta-glucuronidase enzyme heparanase. Processing of HS by heparanase releases both a host of bioactive growth factors anchored within the mesh of the ECM as well as defined fragments of HS capable of promoting cellular proliferation. The finding that heparanase is elevated in a wide variety of tumor types and is subsequently linked to the development of pathological processes has led to an explosion of therapeutic strategies to inhibit its enzyme activity. So far only one compound, the sulphated oligosaccharide PI88, which both inhibits heparanase activity and has effects on growth factor binding has reached clinical trials where it has shown to have promising efficacy. The scene has clearly been set however for a new generation of compounds, either specific to the enzyme or with dual roles, to emerge from the lab and enter the clinic. The aim of this review is to describe the current drug discovery status of small molecule, sugar and neutralising antibody inhibitors of heparanase enzyme activity. Potential strategies will also be discussed on the selection of suitable biomarker strategies for specific monitoring of in vivo heparanase inhibition which will be crucial for both animal model and clinical trial testing.

The centromere and promoter factor, 1, CPF1, of Saccharomyces cerevisiae modulates gene activity through a family of factors including SPT21, RPD1 (SIN3), RPD3 and CCR4.

In Saccharomyces cerevisiae, the CPF1 gene encodes a centromere binding protein that also plays a role in transcription; cpf1 strains are methionine auxotrophs. In this paper we describe four strains that are methionine prototrophs despite containing a defective CPF1 gene. These strains, which contain mutations at either the SPT21, RPD1 (SIN3), RPD3 or CCR4 loci, have defective centromere function and a chromatin structure around the CDEI elements in the MET25 promoter characteristic of strains lacking CPF1. This indicates that the roles of CPF1 in transcription, centromere function and chromatin modulation around CDEI sites are different. We propose that CPF1 functions to overcome the repressing action, mediated via inactive chromatin, of proteins such as SPT21 or RPD1 (SIN3) on gene expression. The absence of proteins such as SPT21 or RPD1 (SIN3) relieves this repression and explains how methionine prototrophy is restored in the absence of CPF1.

Sunlight-induced mutagenicity of a common sunscreen ingredient.

We have tested the mutagenicity of a UV-B sunscreen ingredient called Padimate-O or octyl dimethyl PABA, which, chemically speaking, is identical to an industrial chemical that generates free radicals when illuminated. It is harmless in the dark but mutagenic in sunlight, attacking DNA directly. A commercial sunscreen containing Padimate-O behaves in the same way. UV-A in sunlight also excites Padimate-O, although less than UV-B. Some related compounds, including a known carcinogen, behave similarly. As mutagens may be carcinogenic, our results suggest that some sunscreens could, while preventing sunburn, contribute to sunlight-related cancers.

Activation of chromosomal vitellogenin genes in Xenopus oocytes by pure estrogen receptor and independent activation of albumin genes.

We generate pure estrogen receptor protein in Xenopus oocytes by injecting them with estrogen receptor mRNA synthesized in vitro. A chromosomal vitellogenin gene, which normally responds to estrogen only in liver cells, is activated. Primer extension shows that initiation is accurate, and ribonuclease mapping shows that the first exon is correctly spliced out of the initial transcript. Long transcripts are produced, one being equal in length to poly(A)- vitellogenin mRNA. Immunochemical estimates of receptor levels in the oocyte nuclei suggest that pure receptor, acting alone, cannot activate oocyte vitellogenin genes unless unusually large amounts are present. However, when a receptor-free extract from liver cells is also injected, the amount of receptor required is reduced. Such an extract, but not pure receptor, can also activate albumin genes in oocytes.

High concentrations of estrogen stabilize vitellogenin mRNA against cytoplasmic degradation but physiological concentrations do not.

Using DNA excess filter hybridization to pulse-labeled cellular RNA, we examined the stability of vitellogenin mRNA in Xenopus liver in relation to estrogen concentration. We showed that pharmacological concentrations of estrogen stabilize vitellogenin mRNA against degradation but that physiological concentrations do not. We concluded that there is little foundation for the common belief that estrogen stabilizes vitellogenin mRNA in normal liver cells and that such stabilization contributes to the normal expression of vitellogenin genes. We also discuss the importance of steroid concentration in other contexts, and show that the widespread tendency to use artificially high concentrations may lead to questionable conclusions.

Selective photochemical treatment of oestrogen receptor in a Xenopus liver extract destroys hormone binding and transcriptional activation but not DNA binding.

Photochemical excitation of a simple derivative of oestradiol using light in the UV-A range totally, permanently and selectively inactivates the oestrogen receptor protein present in a Xenopus liver extract without affecting its overall size. Inactivation of the binding site proceeds to completion with simple, first-order kinetics. Inactivation is prevented by excess oestradiol but not by non-oestrogenic steroids. Using an in vitro transcription system, we show that the treatment eliminates transcription of vitellogenin genes, which are normally oestrogen-responsive, but has no effect on the transcription of albumin genes, which are not. Native receptor binds to the two imperfectly palindromic sequences in the vitellogenin B2 gene which together constitute an oestrogen-response unit. Its affinity for one sequence is greater than its affinity for the other, suggesting that a compulsory binding order operates when receptor interacts with the B2 gene. Photoinactivated receptor still binds to both sequences, but with reduced affinity. We also discuss our findings in the context of the current concern over the effects of UV-A on human tissues.