Polycomb safeguards imaginal disc specification through control of the Vestigial-Scalloped complex.

A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are misregulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex, which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high-throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate.

BoGH13ASus from Bacteroides ovatus represents a novel α-amylase used for Bacteroides starch breakdown in the human gut.

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98-CBM48-GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut.

Single-molecule dynamics of surface lipoproteins in bacteroides indicate similarities and cooperativity.

The gut microbiota comprises hundreds of species with a composition shaped by the available glycans. The well-studied starch utilization system (Sus) is a prototype for glycan uptake in the human gut bacterium Bacteroides thetaiotaomicron (Bt). Each Sus-like system includes outer-membrane proteins, which translocate glycan into the periplasm, and one or more cell-surface glycoside hydrolases, which break down a specific (cognate) polymer substrate. Although the molecular mechanisms of the Sus system are known, how the Sus and Sus-like proteins cooperate remains elusive. Previously, we used single-molecule and super-resolution fluorescence microscopy to show that SusG is mobile on the outer membrane and slows down in the presence of starch. Here, we compare the dynamics of three glycoside hydrolases: SusG, Bt4668, and Bt1760, which target starch, galactan, and levan, respectively. We characterized the diffusion of each surface hydrolase in the presence of its cognate glycan and found that all three enzymes are mostly immobile in the presence of the polysaccharide, consistent with carbohydrate binding. Moreover, experiments in glucose versus oligosaccharides suggest that the enzyme dynamics depend on their expression level. Furthermore, we characterized enzyme diffusion in a mixture of glycans and found that noncognate polysaccharides modify the dynamics of SusG and Bt1760 but not Bt4668. We investigated these systems with polysaccharide mixtures and genetic knockouts and found that noncognate polysaccharides modify hydrolase dynamics through some combination of nonspecific protein interactions and downregulation of the hydrolase. Overall, these experiments extend our understanding of how Sus-like lipoprotein dynamics can be modified by changing carbohydrate conditions and the expression level of the enzyme.

Midline axon guidance in the Drosophila embryonic central nervous system.

Studies in the fruit fly Drosophila melanogaster have provided many fundamental insights into the genetic regulation of neural development, including the identification and characterization of evolutionarily conserved axon guidance pathways and their roles in important guidance decisions. Due to its highly organized and fast-developing embryonic nervous system, relatively small number of neurons, and molecular and genetic tools for identifying, labeling, and manipulating individual neurons or small neuronal subsets, studies of axon guidance in the Drosophila embryonic CNS have allowed researchers to dissect these genetic mechanisms with a high degree of precision. In this review, we discuss the major axon guidance pathways that regulate midline crossing of axons and the formation and guidance of longitudinal axon tracts, two processes that contribute to the development of the precise three-dimensional structure of the insect nerve cord. We focus particularly on recent insights into the roles and regulation of canonical midline axon guidance pathways, and on additional factors and pathways that have recently been shown to contribute to axon guidance decisions at and near the midline.

Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii.

The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1-CBM22.2-Xyn10C construct, indicating a compact arrangement at room temperature.

Structure and substrate recognition by the Ruminococcus bromii amylosome pullulanases.

Pullulanases are glycoside hydrolase family 13 (GH13) enzymes that target α1,6 glucosidic linkages within starch and aid in the degradation of the α1,4- and α1,6- linked glucans pullulan, glycogen and amylopectin. The human gut bacterium Ruminococcus bromii synthesizes two extracellular pullulanases, Amy10 and Amy12, that are incorporated into the multiprotein amylosome complex that enables the digestion of granular resistant starch from the diet. Here we provide a comparative biochemical analysis of these pullulanases and the x-ray crystal structures of the wild type and the nucleophile mutant D392A of Amy12 complexed with maltoheptaose and 63-α-D glucosyl-maltotriose. While Amy10 displays higher catalytic efficiency on pullulan and cleaves only α1,6 linkages, Amy12 has some activity on α1,4 linkages suggesting that these enzymes are not redundant within the amylosome. Our structures of Amy12 include a mucin-binding protein (MucBP) domain that follows the C-domain of the GH13 fold, an atypical feature of these enzymes. The wild type Amy12 structure with maltoheptaose captured two oligosaccharides in the active site arranged as expected following catalysis of an α1,6 branch point in amylopectin. The nucleophile mutant D392A complexed with maltoheptaose or 63-α-D glucosyl-maltotriose captured ß-glucose at the reducing end in the -1 subsite, facilitated by the truncation of the active site aspartate and stabilized by stacking with Y279. The core interface between the co-crystallized ligands and Amy12 occurs within the -2 through + 1 subsites, which may allow for flexible recognition of α1,6 linkages within a variety of starch structures.

