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Poor translation from animal studies to human clinical trials is one of the main hurdles in the development of new drugs. Here, we used precision-cut kidney slices (PCKS) as a translational model to study renal fibrosis and to investigate whether inhibition of tyrosine kinase receptors, with the selective inhibitor nintedanib, can halt fibrosis in murine and human PCKS. We used renal tissue of murine and human origins to obtain PCKS. Control slices and slices treated with nintedanib were studied to assess viability, activation of tyrosine kinase receptors, cell proliferation, collagen type I accumulation, and gene and protein regulation. During culture, PCKS spontaneously develop a fibrotic response that resembles in vivo fibrogenesis. Nintedanib blocked culture-induced phosphorylation of platelet-derived growth factor receptor and vascular endothelial growth factor receptor. Furthermore, nintedanib inhibited cell proliferation and reduced collagen type I accumulation and expression of fibrosis-related genes in healthy murine and human PCKS. Modulation of extracellular matrix homeostasis was achieved already at 0.1 μM, whereas high concentrations (1 and 5 μM) elicited possible nonselective effects. In PCKS from human diseased renal tissue, nintedanib showed limited capacity to reverse established fibrosis. In conclusion, nintedanib attenuated the onset of fibrosis in both murine and human PCKS by inhibiting the phosphorylation of tyrosine kinase receptors; however, the reversal of established fibrosis was not achieved.

Objective: AIDS Drug Assistance Programs (ADAPs) are state-administered programs that pay for medical care and medication for people living with HIV (PLWH) in the United States. In October 2021, the federal policy requiring that clients recertify for the program every 6 months was repealed, giving states the authority to set their own recertification policies. However, little data exist on the costs and health effects of alternative recertification schedules. We assessed the cost of changing the legacy 6-month recertification to a 12-month schedule in Washington State to inform policy decisions on recertification.Methods: We used a Markov model to simulate the population of PLWH in Washington State who are eligible or enrolled in ADAP. We obtained model inputs and validation data from the Washington State Ryan White database. We estimated the cost of 12-month and 6-month criteria over a 5-year time horizon. Model outputs included annual program costs, population sizes, and number of people virally suppressed, by scenario.Results: Under a continuation of the legacy 6-month recertification criteria, the annual cost of Washington ADAP would be $37 663 000 (95% CI, $34 570 000-$41 686 000) during the next 5 years, with a per-client cost of $7966 (95% CI, $7478-$8494). Under 12-month criteria, the annual cost would be $40 217 000 (95% CI, $36 243 000-$44 401 000) and the per-client cost would be $7543 (95% CI, $7084-$8042). Under the 12-month scenario, 245 more people will have been virally suppressed by the end of 2025.Conclusions: Switching to a less frequent recertification process may improve health outcomes at a modest increase in cost in Washington State.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype–specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.

The Biology/Disease-driven (B/D) working groups of the Human Proteome Project are alliances of research groups aimed at developing or improving proteomic tools to support specific biological or disease-related research areas. Here, we describe the activities and progress to date of the B/D working group focused on protein aggregation diseases (PADs). PADs are characterized by the intra- or extracellular accumulation of aggregated proteins and include devastating diseases such as Parkinson’s and Alzheimer’s disease and systemic amyloidosis. The PAD B/D working group aims for the development of proteomic assays for the quantification of aggregation-prone proteins involved in PADs to support basic and clinical research on PADs. Because the proteins in PADs undergo aberrant conformational changes, a goal is to quantitatively resolve altered protein structures and aggregation states in complex biological specimens. We have developed protein-extraction protocols and a set of mass spectrometric (MS) methods that enable the detection and quantification of proteins involved in the systemic and localized amyloidosis and the probing of aberrant protein conformational transitions in cell and tissue extracts. In several studies, we have demonstrated the potential of MS-based proteomics approaches for specific and sensitive clinical diagnoses and for the subtyping of PADs. The developed methods have been detailed in both protocol papers and manuscripts describing applications to facilitate implementation by nonspecialized laboratories, and assay coordinates are shared through public repositories and databases. Clinicians actively involved in the PAD working group support the transfer to clinical practice of the developed methods, such as assays to quantify specific disease-related proteins and their fragments in biofluids and multiplexed MS-based methods for the diagnosis and typing of systemic amyloidosis. We believe that the increasing availability of tools to precisely measure proteins involved in PADs will positively impact research on the molecular bases of these diseases and support early disease diagnosis and a more-confident subtyping.

