Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing Abs target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with a human adenovirus type 5 vector expressing the SARS-CoV-2 nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and K18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral Ags in SARS-CoV-2 vaccines, even if they are not a target of neutralizing Abs, to broaden epitope coverage and immune effector mechanisms.

Stability of Smyd1 in endothelial cells is controlled by PML-dependent SUMOylation upon cytokine stimulation.

Smyd1 is an epigenetic modulator of gene expression that has been well-characterized in muscle cells. It was recently reported that Smyd1 levels are modulated by inflammatory processes. Since inflammation affects the vascular endothelium, this study aimed to characterize Smyd1 expression in endothelial cells. We detected Smyd1 in human endothelial cells (HUVEC and EA.hy926 cells), where the protein was largely localized in PML nuclear bodies (PML-NBs). By transfection of EA.hy926 cells with expression vectors encoding Smyd1, PML, SUMO1, active or mutant forms of the SUMO protease SuPr1 and/or the SUMO-conjugation enzyme UBC9, as well as Smyd1- or PML-specific siRNAs, in the presence or absence of the translation blocker cycloheximide or the proteasome-inhibitor MG132, and supported by computational modeling, we show that Smyd1 is SUMOylated in a PML-dependent manner and thereby addressed for degradation in proteasomes. Furthermore, transfection with Smyd1-encoding vectors led to PML up-regulation at the mRNA level, while PML transfection lowered Smyd1 protein stability. Incubation of EA.hy926 cells with the pro-inflammatory cytokine TNF-α resulted in a constant increase in Smyd1 mRNA and protein over 24 h, while incubation with IFN-γ induced a transient increase in Smyd1 expression, which peaked at 6 h and decreased to control values within 24 h. The IFN-γ-induced increase in Smyd1 was accompanied by more Smyd1 SUMOylation and more/larger PML-NBs. In conclusion, our data indicate that in endothelial cells, Smyd1 levels are regulated through a negative feedback mechanism based on SUMOylation and PML availability. This molecular control loop is stimulated by various cytokines.

The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway.

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.

Promyelocytic leukemia protein promotes the phenotypic switch of smooth muscle cells in atherosclerotic plaques of human coronary arteries.

Promyelocytic leukemia protein (PML) is a constitutive component of PML nuclear bodies (PML-NBs), which function as stress-regulated SUMOylation factories. Since PML can also act as a regulator of the inflammatory and fibroproliferative responses characteristic of atherosclerosis, we investigated whether PML is implicated in this disease. Immunoblotting, ELISA and immunohistochemistry showed a stronger expression of PML in segments of human atherosclerotic coronary arteries and sections compared with non-atherosclerotic ones. In particular, PML was concentrated in PML-NBs from α-smooth muscle actin (α-SMA)-immunoreactive cells in plaque areas. To identify possible functional consequences of PML-accumulation in this cell type, differentiated human coronary artery smooth muscle cells (dHCASMCs) were transfected with a vector containing the intact PML-gene. These PML-transfected dHCASMCs showed higher levels of small ubiquitin-like modifier (SUMO)-1-dependent SUMOylated proteins, but lower levels of markers for smooth muscle cell (SMC) differentiation and revealed more proliferation and migration activities than dHCASMCs transfected with the vector lacking a specific gene insert or with the vector containing a mutated PML-gene coding for a PML-form without SUMOylation activity. When dHCASMCs were incubated with different cytokines, higher PML-levels were observed only after interferon γ (IFN-γ) stimulation, while the expression of differentiation markers was lower. However, these phenotypic changes were not observed in dHCASMCs treated with small interfering RNA (siRNA) suppressing PML-expression prior to IFN-γ stimulation. Taken together, our results imply that PML is a previously unknown functional factor in the molecular cascades associated with the pathogenesis of atherosclerosis and is positioned in vascular SMCs (VSMCs) between upstream IFN-γ activation and downstream SUMOylation.

Biology and Biochemistry of Bacterial Proteasomes.