Host glycan utilization within the Bacteroidetes Sus-like paradigm.

The Bacteroidetes are numerically abundant Gram-negative organisms of the distal human gut with a greatly expanded capacity to degrade complex glycans. A subset of these are adept at scavenging host glycans within this environment, including mucin O-linked glycans, N-linked glycoproteins and highly sulfated glycosaminoglycans (GAGs) such as heparin (Hep) and chondroitin sulfate (CS). Several recent biochemical studies have revealed the specific polysaccharide utilization loci (PULs) within the model symbiont Bacteroides thetaiotaomicron for the deconstruction of these host glycans. Here we discuss the Sus-like paradigm that defines glycan uptake by the Bacteroidetes and the salient details of the PULs that target heparin/heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (DS)/hyaluronic acid (HA), respectively, in B. thetaiotaomicron. The ability of the Bacteroidetes to target highly sulfated host glycans is key to their success in the gut environment but can lead to inflammation in susceptible hosts. Therefore, our continued understanding of the molecular strategies employed by these bacteria to scavenge carbohydrate nutrition is likely to lead to novel ways to alter their metabolism to promote host health.

Biosynthesis of Nucleoside Diphosphoramidates in Campylobacter jejuni.

Campylobacter jejuni is a pathogenic Gram-negative bacterium and a leading cause of food-borne gastroenteritis. C. jejuni produces a capsular polysaccharide (CPS) that contains a unique O-methyl phosphoramidate modification (MeOPN). Recently, the first step in the biosynthetic pathway for the assembly of the MeOPN modification to the CPS was elucidated. It was shown that the enzyme Cj1418 catalyzes the phosphorylation of the amide nitrogen of l-glutamine to form l-glutamine phosphate. In this investigation, the metabolic fate of l-glutamine phosphate was determined. The enzyme Cj1416 catalyzes the displacement of pyrophosphate from MgCTP by l-glutamine phosphate to form CDP-l-glutamine. The enzyme Cj1417 subsequently catalyzes the hydrolysis of CDP-l-glutamine to generate cytidine diphosphoramidate and l-glutamate. The structures of the two novel intermediates, CDP-l-glutamine and cytidine diphosphoramidate, were confirmed by 31P nuclear magnetic resonance spectroscopy and mass spectrometry. It is proposed that the enzyme Cj1416 be named CTP:phosphoglutamine cytidylyltransferase and that the enzyme Cj1417 be named γ-glutamyl-CDP-amidate hydrolase.

Discovery of a Glutamine Kinase Required for the Biosynthesis of the O-Methyl Phosphoramidate Modifications Found in the Capsular Polysaccharides of Campylobacter jejuni.

Bacterial capsular polysaccharides (CPS) are complex carbohydrate structures that play a role in the overall fitness of the organism. Campylobacter jejuni, known for being a major cause of bacterial gastroenteritis worldwide, produces a CPS with a unique O-methyl phosphoramidate (MeOPN) modification on specific sugar residues. The formation of P-N bonds in nature is relatively rare, and the pathway for the assembly of the phosphoramidate moiety in the CPS of C. jejuni is unknown. In this investigation we discovered that the initial transformation in the biosynthetic pathway for the MeOPN modification of the CPS involves the direct phosphorylation of the amide nitrogen of l-glutamine with ATP by the catalytic activity of Cj1418. The other two products are AMP and inorganic phosphate. The l-glutamine-phosphate product was characterized using 31P NMR spectroscopy and mass spectrometry. We suggest that this newly discovered enzyme be named l-glutamine kinase.

Examination of Lifestyle Behaviors and Cardiometabolic Risk Factors in University Students Enrolled in Kinesiology Degree Programs.