A recent paper in The Anthropocene Reviewintroduces an ‘Anthropocene equation’, which is assumed to model the Anthropocene. While welcoming mathematical treatment as such, we criticize the specific approach for being sloppy, wrong and empty. While the use of mathematics in the criticized paper suggests a high level of scientific rigor, it actually weakens the message rather than strengthens it.

Abstract:Whiteness has always been visible and marked in Africa. This is what makes whiteness in Africa distinct from whiteness in the West. This article explores the question of how the visibility of whiteness matters for its politics by focusing on the case of Zimbabwe. Much of the work on whiteness in this country, concentrating solely on the white farming community, presents the white population as a homogeneous group. This article uses the urban–rural divide to challenge such a portrayal and to explore the relationship in Zimbabwe between the politics of representation and the politics of whiteness in the postcolonial era. Based on four years of ethnographic research, it investigates urban and rural whiteness together because they are interrelated. We make two specific observations: first, that urban privilege has remained invisible because white Zimbabweans and white privilege are imagined to be connected to the land and to being a farmer. Urban whites have perpetuated this stereotype, which helped mask their own privileged lives. Second, we demonstrate that the defence of white privilege happens through means other than simple denial. Our interview data shows that, despite urban whites' acknowledgement and understanding of white privilege, they still defend and try to legitimize it. Finally, we conclude that raising awareness and demanding acknowledgement of white privilege might be a necessary but insufficient condition to end it.Résumé:La blanchité a toujours été visible et marquée en Afrique. C'est ce qui distingue la blanchité en Afrique de la blanchité en Occident. Cet article explore la question de savoir en quoi la visibilité de la blanchité importe pour sa politique en se concentrant sur le cas du Zimbabwe. L'essentiel de la recherche sur la blanchité dans ce pays, exclusivement axée sur le paysannat blanc, présente la population blanche comme un groupe homogène. Cet article utilise le clivage urbain–rural pour remettre en cause cette description et explorer la relation, au Zimbabwe, entre la politique de la représentation et la politique de la blanchité au cours de la période postcoloniale. Basé sur quatre années de recherche ethnographique, il examine la blanchité urbaine et la blanchité rurale ensemble parce qu'elles sont liées. Les auteurs font deux observations spécifiques : d'abord, que le privilège urbain est resté invisible parce que les Zimbabwéens blancs et le privilège blanc sont imaginés liés à la terre et au fait d'être agriculteur. Les blancs urbains ont perpétué ce stéréotype qui a contribué à masquer leur existence privilégiée. Ensuite, les auteurs démontrent que la défense du privilège blanc se fait par des moyens autres que le simple déni. Les données d'entretiens montrent qu'en dépit de leur reconnaissance et de leur compréhension du privilège blanc, les blancs urbains continuent de le défendre et de tenter de le légitimer. Enfin, les auteurs concluent que la sensibilisation et la demande de reconnaissance du privilège blanc peuvent être une condition nécessaire mais insuffisante pour y mettre fin.