Proteasomes are a class of protease that carry out the degradation of a specific set of cellular proteins. While essential for eukaryotic life, proteasomes are found only in a small subset of bacterial species. In this chapter, we present the current knowledge of bacterial proteasomes, detailing the structural features and catalytic activities required to achieve proteasomal proteolysis. We describe the known mechanisms by which substrates are doomed for degradation, and highlight potential non-degradative roles for components of bacterial proteasome systems. Additionally, we highlight several pathways of microbial physiology that rely on proteasome activity. Lastly, we explain the various gaps in our understanding of bacterial proteasome function and emphasize several opportunities for further study.

Release of protein A from the cell wall of Staphylococcus aureus.

Staphylococcal protein A (SpA) is anchored to the cell wall envelope of Staphylococcus aureus by sortase A, which links the threonyl (T) of its C-terminal LPXTG motif to peptidoglycan cross-bridges (i.e., Gly5). SpA binds the Fcγ domains of IgG and protects staphylococci from opsonophagocytic clearance. Moreover, SpA cross-links B-cell receptors to modify host adaptive immune responses. The mechanisms whereby SpA is released from the bacterial surface to access the host's immune system are not known. Here we demonstrate that SpA is released with murein tetrapeptide-tetraglycyl [L-Ala-D-iGln-(SpA-Gly5)L-Lys-D-Ala-Gly4] linked to its C-terminal threonyl. LytN, a cross-wall murein hydrolase, contributes to the release of SpA by removing amino sugars [i.e., N-acetylmuramic acid-N-acetylglucosamine (MurNAc-GlcNAc)] from attached peptidoglycan, whereas LytM, a pentaglycyl-endopeptidase, triggers polypeptide release from the bacterial envelope. A model is proposed whereby murein hydrolases cleave the anchor structure of released SpA to modify host immune responses.

N-acetylglucosaminylation of serine-aspartate repeat proteins promotes Staphylococcus aureus bloodstream infection.

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.

Liquid-core polymer nanocapsules prepared using flash nanoprecipitation.

Hypothesis: Nanocapsules, consisting of a solid shell and a liquid core, are an interesting class of materials with numerous applications and various methods of synthesis. One common method for synthesis of nanoparticles is flash nanoprecipitation. For a multicomponent system consisting of a liquid (n-hexadecane) and solid (polystyrene), we hypothesize that nanocapsules will form from droplets created by the turbulent mixing in the nanoprecipitation process. We anticipate n-hexadecane molecules should phase-separate more rapidly from the non-solvent, thus becoming the core, while the more slowly diffusing polystyrene forms the shell. Additionally, we predict that the amount of both n-hexadecane and polystyrene used in creating these nanocapsules will influence capsule size. Experiments: Using flash nanoprecipitation, we synthesized nanocapsules of a polystyrene shell and liquid core of n-hexadecane. We varied the concentrations of both polystyrene and n-hexadecane and characterized the resulting dispersions using dynamic light scattering and scanning electron microscopy. Findings: Our experiments demonstrate that flash nanoprecipitation can be employed to create nanocapsules with radii ranging from 50 to 200 nm, with radii of the n-hexadecane cores between 35 and 175 nm and polystyrene shells with thickness ranging from 7 to 62 nm. We used various methods of analysis to confirm this core/shell morphology and applied a droplet model to explain the dependence of particle size on initial concentrations of n-hexadecane and polystyrene.

Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression.

Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the functions of these TF condensates in 3D genome organization and gene regulation remain elusive. In response to methionine (met) starvation, budding yeast TF Met4 and a few co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form co-localized puncta-like structures in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes are clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4 binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4 binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to methionine depletion.

Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) orchestrates a suppressive tumor microenvironment that fosters immunotherapy resistance. Tumor-associated macrophages (TAMs) are the principal immune cell infiltrating PDA and are heterogeneous. Here, by employing macrophage fate-mapping approaches and single-cell RNA sequencing, we show that monocytes give rise to most macrophage subsets in PDA. Tumor-specific CD4, but not CD8, T cells promote monocyte differentiation into MHCIIhi anti-tumor macrophages. By conditional major histocompatibility complex (MHC) class II deletion on monocyte-derived macrophages, we show that tumor antigen presentation is required for instructing monocyte differentiation into anti-tumor macrophages, promoting Th1 cells, abrogating Treg cells, and mitigating CD8 T cell exhaustion. Non-redundant IFNγ and CD40 promote MHCIIhi anti-tumor macrophages. Intratumoral monocytes adopt a pro-tumor fate indistinguishable from that of tissue-resident macrophages following loss of macrophage MHC class II or tumor-specific CD4 T cells. Thus, tumor antigen presentation by macrophages to CD4 T cells dictates TAM fate and is a major determinant of macrophage heterogeneity in cancer.

Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.

The role of local steroid injection for nasal polyposis.

Sinonasal polyps affect a small but significant percentage of patients with chronic sinusitis. Treatments vary and range from oral and topical medical treatments to surgical removal. Corticosteroids typically have been regarded as the gold standard medical treatment for sinonasal polyps. Delivery of steroids is traditionally via oral or topical means. Over the years, otolaryngologists have also found that intrapolyp injection of corticosteroids is an effective means to treat some patients with sinonasal polyps. This article reviews the prevalence, pathophysiology, and medical treatment options for sinonasal polyps. Focused attention is paid to treatment with steroid injections, including a review of its associated risks and benefits.

Structural Microangiopathies in Skeletal Muscle Related to Systemic Vascular Pathologies in Humans.

It is unclear how microangiopathic changes in skeletal muscle vary among systemic vascular pathologies. We therefore analyzed the capillary fine structure in skeletal muscle from patients with arterial hypertension (HYPT), diabetes mellitus type 2 (T2DM) or intermittent claudication - peripheral arterial disease (IC/PAD). Tablet-based image analysis (TBIA) was carried out to largely re-evaluate 5,000 transmission electron micrographs of capillaries from 126 vastus lateralis biopsies of 75 individuals (HYPT, T2DM or IC/PAD patients as well as healthy individuals before and after endurance exercise training) used in previous morphometric studies, but assessed using stereological counting grids of different sizes. Serial block-face scanning electron microscopy (SBFSEM) of mouse skeletal muscle was used for validation of the particular fine structural events observed in human biopsies. The peri-capillary basement membrane (BM) was 38.5 and 45.5% thicker (P < 0.05) in T2DM and IC/PAD patients than in the other groups. A 17.7-39.6% lower (P < 0.05) index for intraluminal endothelial cell (EC) surface enlargement by projections was exclusively found in T2DM patients by TBIA morphometry. The proportion of capillaries with disrupted BM between pericytes (PC) and EC was higher (P < 0.05) in HYPT (33.2%) and T2DM (38.7%) patients than in the control group. Empty EC-sockets were more frequent (P < 0.05) in the three patient groups (20.6% in HYPT, 27.1% in T2DM, 30.0% in IC/PAD) than in the healthy individuals. SBFSEM confirmed that EC-sockets may exhibit close proximity to the capillary lumen. Our comparative morphometric analysis demonstrated that structural arrangement of skeletal muscle capillaries is more affected in T2DM than in HYPT or IC/PAD, although some similar elements of remodeling were found. The increased frequency of empty EC-sockets in the three patient groups indicates that the PC-EC interaction is commonly disturbed in these systemic vascular pathologies.

Mycobacterium tuberculosis Rv0991c Is a Redox-Regulated Molecular Chaperone.

The bacterial pathogen Mycobacterium tuberculosis is the leading cause of death by an infectious disease among humans. Here, we describe a previously uncharacterized M. tuberculosis protein, Rv0991c, as a molecular chaperone that is activated by oxidation. Rv0991c has homologs in most bacterial lineages and appears to function analogously to the well-characterized Escherichia coli redox-regulated chaperone Hsp33, despite a dissimilar protein sequence. Rv0991c is transcriptionally coregulated with hsp60 and hsp70 chaperone genes in M. tuberculosis, suggesting that Rv0991c functions with these chaperones in maintaining protein quality control. Supporting this hypothesis, we found that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded protein in vitro and promotes its refolding by the M. tuberculosis Hsp70 chaperone system. Furthermore, Rv0991c interacts with DnaK and can associate with many other M. tuberculosis proteins. We therefore propose that Rv0991c, which we named "Ruc" (redox-regulated protein with unstructured C terminus), represents a founding member of a new chaperone family that protects M. tuberculosis and other species from proteotoxicity during oxidative stress.IMPORTANCEM. tuberculosis infections are responsible for more than 1 million deaths per year. Developing effective strategies to combat this disease requires a greater understanding of M. tuberculosis biology. As in all cells, protein quality control is essential for the viability of M. tuberculosis, which likely faces proteotoxic stress within a host. Here, we identify an M. tuberculosis protein, Ruc, that gains chaperone activity upon oxidation. Ruc represents a previously unrecognized family of redox-regulated chaperones found throughout the bacterial superkingdom. Additionally, we found that oxidized Ruc promotes the protein-folding activity of the essential M. tuberculosis Hsp70 chaperone system. This work contributes to a growing body of evidence that oxidative stress provides a particular strain on cellular protein stability.

