Puromycin reveals a distinct conformation of neuronal ribosomes.

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.

Critical steps in the assembly process of the bacterial 50S ribosomal subunit.

During assembly, ribosomal particles in bacteria fold according to energy landscapes comprised of multiple parallel pathways. Cryo-electron microscopy studies have identified a critical maturation step that occurs during the late assembly stages of the 50S subunit in Bacillus subtilis. This step acts as a point of convergency for all the parallel assembly pathways of the subunit, where an assembly intermediate accumulates in a 'locked' state, causing maturation to pause. Assembly factors then act on this critical step to 'unlock' the last maturation steps involving the functional sites. Without these factors, the 50S subunit fails to complete its assembly, causing cells to die due to a lack of functional ribosomes to synthesize proteins. In this review, we analyze these findings in B. subtilis and examine other cryo-EM studies that have visualized assembly intermediates in different bacterial species, to determine if convergency points in the ribosome assembly process are a common theme among bacteria. There are still gaps in our knowledge, as these methodologies have not yet been applied to diverse species. However, identifying and characterizing these convergency points can reveal how different bacterial species implement unique mechanisms to regulate critical steps in the ribosome assembly process.

Ribosomes in RNA Granules Are Stalled on mRNA Sequences That Are Consensus Sites for FMRP Association.

Local translation in neurons is partly mediated by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the granule fraction, defined as the pellet of sucrose gradients used to separate polysomes from monosomes. The mechanism of how elongating ribosomes are reversibly stalled and unstalled on mRNAs is still unclear. In the present study, we characterize the ribosomes in the granule fraction using immunoblotting, cryogenic electron microscopy (cryo-EM), and ribosome profiling. We find that this fraction, isolated from 5-d-old rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homologue. Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (1) an enrichment for footprint reads of mRNAs that interact with FMRPs and are associated with stalled polysomes, (2) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development, and (3) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared with those usually found in ribosome profiling studies, the footprint reads were longer and were mapped to reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the granule fraction to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall ribosomes during translation elongation in neurons.SIGNIFICANCE STATEMENT Neurons send mRNAs to synapses in RNA granules, where they are not translated until an appropriate stimulus is given. Here, we characterize a granule fraction obtained from sucrose gradients and show that polysomes in this fraction are stalled on consensus sequences in a specific state of translational arrest with extended ribosome-protected fragments. This finding greatly increases our understanding of how neurons use specialized mechanisms to regulate translation and suggests that many studies on neuronal translation may need to be re-evaluated to include the large fraction of neuronal polysomes found in the pellet of sucrose gradients used to isolate polysomes.

SARS-CoV-2 Protein Nanoparticle Vaccines Formed In Situ From Lyophilized Lipids.

The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) glycoprotein is an appealing immunogen, but associated vaccine approaches must overcome the hapten-like nature of the compact protein and adapt to emerging variants with evolving RBD sequences. Here, a vaccine manufacturing methodology is proposed comprising a sterile-filtered freeze-dried lipid cake formulation that can be reconstituted with liquid proteins to instantaneously form liposome-displayed protein nanoparticles. Mannitol is used as a bulking agent and a small amount of Tween-80 surfactant is required to achieve reconstituted submicron particles that do not precipitate prior to usage. The lipid particles include an E. coli-derived monophosphoryl lipid A (EcML) for immunogenicity, and cobalt porphyrin-phospholipid (CoPoP) for antigen display. Reconstitution of the lipid cake with aqueous protein results in rapid conversion of the RBD into intact liposome-bound format prior to injection. Protein particles can readily be formed with sequent-divergent RBD proteins derived from the ancestral or Omicron strains. Immunization of mice elicits antibodies that neutralize respective viral strains. When K18-hACE2 transgenic mice are immunized and challenged with ancestral SARS-CoV-2 or the Omicron BA.5 variant, both liquid liposomes displaying the RBD and rapid reconstituted particles protect mice from infection, as measured by the viral load in the lungs and nasal turbinates.

RbgA ensures the correct timing in the maturation of the 50S subunits functional sites.