AbstractWhiteness has always been visible and marked in Africa. This is what makes whiteness in Africa distinct from whiteness in the West. This article explores the question of how the visibility of whiteness matters for its politics by focusing on the case of Zimbabwe. Much of the work on whiteness in this country, concentrating solely on the white farming community, presents the white population as a homogeneous group. This article uses the urban–rural divide to challenge such a portrayal and to explore the relationship in Zimbabwe between the politics of representation and the politics of whiteness in the postcolonial era. Based on four years of ethnographic research, it investigates urban and rural whiteness together because they are interrelated. We make two specific observations: first, that urban privilege has remained invisible because white Zimbabweans and white privilege are imagined to be connected to the land and to being a farmer. Urban whites have perpetuated this stereotype, which helped mask their own privileged lives. Second, we demonstrate that the defence of white privilege happens through means other than simple denial. Our interview data shows that, despite urban whites’ acknowledgement and understanding of white privilege, they still defend and try to legitimize it. Finally, we conclude that raising awareness and demanding acknowledgement of white privilege might be a necessary but insufficient condition to end it.

Many intra- and extracellular signals induce structural changes in proteins. Schopper et al., describe a limited proteolysis–based mass spectrometry (LiP-MS) approach to characterizing these changes at a proteome-wide scale.

Objective: To explore the privacy and security of free medication applications (apps) available to Canadian consumers.Methods: The authors searched the Canadian iTunes store for iOS apps and the Canadian Google Play store for Android apps related to medication use and management. Using an Apple iPad Air 2 and a Google Nexus 7 tablet, 2 reviewers generated a list of apps that met the following inclusion criteria: free, available in English, intended for consumer use and related to medication management. Using a standard data collection form, 2 reviewers independently coded each app for the presence/absence of passwords, the storage of personal health information, a privacy statement, encryption, remote wipe and third-party sharing. A Cohen’s Kappa statistic was used to measure interrater agreement.Results: Of the 184 apps evaluated, 70.1% had no password protection or sign-in system. Personal information, including name, date of birth and gender, was requested by 41.8% (77/184) of apps. Contact information, such as address, phone number and email, was requested by 25% (46/184) of apps. Finally, personal health information, other than medication name, was requested by 89.1% (164/184) of apps. Only 34.2% (63/184) of apps had a privacy policy in place.Conclusion: Most free medication apps offer very limited authentication and privacy protocols. As a result, the onus currently falls on patients to input information in these apps selectively and to be aware of the potential privacy issues. Until more secure systems are built, health care practitioners cannot fully support patients wanting to use such apps.

Immigrants are often described as ‘under-participating’ in non-urban outdoor recreation. Our quantitative study among 1057 young adults of Chinese, Turkish and non-immigrant descent in the Netherlands, however, showed strong differences in outdoor recreational behaviour between and within ethnic groups. Participation frequency, participation rate, group size and motivations strongly varied between ethnic groups. In our study, a similar share of Turkish immigrants and non-immigrants recreated in non-urban greenspace, while half as many Chinese immigrants did so. Respondents of Turkish descent mainly participated in group-based activities and respondents of Chinese descent in individual-based activities. Respondents subjectively perceived their ethnicity but also personal identities, age and, to a lesser extent, gender and religion to influence their outdoor recreational behaviour. Cluster analysis of participants in non-urban outdoor recreation resulted in five recreational types: ‘Nature lovers’, ‘Social animals’, ‘Activity lovers’, ‘Group-based quiet seekers’ and ‘Individual quiet seekers’. These recreational types were correlated with ethnicity, but also with various other socio-demographic variables. We therefore argue that ‘under-participation’ is a misleading term to typify non-urban outdoor recreational behaviour of immigrants, and that multiple identities should be taken into account to understand this behaviour.