Preventing physical inactivity and weight gain during college is critical in decreasing lifelong obesity and associated disease risk. As such, we sought to compare cardiometabolic risk factors and lifestyle behaviors between college students enrolled in kinesiology and non-kinesiology degree programs to assess whether health and exercise degree programs may influence health behaviors and associated disease risk outcomes. Anthropometrics, fasting blood glucose, insulin, lipid profiles and HbA1c%, blood pressure, and peak oxygen consumption (V[Combining Dot Above]O2peak) were assessed in 247 healthy college students. The homeostasis model assessment of insulin sensitivity (HOMA) was calculated using glucose and insulin levels. Self-reported physical activity from the Paffenbarger questionnaire was collected to estimate the average caloric expenditure due to different types of physical activities. Despite no significant differences in body mass index or waist circumference between groups, kinesiology majors presented with ∼20% lower fasting insulin levels and HOMA (p = 0.01; p < 0.01, respectively) relative to nonmajors. Kinesiology majors reported increased weekly participation in vigorous-intensity sport and leisure activities and, on average, engaged in >300 metabolic equivalent-h·wk, whereas non-kinesiology majors engaged in <300 MET-h wk (p = 0.01). Our data suggest that students enrolled in kinesiology degree programs display improved healthy behaviors and associated outcomes (parameters of glucose homeostasis). Practical outcomes of this research indicate that implementing components of a comprehensive kinesiology curriculum encourages improved health behaviors and associated cardiometabolic risk factors.

Development of an improved mammalian overexpression method for human CD62L.

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5-30mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4-6months to produce. To shorten the construction time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ∼5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.

Acarbose Impairs Gut Bacteroides Growth by Targeting Intracellular GH97 Enzymes.

Acarbose is a type-2 diabetes medicine that inhibits dietary starch breakdown into glucose by inhibiting host amylase and glucosidase enzymes. Numerous gut species in the Bacteroides genus enzymatically break down starch and change in relative abundance within the gut microbiome in acarbose-treated individuals. To mechanistically explain this observation, we used two model starch-degrading Bacteroides, Bacteroides ovatus (Bo) and Bacteroides thetaiotaomicron (Bt). Bt growth is severely impaired by acarbose whereas Bo growth is not. The Bacteroides use a starch utilization system (Sus) to grow on starch. We hypothesized that Bo and Bt Sus enzymes are differentially inhibited by acarbose. Instead, we discovered that although acarbose primarily targets the Sus periplasmic GH97 enzymes in both organisms, the drug affects starch processing at multiple other points. Acarbose competes for transport through the Sus beta-barrel proteins and binds to the Sus transcriptional regulators. Further, Bo expresses a non-Sus GH97 (BoGH97D) when grown in starch with acarbose. The Bt homolog, BtGH97H, is not expressed in the same conditions, nor can overexpression of BoGH97D complement the Bt growth inhibition in the presence of acarbose. This work informs us about unexpected complexities of Sus function and regulation in Bacteroides, including variation between related species. Further, this indicates that the gut microbiome may be a source of variable response to acarbose treatment for diabetes.

Socio-ecological impacts of extreme weather events in two informal settlements in Nairobi, Kenya.

Climate change is expected to profoundly impact health and coping and widen social and environmental inequalities. People living in informal settlements are especially vulnerable to climate change as they are often located in ecologically sensitive areas more susceptible to extreme weather events (EWEs), such as floods, droughts, and heat waves. Women residing in informal settlements are especially vulnerable to climate change and related EWEs because they are more likely to experience worse health-related impacts than men but are less likely to have access to health-related services. Despite this inequality, there is a dearth of research that focuses on the impacts of EWEs on women in informal settlements. This study aims to explore the multidimensional impacts of EWEs on the daily lives of women in informal settlements through the lens of socio-ecological theory. Study data is from six monthly surveys (1 September 2022-28 February 2023) collected from a probability sample of 800 women living in two of the largest informal settlements in Nairobi, Kenya. This data is part of an ongoing longitudinal study that uses community participatory methods to investigate the effects of climate change on health and wellbeing in informal settlements by a team of 16 community health volunteers who lead data collection and provide expertise in ongoing analysis. Findings show profound impacts on women's health and wellbeing across individual, micro-, meso-, exo-, and macrosystems. These include physical and mental health, financial disruptions, property issues, social impacts, and impacts on their surrounding physical environment, such as disrupted food or water access, poor air quality, drainage issues, and safety concerns. In addition, findings highlight the critical importance of the chrono- and biosphere systems in research focused on the impacts of climate change and related EWEs among climate-vulnerable communities and marginalized populations within them.