RbgA is an essential protein for the assembly of the 50S subunit in Bacillus subtilis. Depletion of RbgA leads to the accumulation of the 45S intermediate. A strain expressing a RbgA variant with reduced GTPase activity generates spontaneous suppressor mutations in uL6. Each suppressor strain accumulates a unique 44S intermediate. We reasoned that characterizing the structure of these mutant 44S intermediates may explain why RbgA is required to catalyze the folding of the 50S functional sites. We found that in the 44S particles, rRNA helices H42 and H97, near the binding site of uL6, adopt a flexible conformation and allow the central protuberance and functional sites in the mutant 44S particles to mature in any order. Instead, the wild-type 45S particles exhibit a stable H42-H97 interaction and their functional sites always mature last. The dependence on RbgA was also less pronounced in the 44S particles. We concluded that the binding of uL6 pauses the maturation of the functional sites, but the central protuberance continues to fold. RbgA exclusively binds intermediates with a formed central protuberance and licenses the folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.

Ribosomal subunits in bacteria assemble according to energy landscapes comprised of multiple parallel pathways. In this study, the authors identified a critical maturation step in the late assembly stages of the large 50S ribosomal subunit in bacteria. This step represents a merging point where all parallel assembly pathways of the ribosomal particles converge. At this critical step, the convergent assembly intermediate that accumulates in cells exists in a 'locked' state, and its maturation is paused. The RbgA protein acts on this critical step to 'unlock' the last maturation steps involving folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.

A liposome-displayed hemagglutinin vaccine platform protects mice and ferrets from heterologous influenza virus challenge.

Recombinant influenza virus vaccines based on hemagglutinin (HA) hold the potential to accelerate production timelines and improve efficacy relative to traditional egg-based platforms. Here, we assess a vaccine adjuvant system comprised of immunogenic liposomes that spontaneously convert soluble antigens into a particle format, displayed on the bilayer surface. When trimeric H3 HA was presented on liposomes, antigen delivery to macrophages was improved in vitro, and strong functional antibody responses were induced following intramuscular immunization of mice. Protection was conferred against challenge with a heterologous strain of H3N2 virus, and naive mice were also protected following passive serum transfer. When admixed with the particle-forming liposomes, immunization reduced viral infection severity at vaccine doses as low as 2 ng HA, highlighting dose-sparing potential. In ferrets, immunization induced neutralizing antibodies that reduced the upper respiratory viral load upon challenge with a more modern, heterologous H3N2 viral strain. To demonstrate the flexibility and modular nature of the liposome system, 10 recombinant surface antigens representing distinct influenza virus strains were bound simultaneously to generate a highly multivalent protein particle that with 5 ng individual antigen dosing induced antibodies in mice that specifically recognized the constituent immunogens and conferred protection against heterologous H5N1 influenza virus challenge. Taken together, these results show that stable presentation of recombinant HA on immunogenic liposome surfaces in an arrayed fashion enhances functional immune responses and warrants further attention for the development of broadly protective influenza virus vaccines.

Respiratory Vaccination with Hemagglutinin Nanoliposomes Protects Mice from Homologous and Heterologous Strains of Influenza Virus.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

Molecular Sieving with PEGylated Dendron-Protein Conjugates.

A series of generation 3-5 dendrons based on a bis(2,2-hydroxymethylpropionic acid) (bis-MPA) scaffold bearing three respective lengths of linear poly(ethylene glycol) at their periphery and a dibenzocyclooctyne unit at their core was prepared. These dendrons were appended to the surface of azide-decorated α-chymotrypsin (α-CT) via strain-promoted azide-alkyne cycloaddition to yield a library of dendron-protein conjugates. These conjugates were characterized by FT-IR and NMR spectroscopy and were imaged using cryo-electron microscopy. The activity of the PEGylated α-CT-dendron conjugates was investigated using a small molecule (benzoyl-l-tyrosine p-nitroanilide) as well as different proteins of different sizes and crystallinities (casein and bovine serum albumin) as substrates. It was found that the activity of the conjugates toward the small molecule was largely retained, while the activity toward the proteins was significantly diminished. Furthermore, the results indicate that for most of the conjugates the PEG length had a more pronounced impact on enzyme activity than the dendron generation. Overall, the highest sieving ratios were found for α-CT-dendron conjugates decorated with G3-PEG2000, G4-PEG2000, and G5-PEG1000, with the latter two structures offering the best combination of sieving ratio and small molecule activity.

Clinical and cost outcomes of a polyethylene glycol (PEG)-coated patch versus drainage after axillary lymph node dissection in breast cancer: results from a multicentre randomized clinical trial.

