The complex circumstellar environment of supernova 2023ixf.

The early evolution of a supernova (SN) can reveal information about the environment and the progenitor star. When a star explodes in vacuum, the first photons to escape from its surface appear as a brief, hours-long shock-breakout flare1,2, followed by a cooling phase of emission. However, for stars exploding within a distribution of dense, optically thick circumstellar material (CSM), the first photons escape from the material beyond the stellar edge and the duration of the initial flare can extend to several days, during which the escaping emission indicates photospheric heating3. Early serendipitous observations2,4 that lacked ultraviolet (UV) data were unable to determine whether the early emission is heating or cooling and hence the nature of the early explosion event. Here we report UV spectra of the nearby SN 2023ixf in the galaxy Messier 101 (M101). Using the UV data as well as a comprehensive set of further multiwavelength observations, we temporally resolve the emergence of the explosion shock from a thick medium heated by the SN emission. We derive a reliable bolometric light curve that indicates that the shock breaks out from a dense layer with a radius substantially larger than typical supergiants.

Improving health equity for ethnic minority women in Thai Nguyen, Vietnam: qualitative results from an mHealth intervention targeting maternal and infant health service access.

Background: Ethnic minority women (EMW) in Vietnam experience disproportionately high infant and maternal mortality rates due to low social status, poverty and remoteness from health centres. This project piloted and evaluated a low-cost mobile health (mHealth) intervention called mMom utilizing behaviour change communication (BCC) to improve access to maternal, newborn and child health (MNCH) services and health equity among EMW living in remote areas. Methods: The mMom intervention built an integrated mHealth platform which sent timely MNCH information and BCC text messages to participants, and engaged health workers towards increasing their interaction and building demand for quality natal care. Mid-term and final qualitative evaluations were conducted to assess the intervention's acceptability and impact. Results: In evaluations, all participants expressed satisfaction with the quality, timeliness and convenience of the messages, and health workers reported increased efficiency and quality of care. The use of BCC increased care-seeking from EMW and strengthened relationships with health providers. Conclusion: The mMom project demonstrated the acceptability of mHealth in a remote Vietnamese region with a high proportion of disadvantaged EMW. The messages promoted increased contact between participants and health providers, which holds potential to address the marginalization of EMW from the health system. Keywords: behaviour change communication, eHealth, ethnic minorities, health equity, mHealth, MNCH, mobile health, Vietnam.

MAPK p38 regulates inflammatory gene expression via tristetraprolin: Doing good by stealth.

Tristetraprolin (TTP) is an RNA-destabilizing protein that exerts profound anti-inflammatory effects by inhibiting the expression of tumour necrosis factor and many other inflammatory mediators. The mitogen-activated protein kinase (MAPK) p38 signaling pathway controls the strength and duration of inflammatory responses by regulating both the expression and function of TTP. The kinase MK2 (MAPK activated kinase 2) is activated by MAPK p38, and in turn phosphorylates TTP at two critical serine residues. One consequence of these phosphorylations is the protection of TTP from proteasome-mediated degradation. Another consequence is the loss of mRNA destabilizing activity. The control of TTP expression and function by the MAPK p38 pathway provides an elegant mechanism for coupling the on and off phases of inflammatory responses, and dictating the precise kinetics of expression of individual inflammatory mediators.

Treatment of inflammatory arthritis via targeting of tristetraprolin, a master regulator of pro-inflammatory gene expression.

OBJECTIVES: Tristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation of two serine residues. We wished to test the hypothesis that these phosphorylations contribute to the development of inflammatory arthritis, and that, conversely, joint inflammation may be inhibited by promoting the dephosphorylation and activation of TTP. METHODS: The expression of TTP and its relationship with MAPK p38 activity were examined in non-inflamed and rheumatoid arthritis (RA) synovial tissue. Experimental arthritis was induced in a genetically modified mouse strain, in which endogenous TTP cannot be phosphorylated and inactivated. In vitro and in vivo experiments were performed to test anti-inflammatory effects of compounds that activate the protein phosphatase 2A (PP2A) and promote dephosphorylation of TTP. RESULTS: TTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some fibroblasts and co-localised with MAPK p38 activation. Substitution of TTP phosphorylation sites conferred dramatic protection against inflammatory arthritis in mice. Two distinct PP2A agonists also reduced inflammation and prevented bone erosion. In vitro anti-inflammatory effects of PP2A agonism were mediated by TTP activation. CONCLUSIONS: The phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammatory treatments.