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PHOTO (COLOR) PHOTO (COLOR) PHOTO (COLOR) Dear Editor: Joanne Hammond's article "Stop and Witness" [Winter 2020], is bang-on about the historical inaccuracy of government road plaques that attempt to signpost historical points of interest for the travelling public. INBOX Dear Editor: I am heartened to learn you and others have enjoyed reading Union Laundry: The Story of Harry and May Yuen [in our Winter 2020 issue, 53.4]. [Extracted from the article]

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non–FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitroexperiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Cells secrete a large number of proteins to communicate with their surroundings. Furthermore, plasma membrane proteins and intracellular proteins can be released into the extracellular space by regulated or non-regulated processes. Here, we profiled the supernatant of 11 cell lines that are representative of different stages of breast cancer development by specifically capturing N-glycosylated peptides using the N-glyco FASP technology. For accurate quantification we developed a super-SILAC mix from several labeled breast cancer cell lines and used it as an internal standard for all samples. In total, 1398 unique N-glycosylation sites were identified and quantified. Enriching for N-glycosylated peptides focused the analysis on classically secreted and membrane proteins. N-glycosylated secretome profiles correctly clustered the different cell lines to their respective cancer stage, suggesting that biologically relevant differences were detected. Five different profiles of glycoprotein dynamics during cancer development were detected, and they contained several proteins with known roles in breast cancer. We then used the super-SILAC mix in plasma, which led to the quantification of a large number of the previously identified N-glycopeptides in this important body fluid. The combination of quantifying the secretome of cancer cell lines and of human plasma with a super-SILAC approach appears to be a promising new approach for finding markers of disease.

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft‐derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation‐induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low‐density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf β1and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf β1and Pdgfrbwas upregulated. Gene expression of Klf4was upregulated as well, while expression of Sm22αwas downregulated. Finally, PDGF‐BB‐stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitrowhich was accompanied by increased Klf4and Col1α1,and reduced α‐Smaand Sm22αexpression. In transplant vasculopathy, neointimal PDGF‐BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft-derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation-induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low-density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf β1and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf β1and Pdgfrbwas upregulated. Gene expression of Klf4was upregulated as well, while expression of Sm22αwas downregulated. Finally, PDGF-BB-stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitrowhich was accompanied by increased Klf4and Col1α1,and reduced α-Smaand Sm22αexpression. In transplant vasculopathy, neointimal PDGF-BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Abstract: Objectives: This study evaluated the waiting list for elective electrical cardioversion (ECV) for persistent atrial fibrillation (AF), focusing on when and why procedures were postponed. We compared the effects of management of the waiting list conducted by physicians versus management by nurse practitioners (NPs) and we evaluated the safety of our anticoagulating policy by means of bleeding or thromboembolic complications during and after ECV. Background: Not all patients selected for ECV receive their treatment at the first planned instance due to a variety of reasons. These reasons are still undocumented. Methods: We evaluated 250 consecutive patients with persistent AF admitted to our clinic for elective ECV.Results: Within 5 to 6 weeks, 186 of 242 patients (77%) received ECV. The main reason for postponing an ECV was an inadequate international normalised ratio (INR); other reasons included spontaneous sinus rhythm and switch to rate control. A total of 23 of the 147 patients (16%) managed by the research physician were postponed due to an inadequate INR at admission versus 4 out of 98 patients (4%) managed by NPs (p = 0.005) Conclusion: An inadequate INR is the main reason for postponing an ECV. Management of ECV by NPs is safe and leads to less postponing on admission.

Symbol signs – signs without words – are often used to communicate safety or public information messages. The International Organization for Standardization (ISO) provides standards ISO 3864-1 and ISO 3864-3 giving design principles for both graphical symbols and completed signs in the safety arena. The present study shows that for difficult signage circumstances a two-part sign, showing the desired and undesired circumstances with a tick and a cross, can be much more effective than the normal single-part sign provided for by these ISO standards. The present study also suggests that ISO's sign assessment methods could be efficiently combined and simplified.

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

Chronic transplant dysfunction is the leading cause of long-term renal allograft loss. One of the histologic hallmarks of chronic transplant dysfunction is transplant vasculopathy characterized by accumulation of smooth muscle cells (SMCs) in the arterial subendothelial space, leading to ischemic graft failure. Currently, no therapy is available for transplant vasculopathy, and knowledge of the origin (donor vs. recipient) of neointimal cells may contribute to develop adequate strategies.