BACKGROUND: The aim of this study was to compare the clinical outcomes between breast cancer patients who underwent axillary lymph node dissection with postoperative management using a polyethylene glycol-coated patch versus axillary drainage. The direct costs associated with both postoperative management strategies were also evaluated. METHODS: This was a multicentre RCT in women with breast cancer who underwent axillary lymph node dissection (ClinicalTrials.gov identifier: NCT04487561). Patients were randomly assigned (1 : 1) to receive either drainage or a polyethylene glycol-coated patch as postoperative management. The primary endpoints were the need for an emergency department visit for any event related to the surgery and the rate of seroma development. RESULTS: A total of 227 patients were included , 115 in the patch group (50.7 per cent) and 112 (29.4 per cent ) in the drainage group. The incidence of emergency department visits was significantly greater for patients with drainage versus a polyethylene glycol-coated patch (incidence rate difference 26.1 per cent, 95 per cent c.i. 14.5 to 37.7 per cent; P < 0.001). Conversely, the seroma rate was significantly higher in the polyethylene glycol-coated patch group (incidence rate difference 22.8 per cent, 95 per cent c.i. 6.7 to 38.9 per cent; P < 0.0055). Compared with drainage, using a polyethylene glycol-coated patch resulted in cost savings of €100.41 per patient. An incremental cost-effectiveness ratio analysis found that drainage was associated with an incremental cost-effectiveness ratio of €7594.4 for no need for hospital admission and €491.7 for no need for an emergency department visit. CONCLUSION: Compared with patients who received drainage after axillary lymph node dissection, the use of a polyethylene glycol-coated patch resulted in a higher rate of seroma, but a lower number of postoperative outpatient or emergency department visits and thus a reduction in overall costs.

Aneurysm Associated with Vascular Wall Degeneration in Bearded Dragons (Pogona vitticeps).

This study describes the clinical, gross, and histologic findings in 17 cases of aneurysms in bearded dragons (Pogona vitticeps). The clinical presentation ranged from incidental to sudden and unexpected death. The affected vasculature was predominantly arterial; however, based on the topographical locations of the lesions, gross structure, and drainage, some veins were likely involved. Magnetic resonance imaging and computerized tomography scans of 1 animal showed a large aneurysm of the internal carotid artery extending from near its aortic origin into the caudal head. Aneurysms were organized in 5 groups based on their anatomical locations: cephalic, cranial coelom (for all near the heart), caudal coelom (for the mesenteric vessels and descending aorta), limbs, and tail. The cranial coelomic region was the most prevalent location. Gross findings were large hematomas or red serosanguineous fluid filling the adjacent area, as most of the aneurysms (94%) were ruptured at the time of the study. The main histological findings were degenerative changes of the vessel walls characterized by moderate to severe disruption of the collagen and elastic fibers of the tunica media and adventitia (100%), followed by thickening of the intima with thrombi formation (54%) and dissecting hematoma of the vessel wall (47%). Vasculitis (29%), mineralization (6%), and lipid deposits (6%) in the vessel wall were observed occasionally. Based on these findings, the vascular dilations and ruptures observed in bearded dragons likely are associated with weakness of the vessel walls caused by degenerative changes in the intimal and medial tunics.

Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome.

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.

Alternative conformations and motions adopted by 30S ribosomal subunits visualized by cryo-electron microscopy.

It is only after recent advances in cryo-electron microscopy that it is now possible to describe at high-resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between an "active" and "inactive" conformation. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains unsolved. Here we used cryo-electron microscopy to obtain the structure of the inactive conformation of the 30S subunit to 3.6 Å resolution and study its motions. In the inactive conformation, an alternative base-pairing of three nucleotides causes the region of helix 44, forming the decoding center to adopt an unlatched conformation and the 3' end of the 16S rRNA positions similarly to the mRNA during translation. Incubation of inactive 30S subunits at 42°C reverts these structural changes. The air-water interface to which ribosome subunits are exposed during sample preparation also peel off some ribosomal proteins. Extended exposures to low magnesium concentrations make the ribosomal particles more susceptible to the air-water interface causing the unfolding of large rRNA structural domains. Overall, this study provides new insights about the conformational space explored by the 30S ribosomal subunit when the ribosomal particles are free in solution.

Is Axillary Assessment of Ductal Carcinoma In Situ of the Breast Necessary in All Cases?