The EBV-encoded latent membrane proteins, LMP2A and LMP2B, limit the actions of interferon by targeting interferon receptors for degradation.

Although frequently expressed in Epstein-Barr virus (EBV)-positive malignancies, the role that latent membrane protein 2A and 2B (LMP2A and LMP2B) have in the oncogenic process remains obscure. Here we show a novel function for these proteins in epithelial cells, namely, their ability to modulate signalling from type I/II interferon receptors (IFNRs). We show that LMP2A- and LMP2B-expressing epithelial cells show decreased responsiveness to interferon (IFN)alpha and IFNgamma, as assessed by STAT1 phosphorylation, ISGF3 and GAF-mediated binding to IFN-stimulated response element and IFNgamma-activated factor sequence elements and luciferase reporter activation. Transcriptional profiling highlighted the extent of this modulation, with both viral proteins impacting 'globally' on IFN-stimulated gene expression. Although not affecting the levels of cell-surface IFNRs, LMP2A and LMP2B accelerated the turnover of IFNRs through processes requiring endosome acidification. This function may form part of EBV's strategy to limit anti-viral responses and define a novel function for LMP2A and LMP2B in modulating signalling from receptors that participate in innate immune responses.

Epstein-Barr virus-encoded EBNA1 regulates cellular gene transcription and modulates the STAT1 and TGFbeta signaling pathways.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumors where it plays an essential role in the maintenance, replication and transcription of the EBV genome. Transcriptional profiling of EBNA1-expressing carcinoma cells demonstrated that EBNA1 also influences the expression of a range of cellular genes including those involved in translation, transcription and cell signaling. Of particular interest was the ability of EBNA1 to enhance expression of STAT1 and sensitize cells to interferon-induced STAT1 activation with resultant enhancement of major histocompatibility complex expression. A negative effect of EBNA1 on the expression of TGFbeta1-responsive betaig-h3 and PAI-1 genes was confirmed at the protein level in EBV-infected carcinoma cells. This effect resulted from the ability of EBNA1 to repress TGFbeta1-induced transcription via a reduction in the interaction of SMAD2 with SMAD4. More detailed analysis revealed that EBNA1 induces a lower steady-state level of SMAD2 protein as a consequence of increased protein turnover. These data show that EBNA1 can influence cellular gene transcription resulting in effects that may contribute to the development of EBV-associated tumors.

Bone density and bone area in Canadian Aboriginal women: the First Nations Bone Health Study.

INTRODUCTION: Canadian Aboriginal women are at increased risk of fracture compared with the general population. HYPOTHESIS: There is disproportionately reduced bone density in Aboriginal women as compared to white females of similar age. METHODS: A random age-stratified (25-39, 40-59 and 60-75) sample of Aboriginal women (n=258) and white women (n=181) was recruited. All subjects had calcaneus and distal forearm bone density measurements, and urban participants (n=397 [90.4%]) also had measurements of the lumbar spine, hip and total body. RESULTS: Unadjusted measurements were similar in the two groups apart from the distal forearm which showed a significantly lower mean Z-score in the Aboriginal women (p=0.03). Aboriginal women were heavier than white women (81.0+/-18.0 kg vs. 76.0+/-18.0 kg, p=0.02). Weight was directly associated with BMD at all measurement sites (p<0.00001) and potentially confounded the assessment of ethnicity on bone mass measurements. Weight-adjusted ANCOVA models demonstrated significantly lower bone density in Aboriginal than white women for the calcaneus, distal forearm, and total body (all p<0.05), but not at the other sites. ANCOVA models (adjusted for age, height and weight) were used to explore differences in bone area and bone mineral content (BMC). There was a significant effect of ethnicity on bone area with Aboriginal women having larger adjusted mean values than white women (lumbar spine p=0.038, total hip p=0.0004, total body p=0.020). In contrast, there was no detectable effect of ethnicity on BMC (all p>0.2). CONCLUSIONS: We identified significant site-specific differences in age-and weight-adjusted bone density for Aboriginal and white women. Larger bone area, rather than a reduction in BMC, appeared to be primarily responsible. Further work is needed to define how these differences in bone density and geometry affect indices of bone strength.