The ethmo-frontal angle: a new anatomic and radiologic landmark for use in sinus surgery.

OBJECTIVE: To identify anatomic and radiologic landmarks to assist with frontal sinus surgery. STUDY DESIGN: Retrospective review. SUBJECTS AND METHODS: Sinus CT scans of 50 patients were evaluated with respect to a new radiologic and anatomic landmark, the ethmo-frontal angle (EFA). RESULTS: Right-sided EFA ranged from 135 to 171 degrees. Left-sided EFA ranged from 136 to 167 degrees. Measurements of both sides displayed a normal distribution. When right and left sides within individuals were compared, there was no correlation to indicate a high degree of variation between any particular patient's right and left side EFA. CONCLUSION: The EFA is a new landmark to assist otolaryngologists during surgery on and around the frontal sinus. Normal values for this angle have been presented. Surgeons should be aware that asymmetry in a patient's EFA is common, and each side should be examined individually.

Balloon sinuplasty for the surgical management of immunocompromised and critically ill patients with acute rhinosinusitis.

OBJECTIVE: The purpose of this study was to review use of balloon sinuplasty for surgical treatment in critically ill patients with acute sinusitis. STUDY DESIGN: Case series with chart review. SUBJECTS AND METHODS: Patients who underwent balloon sinuplasty between October 2007 and March 2008 were identified. Medical records of the subset of patients who were immunocompromised or otherwise critically ill were analyzed. RESULTS: Thirty-one patients underwent balloon sinuplasty at our institution between October 2007 and March 2008. We identified five critically ill patients with sinus disease within this group. Patient ages ranged from 15 to 51 years with no sex preponderance. All patients had focal findings on a sinus CT scan. In all cases, purulent drainage was noted intraoperatively. All patients returned to baseline health meeting discharge criteria after treatment. CONCLUSION: Balloon sinuplasty represents a potentially less invasive surgical option than standard Functional Endoscopic Sinus Surgery (FESS) and should be considered in the treatment of critically ill or immunocompromised patients.

Revisiting the interpretation of positive sinus CT findings: a radiological and symptom-based review.

OBJECTIVE: It is widely believed that a high percentage of normal, healthy patients without sinusitis symptoms have abnormal findings on sinus CT. Experiences of the authors of this study suggest otherwise. STUDY DESIGN: Cross-sectional survey. SUBJECTS AND METHODS: Head/sinus CT scans of 50 consecutive patients from each of three study groups were reviewed. Group 1 consisted of patients without any sinus symptoms. Group 2 consisted of patients with acute headache symptoms. Group 3 consisted of patients with complaints consistent with chronic sinusitis. CT scans were evaluated with the Lund-Mackay scoring system. RESULTS: In the asymptomatic patient group (group 1), six (3%) patients had positive sinus CT scan findings, compared with 11 (5.5%) in the acutely symptomatic group (group 2), and 32 (64%) in the chronically symptomatic group (group 3). In the chronically symptomatic group (group 3), 64 percent of patients were allergic compared with 18% of the acute headache group (group 2) and 8 percent of the asymptomatic patient group (group 1). CONCLUSION: Results of this study suggest that symptomatic sinus patients are much more likely to have positive sinus CT scan findings than asymptomatic patients. Conversely, normal healthy patients should not be expected to have abnormal sinus CT scans.

Endoscopic localization of the anterior and posterior ethmoid arteries.

