Uncoupling the Hsp90 and DnaK chaperone activities revealed the in vivo relevance of their collaboration in bacteria.

Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli. Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.

The YmgB-SpoT interaction triggers the stringent response in Escherichia coli.

Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent of its proposed role in the two-component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.

Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

Salmonella antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of EF-Tu P-loop.

Rearrangement hot spot (Rhs) proteins are members of the broad family of polymorphic toxins. Polymorphic toxins are modular proteins composed of an N-terminal region that specifies their mode of secretion into the medium or into the target cell, a central delivery module, and a C-terminal domain that has toxic activity. Here, we structurally and functionally characterize the C-terminal toxic domain of the antibacterial Rhsmain protein, TreTu, which is delivered by the type VI secretion system of Salmonella enterica Typhimurium. We show that this domain adopts an ADP-ribosyltransferase fold and inhibits protein synthesis by transferring an ADP-ribose group from NAD+ to the elongation factor Tu (EF-Tu). This modification is specifically placed on the side chain of the conserved D21 residue located on the P-loop of the EF-Tu G-domain. Finally, we demonstrate that the TriTu immunity protein neutralizes TreTu activity by acting like a lid that closes the catalytic site and traps the NAD+.

Evidence for [2Fe-2S]2+ and Linear [3Fe-4S]1+ Clusters in a Unique Family of Glycine/Cysteine-Rich Fe-S Proteins from Megavirinae Giant Viruses.

We have discovered a protein with an amino acid composition exceptionally rich in glycine and cysteine residues in the giant virus mimivirus. This small 6 kDa protein is among the most abundant proteins in the icosahedral 0.75 µm viral particles; it has no predicted function but is probably essential for infection. The aerobically purified red-brownish protein overproduced inEscherichia coli contained both iron and inorganic sulfide. UV/vis, EPR, and Mössbauer studies revealed that the viral protein, coined GciS, accommodated two distinct Fe-S clusters: a diamagnetic S = 0 [2Fe-2S]2+ cluster and a paramagnetic S = 5/2 linear [3Fe-4S]1+ cluster, a geometry rarely stabilized in native proteins. Orthologs of mimivirus GciS were identified within all clades of Megavirinae, a Mimiviridae subfamily infecting Acanthamoeba, including the distantly related tupanviruses, and displayed the same spectroscopic features. Thus, these glycine/cysteine-rich proteins form a new family of viral Fe-S proteins sharing unique Fe-S cluster binding properties.

Bacterial adaptation to cold: Conservation of a short J-domain co-chaperone and its protein partners in environmental proteobacteria.

Bacterial genomes are a huge reservoir of genes encoding J-domain protein co-chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J-domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C-terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J-domain proteins that (i) are found in several environmental, mostly aquatic, ß- or É£-proteobacteria and (ii) contain a conserved PX7 W motif in their C-terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7 W motif determines the strong AtcJ-AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low-temperature growth and, given the variation in the atc operons, possibly for other biological functions.

A divergent CheW confers plasticity to nucleoid-associated chemosensory arrays.

Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.

The nucleoid as a scaffold for the assembly of bacterial signaling complexes.

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.

An ArsR/SmtB family member is involved in the regulation by arsenic of the arsenite oxidase operon in Thiomonas arsenitoxydans.

The genetic organization of the aioBA operon, encoding the arsenite oxidase of the moderately acidophilic and facultative chemoautotrophic bacterium Thiomonas arsenitoxydans, is different from that of the aioBA operon in the other arsenite oxidizers, in that it encodes AioF, a metalloprotein belonging to the ArsR/SmtB family. AioF is stabilized by arsenite, arsenate, or antimonite but not molybdate. Arsenic is tightly attached to AioF, likely by cysteine residues. When loaded with arsenite or arsenate, AioF is able to bind specifically to the regulatory region of the aio operon at two distinct positions. In Thiomonas arsenitoxydans, the promoters of aioX and aioB are convergent, suggesting that transcriptional interference occurs. These results indicate that the regulation of the aioBA operon is more complex in Thiomonas arsenitoxydans than in the other aioBA containing arsenite oxidizers and that the arsenic binding protein AioF is involved in this regulation. On the basis of these data, a model to explain the tight control of aioBA expression by arsenic in Thiomonas arsenitoxydans is proposed.

mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.