The article addresses the importance of environmental health, conservation and replenishment in the face of economic and social development. It calls on the British government to involve stakeholders and citizens in environmental decision-making when there is a plan for development. It proposes the need to be more optimistic environmental politics that had begun to emerge in the final decades of the 20th century.

Reversible phosphorylation is a key event in many biological processes and is therefore a much studied phenomenon. The mass spectrometric MS analysis of phosphorylation is challenged by the substoichiometric levels of phosphorylation and the lability of the phosphate group in collisioninduced dissociation CID. Here, we review the fragmentation behaviour of phosphorylated peptides in MS and discuss several MS approaches that have been developed to improve and facilitate the analysis of phosphorylated peptides. CID of phosphopeptides typically results in spectra dominated by a neutral loss of the phosphate group. Several proposed mechanisms for this neutral loss and several factors affecting the extent at which this occurs are discussed. Approaches are described to interpret such neutral lossdominated spectra to identify the phosphopeptide and localize the phosphorylation site. Methods using additional activation, such as MS3and multistage activation MSA, have been designed to generate more sequenceinformative fragments from the ion produced by the neutral loss. The characteristics and benefits of these methods are reviewed together with approaches using phosphopeptide derivatization or specific MS scan modes. Additionally, electrondriven dissociation methods by electron capture dissociation ECD or electron transfer dissociation ETD and their application in phosphopeptide analysis are evaluated. Finally, these techniques are put into perspective for their use in largescale phosphoproteomics studies. Copyright © 2009 John Wiley & Sons, Ltd.

In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applicability for de novo sequencing we next separated an ostrich muscle tissue protein lysate by one-dimensional SDS-PAGE. A protein band at 42 kDa was in-gel digested with Lys-N. Relatively straightforward sequencing resulted in the de novo identification of the two ostrich proteins creatine kinase and actin. We therefore conclude that this method that combines Lys-N, strong cation exchange enrichment, and MALDI-MS/MS analysis provides a valuable alternative proteomics strategy.

In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applicability for de novosequencing we next separated an ostrich muscle tissue protein lysate by one-dimensional SDS-PAGE. A protein band at 42 kDa was in-gel digested with Lys-N. Relatively straightforward sequencing resulted in the de novoidentification of the two ostrich proteins creatine kinase and actin. We therefore conclude that this method that combines Lys-N, strong cation exchange enrichment, and MALDI-MS/MS analysis provides a valuable alternative proteomics strategy.

Accurate quantification of protein expression in biological systems is an increasingly important part of proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput proteomics experiments. Although alternative multiplex quantitative proteomics approaches introduce isotope labels at the organism level ('stable isotope labeling by amino acids in cell culture' (SILAC)) or enable the simultaneous analysis of eight samples (isobaric tagging for relative and absolute quantification (iTRAQ)), stable isotope dimethyl labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample. We describe in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams. The labeling steps take approximately 60–90 min, whereas the full protocol including digestion and (two-dimensional) liquid chromatography-mass spectrometry takes approximately 1.5–3 days to complete.

Chronic transplant dysfunction (CTD) is the leading cause for limited kidney graft survival. Renal CTD is characterized by interstitial and vascular remodeling leading to interstitial fibrosis, tubular atrophy and transplant vasculopathy (TV). The origin of cells and pathogenesis of interstitial and vascular remodeling are still unknown. To study graft-versus-recipient origin of interstitial myofibroblasts, vascular smooth muscle cells (SMCs) and endothelial cells (ECs), we here describe a new rat model for renal CTD using Dark Agouti kidney donors and R26 human placental alkaline phosphatase transgenic Fischer344 recipients. This model showed the development of CTD within 12 weeks after transplantation. In interstitial remodeling, both graft- and recipient-derived cells contributed to a similar extent to the accumulation of myofibroblasts. In arteries with TV, we observed graft origin of neointimal SMCs and ECs, whereas in peritubular and glomerular capillaries, we detected recipient EC chimerism. These data indicate that, within the interstitial and vascular compartments of the transplanted kidney, myofibroblasts, SMCs and ECs involved in chronic remodeling are derived from different sources and suggest distinct pathogenetic mechanisms within the renal compartments.