OBJECTIVES: Understanding the endoscopic locations of the anterior and posterior ethmoid arteries is important during endoscopic sinus or endoscopic skull base procedures so that these arteries can be avoided. Therefore, the objective of this study was to define the endoscopic locations of the ethmoid arteries. METHODS: Twenty-four cadaver heads were used to identify the endoscopic location of the ethmoid arteries via an external incision. An image guidance system was used to record the locations of these arteries. The anterior ethmoid artery was referenced to the axilla of the middle turbinate, and the posterior ethmoid artery to the anterior wall of the sphenoid sinus. The closest lamella to these arteries was identified. RESULTS: Forty-eight nasal cavities were dissected. The mean distance from the axilla to the anterior ethmoid artery was 17.5 mm. The anterior ethmoid artery was located immediately anterior to (31%), at (36%), or immediately posterior to (33%) the superior attachment of the basal lamella. The mean distance from the posterior ethmoid artery to the anterior ethmoid artery was 14.9 mm. The mean distance from the posterior ethmoid artery to the anterior wall of the sphenoid sinus was 8.1 mm. The posterior ethmoid artery was either anterior to (98%) or at (2%) the anterior face of the sphenoid sinus. CONCLUSIONS: Specific endoscopic anatomic relationships and measurements have been presented for the anterior and posterior ethmoid arteries.

Cytokinin Signaling in Mycobacterium tuberculosis.

It was recently reported that the human-exclusive pathogen Mycobacterium tuberculosis secretes cytokinins, which had only been known as plant hormones. While cytokinins are well-established, adenine-based signaling molecules in plants, they have never been shown to participate in signal transduction in other kingdoms of life. M. tuberculosis is not known to interact with plants. Therefore, we tested the hypothesis that cytokinins trigger transcriptional changes within this bacterial species. Here, we show cytokinins induced the strong expression of the M. tuberculosis gene Rv0077c. We found that Rv0077c expression is repressed by a TetR-like transcriptional repressor, Rv0078. Strikingly, cytokinin-induced expression of Rv0077c resulted in a loss of acid-fast staining of M. tuberculosis While acid-fast staining is thought to be associated with changes in the bacterial cell envelope and virulence, Rv0077c-induced loss of acid-fastness did not affect antibiotic susceptibility or attenuate bacterial growth in mice, consistent with an unaltered mycolic acid profile of Rv0077c-expressing cells. Collectively, these findings show cytokinins signal transcriptional changes that can affect M. tuberculosis acid-fastness and that cytokinin signaling is no longer limited to the kingdom Plantae.IMPORTANCE Cytokinins have only previously been known as plant hormones. The discovery that they can be used as signaling molecules outside of plants broadens the repertoire of small molecules that can potentially affect gene expression in all domains of life.

Initial surgical treatment for chronic frontal sinusitis: a pilot study.

OBJECTIVES: The initial surgical treatment for chronic frontal sinusitis is not well defined. Our objective was to determine the effectiveness of anterior ethmoidectomy for chronic frontal sinusitis. METHODS: Patients with chronic frontal sinusitis who underwent anterior ethmoidectomy as initial surgical treatment were reviewed. Data were collected from computed tomography scans with use of the Lund-Mackay scale. Data on demographics, comorbidities, management, postoperative recovery, and follow-up were collected. RESULTS: Seventy-seven patients representing 121 diseased frontal sinuses met the inclusion criteria. The respiratory comorbidities were asthma alone (8.3%), asthma and polyps (6.6%), aspirin triad (5.8%), and cystic fibrosis (0.8%). Nineteen of 121 frontal sinuses (15.7%) belonged to smokers. Fourteen of 121 frontal sinuses (11.5%) exhibited postoperative evidence of disease. Of these 14 frontal sinuses, 10 (8.3%) underwent revision surgery. Frontal sinuses of patients with aspirin triad, with both nasal polyposis and asthma, or with inter-frontal sinus septal cells were more likely to fail Draf I surgery (p < .05). CONCLUSIONS: Anterior ethmoidectomy for drainage of frontal sinuses appears to be effective initial surgical treatment for chronic frontal sinusitis. Patients with aspirin triad, both asthma and polyposis, or inter-frontal sinus septal cells are more likely to fail this procedure.