Observation of the Fe-CN and Fe-CO vibrations in the active site of [NiFe] hydrogenase by nuclear resonance vibrational spectroscopy.

Nuclear inelastic scattering of (57)Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe-CO and Fe-CN bending and stretching vibrations of the active site outside the spectral range of the Fe-S cluster normal modes.

Using Simulation to Develop Clinical Judgment in Undergraduate Nursing Students.

The National Council of State Boards of Nursing plans to update the National Council Licensure Exam for nursing graduates to better measure clinical judgment. It is important that schools of nursing provide opportunities for nursing students to practice and develop clinical judgment skills. Simulation offers nursing students opportunities to use clinical reasoning and clinical judgment to care for patients in a safe environment The purpose of this study was to evaluate an unfolding case study using high-fidelity simulation to develop nursing students' clinical judgment skills across three practice settings: primary care office, acute care hospital, and home healthcare. This was a mixed-method, posttest study design with a convenience sample of 91 nursing students using the Lasater Clinical Judgment Rubric (LCJR) and survey questions. The posttest analysis mean of the LCJR subgroups revealed students felt accomplished following the intervention. Four themes emerged from content analysis of the qualitative data: 1.) increased knowledge of management of diabetes in various clinical settings, 2.) using clinical judgment/critical thinking in the home care setting, 3.) being able to self-reflect on actions, and 4.) a desire for more simulation experiences in the home healthcare setting. The results of the LCJR indicated that students felt accomplished after the simulation experience. This outcome was also evident in the qualitative data, indicating that students felt more confident in managing the care of a patient with a chronic illness using clinical judgment skills in various clinical settings.

A qualitative analysis of women's postnatal experiences of breastfeeding supports during the perinatal period in Ireland.

Ireland has among the lowest rates of breastfeeding worldwide. Despite policies to support breastfeeding, breastfeeding initiation and exclusivity remain low in Ireland. Greater knowledge about support received in the maternity unit may-in part-shed light on why this is so. Our aim was to analyse women's experiences of the breastfeeding supports available in the early postnatal period in Ireland. We conducted an analysis of an open-ended question on a cross-sectional survey about breastfeeding support conducted in the Republic of Ireland in 2022. Participants were asked to provide comments about the breastfeeding support they received in the maternity unit or during your home birth. Data were analysed using Braun and Clarke's six-step Thematic Analysis Framework. There were 5,412 unique responses to the survey and 2,264 responses to the question of interest. Two themes were generated from the data: (i) 'Breastfeeding support in theory but not in practice.' Although breastfeeding was promoted by healthcare professionals antenatally, breastfeeding challenges were rarely mentioned. Participants then felt unsupported in overcoming challenges postnatally. (ii) 'Support was either inaccessible due to lack of staff/time, inadequate; i.e., unhelpful or non-specific, and/or physically inappropriate.' Most participants described receiving supports that were less than optimal in aiding them to establish breastfeeding. While many described difficulties in accessing supports, others found support to be 'non-specific,' 'rushed' and sometimes 'rough.' A lack of knowledge, time and support from healthcare professionals was frequently described, which was often recognised as a failing of the healthcare system. Women require practical, informative, and specific breastfeeding support. Barriers such as lack of time and trained staff in the maternity unit need to be addressed.

Selfish uptake versus extracellular arabinoxylan degradation in the primary degrader Ruminiclostridium cellulolyticum, a new string to its bow.