Chronic transplant dysfunction (CTD) is the leading cause for limited kidney graft survival. Renal CTD is characterized by interstitial and vascular remodeling leading to interstitial fibrosis, tubular atrophy and transplant vasculopathy (TV). The origin of cells and pathogenesis of interstitial and vascular remodeling are still unknown. To study graft-versus-recipient origin of interstitial myofibroblasts, vascular smooth muscle cells (SMCs) and endothelial cells (ECs), we here describe a new rat model for renal CTD using Dark Agouti kidney donors and R26 human placental alkaline phosphatase transgenic Fischer344 recipients. This model showed the development of CTD within 12 weeks after transplantation. In interstitial remodeling, both graft- and recipient-derived cells contributed to a similar extent to the accumulation of myofibroblasts. In arteries with TV, we observed graft origin of neointimal SMCs and ECs, whereas in peritubular and glomerular capillaries, we detected recipient EC chimerism. These data indicate that, within the interstitial and vascular compartments of the transplanted kidney, myofibroblasts, SMCs and ECs involved in chronic remodeling are derived from different sources and suggest distinct pathogenetic mechanisms within the renal compartments.

Provides information on food presentations designed by caterers with the International Special Events Society in 2004. Martini glass design; Chinese takeout box; Popularity of crimson red and chocolate brown colors in presentations in 2004. INSET: Drizzle it on.

Presents answers from several event planners about their profession. Benefits of working with event planners for the hospitality industry; Difference between caterers and event planners.

Discusses trends in catering stations and buffets in the U.S. as of March 2004. Emphasis on elevation and lighting; Use of hot rocks; Trends toward minimalism.

A survey was carried out in The Netherlands to determine the prevalence of benzimidazole resistance in sheep nematodes. It was shown that 47 per cent of the 59 investigated flocks harboured trichostrongylid populations with resistance to benzimidazoles, as demonstrated by an LD50 value of 0·12 μg ml–1thiabendazole or higher in an in vitro egg hatch assay. On these farms Haemonchus contortuswas found to be the dominant species. In two flocks resistant Ostertagiaand, or, Trichostrongylusspecies populations were found. Farms with resistance were revisited to determine the anthelmintic efficacy in vivo by means of treatment and egg count. In only four of the 19 flocks which showed resistance in vitro was an efficacy of less than 95 per cent in vivo achieved. This means that there is a discrepancy between the in vivo and in vitro results. Resistance was found more frequently on pure sheep farms than on mixed farms. It is suggested that mixed farms offer more refuge for susceptible H contortuspopulations.

The effect distracting objects have on visual-search performance in real-life situations cannot readily be predicted from current search theories. The validity of an approach to close this gap was tested by comparing search performance for color slides of scenes in public buildings with performance for simplified computer-generated images derived from these slides. The target was always a blue rectangle in both the original slides of scenes and the computer simulations. The distractors were differently colored rectangles (not blue), and their number was varied systematically. Analysis showed a significant linear increase in search time with number of distractors, which challenges predictions of current search theories. An explanation for this contradiction is proposed. Also, search times for color slides were significantly longer than those for computer images; however, there was no significant interaction between type of stimulus and number of distractors. It is concluded that the simulated scenes yielded adequate predictions of the effect of distracting objects on search performance in real-life situations.