Bifunctional electrophiles cross-link thioredoxins with redox relay partners in cells.

Thioredoxin protects cells against oxidative damage by reducing disulfide bonds in improperly oxidized proteins. Previously, we found that the baker's yeast cytosolic thioredoxin Trx2 undergoes cross-linking to form several protein-protein complexes in cells treated with the bifunctional electrophile divinyl sulfone (DVSF). Here, we report that the peroxiredoxin Tsa1 and the thioredoxin reductase Trr1, both of which function in a redox relay network with thioredoxin, become cross-linked in complexes with Trx2 upon DVSF treatment. Treatment of yeast with other bifunctional electrophiles, including diethyl acetylenedicarboxylate (DAD), mechlorethamine (HN2), and 1,2,3,4-diepoxybutane (DEB), resulted in the formation of similar cross-linked complexes. Cross-linking of Trx2 and Tsa1 to other proteins by DVSF and DAD is dependent on modification of the active site Cys residues within these proteins. In addition, the human cytosolic thioredoxin, cytosolic thioredoxin reductase, and peroxiredoxin 2 form cross-linked complexes to other proteins in the presence of DVSF, although each protein shows different susceptibilities to modification by DAD, HN2, and DEB. Taken together, our results indicate that bifunctional electrophiles potentially disrupt redox homeostasis in yeast and human cells by forming cross-linked complexes between thioredoxins and their redox partners.

Isothermal Titration Calorimetry for Quantification of Protein-Carbohydrate Interactions.

Isothermal titration calorimetry allows the determination of thermodynamic parameters for the interaction between a protein and mono- or oligosaccharides in solution. For the study of protein-carbohydrate interactions, it is a robust way to determine the stoichiometry and affinity, as well as the enthalpic and entropic contributions to this interaction, without the use of labeled proteins or substrates. Here we describe a standard multiple-injection titration experiment for measuring the binding energetics between a carbohydrate-binding protein and an oligosaccharide.

Polycomb safeguards imaginal disc specification through control of the Vestigial-Scalloped complex.

A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are mis-regulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate. Summary Statement: Here we describe a novel mechanism by which Pc promotes an eye fate during normal development and how the eye is reprogrammed into a wing in its absence.

A CUT&RUN protocol to determine patterns of epigenetic marks in imaginal discs of Drosophila.

Cleavage Under Targets & Release Using Nucleases (CUT&RUN) sequencing is a technique used to study gene regulation. The protocol presented here has been used successfully to identify the pattern of histone modifications within the genome of the eye-antennal disc of the fruit fly, Drosophila melanogaster. In its present form, it can be used to analyze genomic features of other imaginal discs. It can be modified for use with other tissues and applications including identifying the pattern of transcription factor occupancy.

Slit-independent guidance of longitudinal axons by Drosophila Robo3.

Drosophila Robo3 is a member of the evolutionarily conserved Roundabout (Robo) receptor family and one of three Drosophila Robo paralogs. During embryonic ventral nerve cord development, Robo3 does not participate in canonical Slit-dependent midline repulsion, but instead regulates the formation of longitudinal axon pathways at specific positions along the medial-lateral axis. Longitudinal axon guidance by Robo3 is hypothesized to be Slit dependent, but this has not been directly tested. Here we create a series of Robo3 variants in which the N-terminal Ig1 domain is deleted or modified, in order to characterize the functional importance of Ig1 and Slit binding for Robo3's axon guidance activity. We show that Robo3 requires its Ig1 domain for interaction with Slit and for proper axonal localization in embryonic neurons, but deleting Ig1 from Robo3 only partially disrupts longitudinal pathway formation. Robo3 variants with modified Ig1 domains that cannot bind Slit retain proper localization and fully rescue longitudinal axon guidance. Our results indicate that Robo3 guides longitudinal axons independently of Slit, and that sequences both within and outside of Ig1 contribute to this Slit-independent activity.