BACKGROUND: Staging of the axilla in women with ductal carcinoma in situ (DCIS) is a point of controversy. We aimed to assess whether there is a group of patients in whom axillary assessment can be avoided and whether the likelihood of underdiagnosis of infiltrating carcinoma is sufficient to justify this evaluation. MATERIALS AND METHODS: This was a multicenter, prospective, observational study of patients who were operated on between 2008 and 2018 in three Spanish hospitals, with a diagnosis by radiological or excisional biopsy of DCIS and clinically and radiologically negative axilla. RESULTS: A total of 530 patients with a preoperative diagnosis of DCIS were studied. An axillary assessment was performed in 77% of the patients. In 397 patients, selective sentinel lymph node biopsy was performed. Axillary involvement was found in 7.2% of all patients, which dropped to 2.15% if we only included DCIS diagnosed after a definitive anatomical pathology analysis. Underdiagnosis was correlated with the type of biopsy performed: the risk was 1.34 times as high if the biopsy was performed with a core needle. The risk of lymph node metastasis was higher when there was lymphovascular invasion and when mastectomy was performed. CONCLUSIONS: We propose an axilla management algorithm in patients with a preoperative diagnosis of DCIS. The patients who would benefit from sentinel lymph node biopsy would be those who are not candidates for breast-conserving surgery, those with a BIRADS 5 lesion biopsied by core-needle biopsy, and those whose definitive diagnosis is lymphovascular invasion.

Preoperative risk factors for early hemorrhagic complications in bariatric surgery: a case-control study.

BACKGROUND: Although a reliable procedure in morbid obesity treatment, bariatric surgery may be associated with serious complications such as leakage or bleeding. We aimed to analyze the preoperative factors involved in patients with early postoperative hemorrhage after any type of bariatric surgery who required conservative treatment or reoperation for this complication. METHODS: Retrospective case-controlled study (1:3) of 2 patient cohorts (postoperative bleeding/controls) matched by type of surgical intervention. RESULTS: Hypertension (Odds Ratio 5.029; 95% Confidence Interval 1.78-14.13) and history of antiplatelet medication (OR 13.263; 95% CI 1.39-125.9) were independent risk factors in the bivariate analyses, confirmed in the logistic regression model on multivariate analysis. CONCLUSIONS: With no between-group differences in Body Mass Index (BMI) and type 2 Diabetes (T2D), early hemorrhagic complications were found to be more frequent in patients with hypertension or antiplatelet drug treatment.

When laparoscopic repair is feasible for diaphragmatic hernia in adults? A retrospective study and literature review.

BACKGROUND: Diaphragmatic hernia (DH), congenital or traumatic, is uncommon but sometimes can lead to a serious surgical emergency. There are no clinical guidelines or approved recommendations for the management of this condition, and most data are from retrospective, single-institution series. The aim is to analyze the management of the DH at our institution and review the indications for laparoscopic repair. METHODS: A retrospective serie of patients diagnosed of DH with surgical treatment at our institution between 2009 and 2019. Literature review was carried out to establish the current indications of laparoscopic repair in each type of DH. RESULTS: Surgery was carried out in 15 patients with DH, 5 congenital and 10 traumatic hernias. Traumatic hernias were classified as acute (n = 2) and chronic (n = 8). 53.4% of all cases (8 patients) required urgent surgery using an abdominal approach (5 open and 3 laparoscopic) and elective surgery was performed in 46.6% of all cases (7 patients) with an abdominal approach (3 open and 4 laparoscopic) and 2 patients with a combined approach. Primary repair was performed in 4 patients (26.6%), closure and mesh reinforcement in 9 cases (60%) and only mesh placement in 2 patients (13.4%). Postoperative morbidity and mortality were 20% and 0%, respectively. No recurrences were detected. CONCLUSIONS: DH may pose different scenarios which require urgent or elective surgical treatment. Laparoscopic approach may be a first option in elective surgery; and in emergency setting taking into account hemodynamic stability and associated injuries.

Influence of Transfer Entropy in the Short-Term Prediction of Financial Time Series Using an ∊-Machine.

Predicting the values of a financial time series is mainly a function of its price history, which depends on several factors, internal and external. With this history, it is possible to build an ∊-machine for predicting the financial time series. This work proposes considering the influence of a financial series through the transfer of entropy when the values of the other financial series are known. A method is proposed that considers the transfer of entropy for breaking the ties that occur when calculating the prediction with the ∊-machine. This analysis is carried out using data from six financial series: two American, the S&P 500 and the Nasdaq; two Asian, the Hang Seng and the Nikkei 225; and two European, the CAC 40 and the DAX. This work shows that it is possible to influence the prediction of the closing value of a series if the value of the influencing series is known. This work showed that the series that transfer the most information through entropy transfer are the American S&P 500 and Nasdaq, followed by the European DAX and CAC 40, and finally the Asian Nikkei 225 and Hang Seng.