BACKGROUND: Primary degraders of polysaccharides play a key role in anaerobic biotopes, where plant cell wall accumulates, providing extracellular enzymes to release fermentable carbohydrates to fuel themselves and other non-degrader species. Ruminiclostridium cellulolyticum is a model primary degrader growing amongst others on arabinoxylan. It produces large multi-enzymatic complexes called cellulosomes, which efficiently deconstruct arabinoxylan into fermentable monosaccharides. RESULTS: Complete extracellular arabinoxylan degradation was long thought to be required to fuel the bacterium during this plant cell wall deconstruction stage. We discovered and characterized a second system of "arabinoxylan" degradation in R. cellulolyticum, which challenged this paradigm. This "selfish" system is composed of an ABC transporter dedicated to the import of large and possibly acetylated arabinoxylodextrins, and a set of four glycoside hydrolases and two esterases. These enzymes show complementary action modes on arabinoxylo-dextrins. Two α-L-arabinofuranosidases target the diverse arabinosyl side chains, and two exo-xylanases target the xylo-oligosaccharides backbone either at the reducing or the non-reducing end. Together, with the help of two different esterases removing acetyl decorations, they achieve the depolymerization of arabinoxylo-dextrins in arabinose, xylose and xylobiose. The in vivo study showed that this new system is strongly beneficial for the fitness of the bacterium when grown on arabinoxylan, leading to the conclusion that a part of arabinoxylan degradation is achieved in the cytosol, even if monosaccharides are efficiently provided by the cellulosomes in the extracellular space. These results shed new light on the strategies used by anaerobic primary degrader bacteria to metabolize highly decorated arabinoxylan in competitive environments. CONCLUSION: The primary degrader model Ruminiclostridium cellulolyticum has developed a "selfish" strategy consisting of importing into the bacterium, large arabinoxylan-dextrin fractions released from a partial extracellular deconstruction of arabinoxylan, thus complementing its efficient extracellular arabinoxylan degradation system. Genetic studies suggest that this system is important to support fitness and survival in a competitive biotope. These results provide a better understanding of arabinoxylan catabolism in the primary degrader, with biotechnological application for synthetic microbial community engineering for the production of commodity chemicals from lignocellulosic biomass.

Binding of two zinc ions promotes liquid-liquid phase separation of Tau.

Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc.

NirD curtails the stringent response by inhibiting RelA activity in Escherichia coli.

Bacteria regulate their metabolism to adapt and survive adverse conditions, in particular to stressful downshifts in nutrient availability. These shifts trigger the so-called stringent response, coordinated by the signaling molecules guanosine tetra and pentaphosphate collectively referred to as (p)ppGpp. In Escherichia coli, accumulation of theses alarmones depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. A tight regulation of these intracellular activities is therefore crucial to rapidly adjust the (p)ppGpp levels in response to environmental stresses but also to avoid toxic consequences of (p)ppGpp over-accumulation. In this study, we show that the small protein NirD restrains RelA-dependent accumulation of (p)ppGpp and can inhibit the stringent response in E. coli. Mechanistically, our in vivo and in vitro studies reveal that NirD directly binds the catalytic domains of RelA to balance (p)ppGpp accumulation. Finally, we show that NirD can control RelA activity by directly inhibiting the rate of (p)ppGpp synthesis.

Mechanism of Zn2+ and Ca2+ Binding to Human S100A1.

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 µm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.

Uridine diphosphate N-acetylglucosamine orchestrates the interaction of GlmR with either YvcJ or GlmS in Bacillus subtilis.

In bacteria, glucosamine-6-phosphate (GlcN6P) synthase, GlmS, is an enzyme required for the synthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a precursor of peptidoglycan. In Bacillus subtilis, an UDP-GlcNAc binding protein, GlmR (formerly YvcK), essential for growth on non-glycolytic carbon sources, has been proposed to stimulate GlmS activity; this activation could be antagonized by UDP-GlcNAc. Using purified proteins, we demonstrate that GlmR directly stimulates GlmS activity and the presence of UDP-GlcNAc (at concentrations above 0.1 mM) prevents this regulation. We also showed that YvcJ, whose gene is associated with yvcK (glmR), interacts with GlmR in an UDP-GlcNAc dependent manner. Strains producing GlmR variants unable to interact with YvcJ show decreased transformation efficiency similar to that of a yvcJ null mutant. We therefore propose that, depending on the intracellular concentration of UDP-GlcNAc, GlmR interacts with either YvcJ or GlmS. When UDP-GlcNAc concentration is high, this UDP-sugar binds to YvcJ and to GlmR, blocking the stimulation of GlmS activity and driving the interaction between GlmR and YvcJ to probably regulate the cellular role of the latter. When the UDP-GlcNAc level is low, GlmR does not interact with YvcJ and thus does not regulate its cellular role but interacts with GlmS to stimulate its activity.