Neoadjuvant cemiplimab for resectable hepatocellular carcinoma: a single-arm, open-label, phase 2 trial.

BACKGROUND: Surgical resection of early stage hepatocellular carcinoma is standard clinical practice; however, most tumours recur despite surgery, and no perioperative intervention has shown a survival benefit. Neoadjuvant immunotherapy has induced pathological responses in multiple tumour types and might decrease the risk of postoperative recurrence in hepatocellular carcinoma. We aimed to evaluate the clinical activity of neoadjuvant cemiplimab (an anti-PD-1) in patients with resectable hepatocellular carcinoma. METHODS: For this single-arm, open-label, phase 2 trial, patients with resectable hepatocellular carcinoma (stage Ib, II, and IIIb) were enrolled and received two cycles of neoadjuvant cemiplimab 350 mg intravenously every 3 weeks followed by surgical resection. Eligible patients were aged 18 years or older, had confirmed resectable hepatocellular carcinoma, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate liver function. Patients were excluded if they had metastatic disease, if the surgery was not expected to be curative, if they had a known additional malignancy requiring active treatment, or if they required systemic steroid treatment or any other immunosuppressive therapy. After resection, patients received an additional eight cycles of cemiplimab 350 mg intravenously every 3 weeks in the adjuvant setting. The primary endpoint was significant tumour necrosis on pathological examination (defined as >70% necrosis of the resected tumour). Secondary endpoints included delay of surgery, the proportion of patients with an overall response, change in CD8+ T-cell density, and adverse events. Tumour necrosis and response were analysed in all patients who received at least one dose of cemiplimab and completed surgical resection; safety and other endpoints were analysed in the intention-to-treat population. Patients underwent pre-treatment biopsies and blood collection throughout treatment. This trial is registered with ClinicalTrials.gov (NCT03916627, Cohort B) and is ongoing. FINDINGS: Between Aug 5, 2019, and Nov 25, 2020, 21 patients were enrolled. All patients received neoadjuvant cemiplimab, and 20 patients underwent successful resection. Of the 20 patients with resected tumours, four (20%) had significant tumour necrosis. Three (15%) of 20 patients had a partial response, and all other patients maintained stable disease. 20 (95%) patients had a treatment-emergent adverse event of any grade during the neoadjuvant treatment period. The most common adverse events of any grade were increased aspartate aminotransferase (in four patients), increased blood creatine phosphokinase (in three), constipation (in three), and fatigue (in three). Seven patients had grade 3 adverse events, including increased blood creatine phosphokinase (in two patients) and hypoalbuminaemia (in one). No grade 4 or 5 events were observed. One patient developed pneumonitis, which led to a delay in surgery by 2 weeks. INTERPRETATION: This report is, to our knowledge, the largest clinical trial of a neoadjuvant anti-PD-1 monotherapy reported to date in hepatocellular carcinoma. The observed pathological responses to cemiplimab in this cohort support the design of larger trials to identify the optimal treatment duration and definitively establish the clinical benefit of preoperative PD-1 blockade in patients with hepatocellular carcinoma. FUNDING: Regeneron Pharmaceuticals.

Enhanced toxicity of the protein cross-linkers divinyl sulfone and diethyl acetylenedicarboxylate in comparison to related monofunctional electrophiles.

Previously, we determined that diethyl acetylenedicarboxylate (DAD), a protein cross-linker, was significantly more toxic than analogous monofunctional electrophiles. We hypothesized that other protein cross-linkers enhance toxicity similarly. In agreement with this hypothesis, the bifunctional electrophile divinyl sulfone (DVSF) was 6-fold more toxic than ethyl vinyl sulfone (EVSF) in colorectal carcinoma cells and greater than 10-fold more toxic in Saccharomyces cerevisiae. DVSF and DAD caused oligomerization of yeast thioredoxin 2 (Trx2p) in vitro and promoted Trx2p cross-linking to other proteins in yeast at cytotoxic doses. Our results suggest that protein cross-linking is considerably more detrimental to cellular homeostasis than simple alkylation.