Expression of the cellular FLICE-inhibitory protein (c-FLIP) protects Hodgkin's lymphoma cells from autonomous Fas-mediated death.

Hodgkin's lymphoma (HL) is characterized by the presence of malignant so-called Hodgkin's/Reed-Sternberg (HRS) cells, which display resistance to certain apoptotic stimuli, including a lack of sensitivity to Fas-mediated cell death. However, the mechanisms responsible for their resistance to apoptosis inducers have not been elucidated. Here we confirm that both HL-derived cell lines and the HRS cells of primary HL tissues express Fas ligand (FasL) along with the inhibitory c-FLIP protein. Down-regulation of cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) through the use of specific small inhibitory RNAs (siRNAs) leads to reduced viability of the L428 and L591 HL-derived cell lines. To determine whether endogenous FasL was responsible for the reduction in cell viability observed after down-regulation of c-FLIP, L428 and L591 cells were treated with c-FLIP-specific siRNAs with and without siRNAs directed to FasL. Treatment of these cells with both c-FLIP- and FasL-specific siRNAs in combination restored cell viability to near control levels. Our results provide a mechanism whereby HRS cells are protected from autonomous FasL-mediated cell death while preserving their ability to evade immunosurveillance. Targeting c-FLIP could provide a novel approach to the treatment of HL.

15N and 31P solid-state NMR investigations on the orientation of zervamicin II and alamethicin in phosphatidylcholine membranes.

The topologies of zervamicin II and alamethicin, labeled with (15)N uniformly, selectively, or specifically, have been investigated by oriented proton-decoupled (15)N solid-state NMR spectroscopy. Whereas at lipid-to-peptide (L/P) ratios of 50 (wt/wt) zervamicin II exhibits transmembrane alignments in 1,2-dicapryl (di-C10:0-PC) and 1,2-dilauroyl (di-C12:0-PC) phosphatidylcholine bilayers, it adopts orientations predominantly parallel to the membrane surface when the lengths of the fatty acyl chains are extended. The orientational order of zervamicin II increases with higher phospholipid concentrations, and considerable line narrowing is obtained in di-C10:0-PC/zervamicin II membranes at L/P ratios of 100 (wt/wt). In contrast to zervamicin, alamethicin is transmembrane throughout most, if not all, of its length when reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. The (31)P solid-state NMR spectra of all phospholipid/peptaibol samples investigated show a high degree of headgroup order, indicating that the peptides do not distort the bilayer structure. The observed differences in peptide orientation between zervamicin and alamethicin are discussed with reference to differences in their lengths, helical conformations, distribution of (hydroxy)proline residues, and hydrophobic moments. Possible implications for peptaibol voltage-gating are also described.

The evaluation of transferred health care services in Wunnimin Lake, Wapekeka and Kingfisher Lake First Nations: a nursing perspective.

The Canadian federal government initiated the policy to transfer administrative control of health services to First Nations communities in the late 1980s. While there are outstanding issues concerning the implementation of the policy, many communities consider this an opportunity to improve the health of First Nations people and the work environment of health care providers. This paper reports on the evaluation of the process of transfer of health services experienced by three communities in northwestern Ontario, Canada, focusing on nursing services. Based on interviews with health care providers and community members, the overall assessment was that transfer had successfully addressed chronic issues relating to the working conditions of nurses and problems of recruitment and retention.

Secondary structure and oligomerization of the E. coli glycerol facilitator.

The Major Intrinsic Proteins are found throughout the bacterial, plant, and animal kingdoms and are responsible for the rapid transport of water and other small, polar solutes across membranes. The superfamily includes the aquaporins, the aquaglyceroporins, and the glycerol facilitators. We have overexpressed and purified the Escherichia coli inner membrane glycerol facilitator. Approximately 7.5 mg of 95% pure protein is obtained from 1 L of Escherichia coli cells using immobilized metal affinity chromatography. Well-resolved matrix-assisted laser desorption ionization mass spectra were obtained by solubilization of the protein in octyl-beta-D-glucopyranoside (M(r) = 33 650.3; error approximately 0.4%). The recombinant glycerol facilitator is inserted into the bacterial inner membrane, is functional, and is inhibited by HgCl(2). Polyacrylamide gel electrophoresis suggests that the facilitator is predominantly monomeric when solubilized with dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, and sodium dodecyl sulfate, but that it self-associates, forming soluble oligomers when urea is used during extraction. Similar oligomeric species are demonstrated to exist in the bacterial membrane by chemical cross-linking experiments. Circular dichroism analysis shows that the protein is predominantly alpha-helical. Helix content is significantly higher in protein prepared in the absence of urea (42-55%) than in its presence (32%). A possible role for the facilitator oligomers in interactions with, and regulation of, the glycerol kinase is discussed.