In vitro selection for benzimidazole (BZ) resistance in the nematode Haemonchus contortus, a parasite of sheep, suggested the involvement of β-tubulin genes. Two distinct β-tubulin isotype classes were analyzed. Selection with stepwise increased concentrations of BZ showed for the first time that the increasing degrees of resistance correlate to a dual phase adaptation mechanism. At lower degrees of developing resistance, selection for a single β-tubulin isotype 1 variant takes place. Subsequently, at higher degrees of drug resistance, an abrupt elimination of individuals carrying β-tubulin isotype 2 genes in their genome is seen. To evaluate the in vitro experiments we carried out a survey of resistant field populations and found that in the most resistant population (RNO) apparently the same mechanism had happened.Copyright 1993, 1999 Academic Press

Overwintering of horse cyathostomes as inhibited third stage larvae (L3) and the effect of repeated oxfendazole (ofz)treatment on strongyle infections were studied in an experiment with two groups of three Shetland ponies. Both groups were grazed together from May 28 to November 11, 1986 and subsequently housed. Treatments with 10 mg ofzkg−1were given on May 26, July 1 and July 28 and again one week before each group was necropsied in December and April, respectively. Worm populations of both groups were dominated by inhibited early L3. The proportion of fourth stage larvae (L4) was significantly higher in April than in December. The faecal egg counts showed that the ofztreatment in May was more effective than those in July. This resulted in high pasture larval counts from the end of August onwards. The final treatments revealed a low efficacy against L3 and L4 but not against adult worms.

Two experiments were performed to study the effect of an oxfendazole pulse release intraruminal device (oprb) on the development of lungworm infection and immunity in calves. Infections were monitored in oPRB-treated and control calves by faecal and pasturai larval counts. Levels of immunity of grazing groups and helminth-free housed groups were compared by challenge infection. Even though signs of parasitic bronchitis were seen only in control animals, sufficient immunity had developed in oprb-treated calves to prevent disease after a substantial challenge infection. However, the degree of immunity was less than in untreated grazing control groups and there were indications that it was related to initial infection levels. These results imply that the oprbmay successfully control parasitic bronchitis, provided that pasture larval infections are sufficient to allow the development of immunity.

Larvae of a fenbendazole (FBZ)-selected, benzimidazole-resistant strain of Haemonchus contortus were conditioned for inhibited development during 7 weeks at 15‡ C. Sheep raised nearly wormfree were infected with nonfrozen larvae (L3). Larvae from the same batch were exsheathed and cryopreserved over liquid nitrogen for 1 year, and another group of sheep was infected with the same dose of these L3 larvae after thawing. Egg hatch assays for all sheep were done between 21 and 30 days post infection (p.i.). Fenbendazole susceptibility was tested by treating 50% of each group of sheep on day 30 p.i. Worm counts of all sheep were done on day 38 p.i. The exsheathment time of the designated frozen L3 larvae at 15‡ C exceeded the usual time span at room temperature. Of the frozen L3 larvae, 60% was motile after thawing. The mean LC50 values (?g tiabendazole/ml) and the mean percentage of efficacy after FBZ treatment against total worm burden, adults and early L4 larvae of nonfrozen and frozen H. contortus were not significantly different. The mean percentages of take (infectivity) for nonfrozen and frozen doses were 35.3% and 5.9%, respectively (P}<0.001). In nontreated sheep, the mean percentages of inhibition for nonfrozen and frozen doses were 91.9%, and 54.2%, respectively (P<0.01). The exsheathment time seems to be temperature-dependent. The induction of inhibited development of L3 larvae during 7 weeks was as effective as during 5 weeks at 15‡ C. Cryopreservation did not affect the susceptibility for benzimidazoles in vivo or in vitro; for this reason, it is a reliable method for storing drug-resistant strains of H. contortus. Impairment of the infectivity of this prolific species was not dramatic. This first report on the influence of cryopreservation on inhibition-proneness shows that deep-freeze storage might influence the level of this characteristic; however, other factors might be involved.