The rockefeller university

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.

Caloric restriction, intermittent fasting, and exercise activate defensive cellular responses such as autophagy, DNA repair, and the induction of antioxidant enzymes. These processes improve health and longevity by protecting cells and organs against damage, mutations, and reactive oxygen species. Consuming a diet rich in vegetables, fruits, and mushrooms can also improve health and longevity. Phytochemicals such as alkaloids, polyphenols, and terpenoids found in plants and fungi activate the same cellular processes as caloric restriction, fasting, and exercise. Many of the beneficial effects of fruits and vegetables may thus be due to activation of stress resistance pathways by phytochemicals. A better understanding of the mechanisms of action of phytochemicals may provide important insights to delay aging and prevent chronic diseases.

Background: Inflammatory activation of CD8(+) T cells can, when left unchecked, drive severe immunopathology. Hyperstimulation of CD8(+) T cells through a broad set of triggering signals can precipitate hemophagocytic lymphohistiocytosis (HLH), a life-threatening systemic inflammatory disorder. Objective: The mechanism linking CD8(+) T-cell hyperactivation to pathology is controversial, with excessive production of IFN-gamma and, more recently, excessive consumption of IL-2, which are proposed as competing hypotheses. We formally tested the proximal mechanistic events of each pathway in a mouse model of HLH. Methods: In addition to reporting a complete autosomal recessive IFN-gamma receptor 1-deficient patient with multiple aspects of HLH pathology, we used the mouse model of perforin (Prf1)(KO) mice infected with lymphocytic choriomeningitis virus to genetically eliminate either IFN-gamma production or CD25 expression and assess the immunologic, hematologic, and physiologic disease measurement. Results: We found a striking dichotomy between the mechanistic basis of the hematologic and inflammatory components of CD8(+) T cell-mediated pathology. The hematologic features of HLH were completely dependent on IFN-gamma production, with complete correction after loss of IFN-gamma production without any role for CD8(+) T cell-mediated IL-2 consumption. By contrast, the mechanistic contribution of the immunologic features was reversed, with no role for IFN-gamma production but substantial correction after reduction of IL-2 consumption by hyperactivated CD8(+) T cells. These results were complemented by the characterization of an IFN-gamma receptor 1-deficient patients with HLH-like disease, in whom multiple aspects of HLH pathology were observed in the absence of IFN-gamma signaling. Conclusion: These results synthesize the competing mechanistic models of HLH pathology into a dichotomous pathogenesis driven through discrete pathways. A holistic model provides a new paradigm for understanding HLH and, more broadly, the consequences of CD8(+) T-cell hyperactivation, thereby paving the way for clinical intervention based on the features of HLH in individual patients.

Protein-protein interactions are central to biological processes. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions between nucleocytoplasmic transport factors and intrinsically disordered FG repeat containing nuclear pore complex proteins as a model system, we examined the utility of three surface-based methods: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the apparent effect of mass transport limitations was demonstrated. Additional experiments with a loss-of-interaction FG repeat mutant variant demonstrated that the binding events that take place on surfaces can be unexpectedly complex, suggesting particular care must be exercised in interpretation of such data.

The Gram-negative outer membrane (OM) is a selectively permeable asymmetric bilayer that allows vital nutrients to diffuse into the cell but prevents toxins and hydrophobic molecules from entering. Functionally and structurally diverse a-barrel outer membrane proteins (OMPs) build and maintain the permeability barrier, making the assembly of OMPs crucial for cell viability. In this work, we characterize an assembly-defective mutant of the maltoporin LamB, LamB(G439D). We show that the folding defect of LaMBG439D results in an accumulation of unfolded substrate that is toxic to the cell when the periplasmic protease DegP is removed. Selection for suppressors of this toxicity identified the novel mutant degS(A323t) allele. The mutant DegS(A323E) protein contains an amino acid substitution at the PDZ/protease domain interface that results in a partially activated conformation of this protein. This activation increases basal levels of downstream sigma(E) stress response signaling. Furthermore, the enhanced sigma(E) activity of DegS(A323E) suppresses a number of other assembly-defective conditions without exhibiting the toxicity associated with high levels of sigma(E) activity. We propose that the increased basal levels of sigma(E) signaling primes the cell to respond to envelope stress before OMP assembly defects threaten cell viability. This finding addresses the importance of envelope stress responses in monitoring the OMP assembly process and underpins the critical balance between envelope defects and stress response activation. IMPORTANCE Gram negative bacteria, such as Escherichia coli, inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the sigma(E) stress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A) from 15 laboratories. Laboratories reported similar to 89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (similar to 78 900 centroids), giving similar to 100% coverage, and similar to 10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of < s(Lab)> <= (0.15 +/- 0.01) Da (1 sigma((x) over bar)). All laboratories achieved < s(Lab)> <= 0.4 Da. For immersions of protein at T-HDx = (3.6 to 25) degrees C and for D2O exchange times of t(HDX) = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is sigma(Laboratories)(reproducibility15) (t(HDX)) = (9.0 +/- 0.9) % (1 sigma). A nine laboratory cohort that immersed samples at T-HDX = 25 degrees C exhibited reproducibility of sigma(25C cohort)(reproducibility) (t(HDX)) = (6.5 +/- 0.6) % for back-exchange corrected, deuterium uptake measurements.

RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degradation. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.