Type 2 diabetes mellitus in Canada's first nations: status of an epidemic in progress.

This review provides a status report on the epidemic of type 2 diabetes mellitus that is affecting many of Canada's First Nations. We focus on the published literature, especially reports published in the past 2 decades, and incorporate data from the Aboriginal Peoples Survey and the First Nations and Inuit Regional Health Survey. We look at the extent and magnitude of the problem, the causes and risk factors, primary prevention and screening, clinical care and education, and cultural concepts and traditional knowledge. The epidemic of type 2 diabetes is still on the upswing, with a trend toward earlier age at onset. Genetic-environmental interactions are the likely cause. Scattered intervention projects have been implemented and evaluated, and some show promise. The current health and social repercussions of the disease are considerable, and the long-term outlook remains guarded. A national Aboriginal diabetes strategy is urgently needed.

Phase I and pharmacologic study of PN401 and fluorouracil in patients with advanced solid malignancies.

PURPOSE: To assess the feasibility of administering PN401, an oral uridine prodrug, as a rescue agent for the toxic effects of fluorouracil (5-FU), and to determine the maximum-tolerated dose of 5-FU when given with PN401, with an 8-hour treatment interval between these agents. PATIENTS AND METHODS: Patients with advanced solid malignancies were treated with escalating doses of 5-FU, given as a rapid intravenous infusion weekly for 3 consecutive weeks every 4 weeks. PN401 was administered orally 8 hours after 5-FU administration, to achieve sustained plasma uridine concentrations of at least 50 micromol/L. Initially, patients received 6 g of PN401 orally every 8 hours for eight doses (schedule 1). When dose-limiting toxicity (DLT) was consistently noted, patients then received 6 g of PN401 every 2 hours for three doses and every 6 hours thereafter for 15 doses (schedule 2). RESULTS: Twenty-three patients received 50 courses of 5-FU and PN401. Among patients on schedule 1, DLT (grade 4 neutropenia complicated by fever and diarrhea) occurred in those receiving 5-FU 1,250 mg/m(2)/wk. Among patients on schedule 2, 5-FU 1,250 mg/m(2)/wk was well tolerated, but grade 4, protracted (> 5 days) neutropenia was consistently noted in those treated with higher doses of the drugs. Nonhematologic effects were uncommon and rarely severe. The pharmacokinetics of 5-FU, assessed in 12 patients on schedule 2, were nonlinear, with the mean area under the time-versus-concentration curve (AUC) increasing from 298 +/- 44 to 962 +/- 23 micromol/L and mean clearance decreasing from 34 +/- 4 to 15.6 +/- 0.38 L/h/m(2) as the dose of 5-FU was increased from 1,250 to 1,950 mg/m(2)/wk. 5-FU AUCs achieved with 5-FU 1,250 mg/m(2)/wk for 6 weeks along with the intensified PN401 dose schedule were approximately five-fold higher than those achieved with 5-FU alone. Plasma uridine concentrations increased with each of the three PN401 doses given every 2 hours, and uridine steady-state concentrations were greater than 50 micromol/L. CONCLUSION: Treatment with oral PN401 beginning 8 hours after 5-FU administration is well tolerated and results in sustained plasma uridine concentrations above therapeutic-relevant levels. The recommended 5-FU dosage for phase II evaluations is 1,250 mg/m(2)/wk for 3 weeks every 4 weeks with the intensified PN401 dose schedule (schedule 2). At this dose, systemic exposure to 5-FU as measured by AUC was five-fold higher than that observed after administration of a conventional 5-FU bolus.

Functional characterization of the conserved "GLK" motif in mitochondrial porin from Neurospora crassa.

