Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.

This study explores the feasibility of using a combination of experimental and theoretical 1-bond C─ C scalar couplings ( J ) to establish structure in organic compounds, including unknowns. Historically, J and J studies have emphasized 2 and 3-bond couplings, yet J couplings exhibit significantly larger variations. Moreover, recent improvements in experimental measurement and data processing methods have made J data more available. Herein, an approach is evaluated in which a collection of theoretical structures is created from a partial nuclear magnetic resonance structural characterization. Computed J values are compared to experimental data to identify candidates giving the best agreement. This process requires knowledge of the error in theoretical methods, thus the B3LYP, B3PW91, and PBE0 functionals are evaluated by comparing to 27 experimental values from INADEQUATE. Respective errors of ±1.2, ±3.8, and ±2.3 Hz are observed. An initial test of this methodology involves the natural product 5-methylmellein. In this case, only a single candidate matches experimental data with high statistical confidence. This analysis establishes the intramolecular hydrogen-bonding arrangement, ring heteroatom identity, and conformation at one position. This approach is then extended to hydroheptelidic acid, a natural product not fully characterized in prior studies. The experimental/theoretical approach proposed herein identifies a single best-fit structure from among 26 candidates and establishes, for the first time, 1 configuration and 3 conformations to complete the characterization. These results suggest that accurate and complete structural characterizations of many moderately sized organic structures (<800 Da) may be possible using only J data.

Simple sugars produced from a solvent-free mechanocatalytic degradation of cellulose were evaluated for suitability as a growth medium carbon source for fungi that produce volatile organic compounds. An endophytic Hypoxylon sp. (CI-4) known to produce volatiles having potential value as fuels was initially evaluated. The growth was obtained on a medium containing the degraded cellulose as the sole carbon source, and the volatile compounds produced were largely the same as those produced from a conventional dextrose/starch diet. A second Hypoxylon sp. (BS15) was also characterized and shown to be phylogenetically divergent from any other named species. The degraded cellulose medium supported the growth of BS15, and approximately the same quantity of the volatile compounds was produced as from conventional diets. Although the major products from BS15 grown on the degraded cellulose were identical to those from dextrose, the minor products differed. Neither CI-4 or BS15 exhibited growth on cellulose that had not been degraded. The extraction of volatiles from the growth media was achieved using solid-phase extraction in order to reduce the solvent waste and more efficiently retain compounds having low vapor pressures. A comparison to more conventional liquid–liquid extraction demonstrated that, for CI-4, both methods gave similar results. The solid-phase extraction of BS15 retained a significantly larger variety of the volatile compounds than did the liquid–liquid extraction. These advances position the coupling of solvent-free cellulose conversion and endophyte metabolism as a viable strategy for the production of important hydrocarbons.

T cell exhaustion is a state of dysfunction that frequently occurs during chronic infections and cancer due to persistent antigen stimulation and inflammation. It is defined by poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. In this state, cells remain in the local environment but lack effector functions necessary to combat the tumor or infection. In the current study, we developed an in vitro human antigen-specific CD8 T cell exhaustion model to evaluate the ability of immune checkpoint inhibitors to restore functionality to exhausted T cells. Using the fully human, autologous MIMIC (Modular IMmune In vitro Construct) effector CD8 T cell assay as a framework, we assessed the effect of antigen dose, timing, and frequency of repeated antigen stimulation with antiviral HLA Class I-restricted peptides to drive the development of Ag-specific CD8 T cells toward an exhausted state. Epitope-specific CD8 T cell responses were monitored by HLA class I-restricted pentamers and establishment of an exhausted profile was evaluated by increased expression of inhibitory co-stimulatory receptors and decreased intracellular expression of antiviral effector cytokine via flow cytometry relative to functional Ag-specific CD8 T cells from the same donor. Repeated antigen exposure led to decreased production of IFN-γ and TNF-α and increased expression of inhibitory co-stimulation markers on antigen-specific CD8 T cells. After establishment of an exhausted phenotype, treatment with various immune checkpoint inhibitor molecules restored functionality to Ag-specific CD8 T cells upon an additional antigen re-stimulation. Moreover, here using this model we characterized distinct transcriptomic signatures of functional and exhausted MIMIC CD8 T cells. Development of an in vitro human viral antigen-specific CD8 T cell exhaustion model with characteristic features of dysfunctional CD8 T cells as observed in vivo and the ability to restore effector function by treatment with immune checkpoint inhibitors, provides a platform to assess the effectiveness of therapeutic mono-specific or bi-specific “checkpoint blockade” antibody candidates and, in general, the potential to evaluate a myriad of immunotherapeutic approaches for cancer treatment. Citation Format: Roberto Carrio, Margot Cucchetti, Mikielia Devonish, Louis Poisson, Mihaela Babiceanu, Andrew Kettring, Yingchun Liu, Donald Jackson, Evan Gomes, Jeremy Baudhuin, Donald R. Drake, Valeria Fantin, Anthony Byers, Ingrid Sassoon. An in vitro human CD8 T cell exhaustion model for the functional screening of immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6378.