HetL, HetR and PatS form a reaction-diffusion system to control pattern formation in the cyanobacterium nostoc PCC 7120.

Local activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns. A number of them involve the production by the developing cell of an inhibitory morphogen, but how this cell becomes immune to self-inhibition is rather unknown. Under combined nitrogen starvation, the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of one heterocyst every 10-12 vegetative cells. Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogens, diffusion of which establishes the differentiation pattern. Here, we show that HetR interacts with HetL at the same interface as PatS, and that this interaction is necessary to suppress inhibition and to differentiate heterocysts. hetL expression is induced under nitrogen-starvation and is activated by HetR, suggesting that HetL provides immunity to the heterocyst. This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum.

Cyanobacteria are the only bacteria on Earth able to draw their energy directly from the sun in the same way that plants do. In addition, some strains are able to 'fix' the nitrogen present in the atmosphere: they can extract this gas and transform it into nitrogen-based compounds necessary for life. However, both processes cannot happen in a given cell at the same time. A strain of cyanobacteria called Nostoc PCC 7120 can organise itself into long filaments of interconnected cells. Under certain conditions, one in every ten cells stops drawing its energy from the sun, and starts fixing atmospheric nitrogen instead. Exactly how the bacteria are able to 'count to ten' and organize themselves in such a precise pattern is still unclear. Cells can communicate and establish patterns by exchanging molecular signals that switch on and off certain cell programs. For instance, a protein called HetR turns on the genetic program that allows cyanobacteria to fix nitrogen; on the other hand, a signal known as PatS binds to HetR and turns it off. Cells starting to specialise in fixing nitrogen produce both HetR and PatS, with the latter diffusing in surrounding cells and preventing them from extracting nitrogen. However, it remained unclear how the nitrogen-fixing cell could ignore its own PatS signal and keep its HetR signal active. HetL ­ another protein produced by the future nitrogen-fixing cell ­ could potentially play this role, but how it acts was unknown. Here, Xu et al. show that HetL cannot diffuse from one cell to the other, and that it binds to HetR at the same place than PatS does. When both PatS and HetL are present, they compete to attach to HetR, which stops PatS from turning off HetR and deactivating the nitrogen-fixing program. Understanding how cyanobacteria fix nitrogen could help to develop new types of natural fertiliser. More generally, dissecting how these simple organisms can create patterns could help to grasp how patterning emerges in more complex creatures.

Assessing Undergraduate Nursing Students' Attitudes Toward the Dying in an End-of-Life Simulation Using an ACE.S Unfolding Case Study.

Caring for the dying patient can be stressful for nursing students. The purpose of this study was to describe a multimodal educational intervention designed to improve nursing students' attitude toward care of the dying patient and the family. Sophomore nursing students participated in an interactive end-of-life (EOL) lecture and simulation. A quasi-experimental, pretest/posttest design with a convenience sample was used for this study. Frommelt Attitudes Toward Care of the Dying version A was used to measure attitudes toward care of the dying patient before and after educational intervention. In addition, students were given an open-ended questionnaire to reflect on their perceptions of the EOL experience and a demographic questionnaire. A paired t test revealed a statistically significant difference between the pretest and posttest (t50 = 3.1, P = .003) on the Frommelt Attitudes Toward Care of the Dying, suggesting that students gained a more positive attitude toward caring for the dying patient. Three themes emerged from the content analysis and included knowing what to say and how to offer presence, becoming emotionally prepared, and learning skills to comfort. The use of lecture and simulation allowed students to assimilate the knowledge and affective skills needed to provide quality EOL care.