Mitochondrial porin facilitates the diffusion of small hydrophilic molecules across the mitochondrial outer membrane. Despite low sequence similarity among porins from different species, a "glycine-leucine-lysine" (GLK) motif is conserved in mitochondrial and Neisseria porins. To investigate the possible roles of these conserved residues, including their hypothesized participation in ATP binding by the protein, we replaced the lysine residue of the GLK motif of Neurospora crassa porin with glutamic acid through site-directed mutagenesis of the corresponding gene. Although the pores formed by this protein have size and gating characteristics similar to those of the wild-type protein, the channels formed by GLEporin are less anion selective than the wild-type pores. The GLEporin retains the ability to be cross linked to [alpha-(32)P]ATP, indicating that the GLK sequence is not essential for ATP binding. Furthermore, the pores formed by both GLEporin and the wild-type protein become more cation selective in the presence of ATP. Taken together, these results support structural models that place the GLK motif in a part of the ion-selective beta-barrel that is not directly involved in ATP binding.

Demystified ... the polymerase chain reaction.

Since its initial description over twenty years ago the PCR has become one of the most valuable and flexible tools available to biomedical research. Subsequently, refinements and modifications to the basic approach, many of which have been described in this review, have enabled the application of the PCR to many areas of diagnostic medicine and have ensured its rapid acceptance as a routine test in many pathology disciplines. The growing importance of molecular approaches to the diagnosis of disease, particularly in histopathology, will continue to secure an ever expanding role for the PCR in diagnostic pathology.

Backbone dynamics of detergent-solubilized alamethicin from amide hydrogen exchange measurements.

Alamethicin is a 20 amino acid antibiotic peptide produced by the soil fungus Trichoderma viride. The peptide inserts into bacterial membranes and self-associates to form ion channels, but the details of this process are unknown. Residue-specific acid- and base-catalyzed exchange data were obtained for 16 of 18 backbone amides of alamethicin dissolved in sodium dodecyl sulfate micelles using high-resolution 2-dimensional heteronuclear nuclear magnetic resonance spectroscopy. To facilitate interpretation of the exchange data, we synthesized N-acetyl-alpha-aminoisobutyric acid-N'-methyl and N-acetyl-alanine-N'-methyl and measured the pD dependence of their hydrogen-deuterium exchange rates to determine the sequence-dependent inductive and steric effects of the alpha-aminoisobutyric acid residue. Intramolecular H-bonding in alamethicin was monitored through the exchange parameters kmin (minimum exchange rate) which indicate that the backbone is significantly more stable than the backbones of alanine-based helical peptides. Rapid exchange at Gly-11 suggests a highly local conformational flexibility in the middle of the peptide. Interactions with the detergent micelle were revealed by the exchange parameters pDmin (pD of minimum exchange) which suggest that the N-terminus of alamethicin interacts more strongly with the detergent micelle than does the C-terminus. A periodicity in pDmin difference data reveals that one surface of the helix interacts more strongly with the micelle. The surface consists of residues 1, 5, 9, 13, 16, and 20. The opposite face of the helix contains several polar residues (two glutamines and a glycine), suggesting that, on average, this face of the helix is directed toward the solvent. These results serve as a model for the interaction of the peptide with membranes containing anionic lipid. In combination with published molecular dynamics simulations [Gibbs et al. (1997) Biophys. J. 72, 2490-2495], the present results also offer insight into the mechanisms of hydrogen-deuterium exchange in helical peptides.

Plasma phospholipid fatty acids in the central Canadian arctic: biocultural explanations for ethnic differences.