HPV-Associated Tumor Eradication by Vaccination with Synthetic Short Peptides and Particle-Forming Liposomes.

Human papilloma virus (HPV)-16 is associated with cervical cancers and induces expression of the E6 and E7 oncogenes. Using a murine cell line that expresses these, the genes are sequenced, and six predicted major histocompatibility complex (MHC) class I (MHC-I) epitopes are identified. A liposomal vaccine adjuvant based on cobalt-porphyrin-phospholipid (CoPoP) is admixed with synthetic 9-mer epitopes appended with three histidine residues, resulting in rapid formation of peptide-liposome particles. Immunization with multivalent peptides leads to protection from tumor challenge. Of the peptides screened, only the previously identified E749-57 epitope is functional. The peptide-liposome particles that form upon mixing E7HHH49-57 with CoPoP liposomes are stable in serum and are avidly taken up by immune cells in vitro. Immunization results in robust protection from tumor challenge and re-challenge. A 100 ng peptide dose protects mice in a therapeutic tumor challenge when admixed with CoPoP liposomes, whereas 200-fold higher peptide doses are ineffective with the polyinosinic-polycytidylic (poly(I:C)) adjuvant. CoPoP induces a strong infiltrating CD8+ T-cell response within the tumor microenvironment with an improved functional profile. Vaccine monotherapy using nanogram dosing of the E7HHH49-57 peptide admixed with CoPoP reverses the growth of large established tumors, eradicating subcutaneous tumors upwards of 100 mm3 . Immunization also eradicates lung tumors in a metastasis model.

The nuclear retinoid-related orphan receptor RORα controls adipose tissue inflammation in patients with morbid obesity and diabetes.

BACKGROUND/AIMS: Inflammation governs adipose tissue (AT) dysfunction in obesity. Retinoic acid receptor-related orphan receptor alpha (RORα) is associated with inflammation and insulin resistance in animal studies, but its role in human obesity remains elusive. We investigated the expression and function of RORα on AT inflammation in patients with morbid obesity with/without diabetes. SUBJECTS/METHODS: We assessed RORα expression in paired biopsies of subcutaneous and omental AT from 41 patients (body mass index (BMI) 43.3 ± 0.8 kg/m2) during Roux-en-Y-gastric surgery and explored the functional consequences of pharmacological RORα blockade in AT ex vivo. RESULTS: RORα expression was significantly higher in omental AT than in subcutaneous AT (p = 0.03) and was positively associated with BMI (r = 0.344, p = 0.027) and homeostasis model assessment of insulin resistance (r = 0.319, p = 0.041). In ex vivo assays, IL-8/CXCL8 and MCP-1/CCL2 chemokine release was significantly higher in omental fat explants from diabetic patients than from non-diabetics and was significantly diminished by RORα blockade (p < 0.05). Inhibition of RORα improved protein kinase B signaling and decreased NF-κB activity in omental AT from patients with diabetes (p < 0.05). Under dynamic flow conditions, RORα blockade prevented mononuclear cell attachment to human dysfunctional endothelial cells. CONCLUSIONS: RORα blockade represents a potential therapy to prevent AT dysfunction and inflammation associated with insulin resistance in human obesity.

Light-Triggered Release of Large Biomacromolecules from Porphyrin-Phospholipid Liposomes.

Liposomes containing small amounts of porphyrin-phospholipid (PoP) have been shown to encapsulate small molecular weight cargos and then release them upon exposure to red light. A putative mechanism involves transient pore formation in the bilayer induced by PoP-mediated photo-oxidation of unsaturated lipids. However, little is known about the properties of such pores. This study assesses whether large carbohydrate and protein molecules could be released from PoP liposomes upon red light exposure. A small fluorophore with ∼0.5 kDa in molecular weight, fluorescently labeled dextrans of ∼5 and ∼500 kDa, and a ∼240 kDa fluorescent protein were passively entrapped in PoP liposomes. When exposed to 665 nm irradiation, liposomes containing PoP, but not liposomes lacking it, released all these cargos in a size-dependent manner that occurred with oxidation of unsaturated lipids included in the bilayer. Thus, this study demonstrates the feasibility of light-triggered release of large biomacromolecules from liposomes.

Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate.

Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.