As part of the Keewatin Health Assessment Study, a comprehensive health interview and examination survey of Inuit and non-Inuit in the central Canadian Arctic during 1990-91, plasma samples were analyzed for phospholipid fatty acid composition. Compared to non-Inuit, the Inuit have reduced levels of dihomo-gamma-linoleic (DGLA) and arachidonic acid (ratios of 0.41 and 0.46) and the sum of all n-6 fatty acids (ratio of 0.65), but increased level of eicosapentaenoic (EPA) acid (ratio of 1.37). These trends are consistent with those reported from other circumpolar Inuit populations, especially the reduced arachidonic acid and increased EPA, although the Inuit excess in EPA is much less pronounced due to the greater importance of caribou rather than sea mammals in most of the Keewatin communities. The high linoleic/arachidonic acid ratio suggests increased inhibition of the metabolic pathway regulated by the enzyme delta-5 desaturase, which can be explained by the presence of high levels of highly unsaturated fatty acids of dietary origin, and/or a genetic deficiency. In multiple linear regression models with the independent variable list consisting of Inuit status, age, sex, education, physical activity, spending time on the land and consumption of wild meat and local fish, Inuit status is independently associated with lower levels of the n-6 acids but not the n-3 acids. This indicates that factors other than diet and lifestyle, perhaps genetic ones, may account for the observed "ethnic" differences. However, for those fatty acids in which Inuit differ from non-Inuit, there is no dose-response relationship in terms of self-reported degree ofnon-Inuit admixture. Dietary fatty acids play an important role in the risk of cardiovascular diseases and diabetes, diseases of increasing importance in the health transition experienced by the Inuit. Association studies of plasma fatty acids and DNA markers of candidate genes for atherosclerosis and insulin resistance may provide a clearer picture of the genetic basis for the observed differences in plasma fatty acid composition between Inuit and non-Inuit.

The interactions with solvent, heat stability, and 13C-labelling of alamethicin, an ion-channel-forming peptide.

The peptide alamethicin was labelled with 13C and 15N by growing the fungus Trichoderma viride in a medium containing [U-13C] glucose and K15NO3. Spin-echo difference spectroscopy showed that 13C was incorporated to a level of about 50% and 15N to about 98%. Incorporation of 13C into the peptide provided residue-specific probes of the interactions with solvent and heat stability of this ion-channel-forming peptide. All of the carbonyl carbons and the alpha-carbons of the alpha-aminoisobutyric acid [Ala(Me)] residues of alamethicin in methanol were assigned using two-dimensional and three-dimensional heteronuclear correlation experiments. Measurements of 1JC'N revealed hydrogen bonding with solvent at residues 1 and 19 at the ends of the peptide and at Gly11 in the middle. The data also support the thesis [see Juranic, N., Ilich, P. K. & Macara, S. (1995) J. Am. Chem. Soc. 117, 405-410 that intramolecular hydrogen bonds in proteins and peptides are weaker than hydrogen bonds to solvent. The sensitivity of alamethicin carbonyl and proton chemical shifts to perturbation by dimethyl sulfoxide correlates well with the calculated solvent accessibilities of the carbonyls in the crystal structures and reveals residues in the middle of the peptide and at the C-terminus which interact with solvent. Taken together with the 1JC'N measurements, the data support a model in which hydrogen bonding to solvent at the Gly11/Leu12 amide could provide a site of hydration in the interior of the alamethicin channel structure. The temperature dependencies of the carbonyl chemical shifts support the suggestion that the peptide is flexible in the regions where solvent interacts with the backbone of the peptide. The linear temperature dependence of the carbonyl chemical shifts and molar ellipticity indicate that, due to steric constraints at the Ala(Me) residues, the peptide folding/unfolding transition is non-cooperative and that the peptide is remarkably heat stable.

Use of a non-porous polyurethane membrane as a sample support for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of proteins and peptides was performed on samples deposited onto non-porous ether-type polyurethane (PU) membranes. Spectra obtained using PU membranes showed that mass resolution and accuracy were equivalent to values observed using a metal target, and superior to those obtained using poly(vinylidene difluoride) (PVDF) membranes. A small apparent increase in the mass of proteins and also loss of resolution were observed at very high laser irradiance due to charging, but were not observed under normal conditions. Analysis of NaCl-doped standards demonstrated that PU membranes yielded better results than a metallic target for salt-containing solutions. Relatively strong hydrophobic interactions between the proteins and peptides and the PU membrane allowed the incorporation of a washing step. This step allowed for the removal of salts and buffer components and thus provided an increase in resolution and mass accuracy. Digestion of citrate synthase (a protein of molecular weight 47,886) with trypsin was performed directly on the surface of the membrane for variable periods of time, and characteristic peptide fragments were observed by MALDI-TOFMS. Delayed extraction was used to increase the resolution and to permit more accurate mass assignments for those fragments. The use of PU membranes for MALDI-TOFMS analysis of proteins with higher molecular weights is also demonstrated.