We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli . For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.

Modic type I change (MC1) are vertebral bone marrow lesions adjacent to degenerated discs that are specific for discogenic low back pain. The etiopathogenesis is unknown, but occult discitis, in particular with Propionibacteria acnes (P. acnes), has been suggested as a possible etiology. If true, antibiotic therapy should be considered for patients with MC1. However, this hypothesis is controversial. While some studies report up to 40% infection rate in herniated discs, others fail to detect infected discs and attribute reports of positive cultures to contamination during sampling procedure. Irrespective of the clinical controversy, whether it is biologically plausible for P. acnes to cause MC1 has never been investigated. Therefore, the objective of this study was to test if P. acnes can proliferate within discs and cause reactive changes in the adjacent bone marrow. P. acnes was aseptically isolated from a symptomatic human L4/5 disc with MC1 and injected into rat tail discs. We demonstrate proliferation of P. acnes and up‐regulation of IL‐1 and IL‐6 within three days of inoculation. At day‐7, disc degeneration was apparent along with fibrotic endplate erosion. TNF‐α immunoreactivity was enhanced within the effected endplates along with cellular infiltrates. The bone marrow appeared normal. At day‐14, endplates and trabecular bone close to the disc were almost completely resorbed and fibrotic tissue extended into the bone marrow. T‐cells and TNF‐α immunoreactivity were identified at the disc/marrow junction. On MRI, bone marrow showed MC1‐like changes. In conclusion, P. acnes proliferate within the disc, induce degeneration, and cause MC1‐like changes in the adjacent bone marrow. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1447–1455, 2016.

The cartilaginous endplates (CEP) are key components of the intervertebral disc (IVD) necessary for sustaining the nutrition of the disc while distributing mechanical loads and preventing the disc from bulging into the adjacent vertebral body. The size, shape, and composition of the CEP are essential in maintaining its function, and degeneration of the CEP is considered a contributor to early IVD degeneration. In addition, the CEP is implicated in Modic changes, which are often associated with low back pain. This review aims to tackle the current knowledge of the CEP regarding its structure, composition, permeability, and mechanical role in a healthy disc, how they change with degeneration, and how they connect to IVD degeneration and low back pain. Additionally, the authors suggest a standardized naming convention regarding the CEP and bony endplate and suggest avoiding the term vertebral endplate. Currently, there is limited data on the CEP itself as reported data is often a combination of CEP and bony endplate, or the CEP is considered as articular cartilage. However, it is clear the CEP is a unique tissue type that differs from articular cartilage, bony endplate, and other IVD tissues. Thus, future research should investigate the CEP separately to fully understand its role in healthy and degenerated IVDs. Further, most IVD regeneration therapies in development failed to address, or even considered the CEP, despite its key role in nutrition and mechanical stability within the IVD. Thus, the CEP should be considered and potentially targeted for future sustainable treatments.

Vertebral endplate damage is associated with intervertebral disc (IVD) degeneration (DD) in vivo as confirmed by in‐vitro investigations. Our aims were to further characterize the process of DD using an in vitro full‐organ culture model and to elucidate whether significant endplate damage or impact loading alone is pivotal for the initiation of DD. Rabbit spinal segments (n = 80) were harvested, subjected to pure axial impact loading (n = 40) using a custom‐made device, and cultured for 28 days. The applied threshold energy (0.76 J) induced endplate fractures in 21 specimens (group A); 19 remained intact (group B). Markers for DD (cell viability, apoptosis, necrosis, matrix remodeling, and inflammation) were monitored for 28 days post‐trauma in the annulus fibrosus (AF) and nucleus pulposus and compared to non‐impacted control discs. Cell viability in both groups stayed at a control level. Group A compared to group B showed enhanced lactate dehydrogenase (LDH) and caspase‐3/7 activity, reduced glycosaminoglycan content, reduced aggrecan mRNA, but elevated mRNA for collagen‐2, catabolic enzymes (MMP‐1/‐3/‐13), and pro‐inflammatory (TNFα, IL‐6, IL‐8, MCP‐1) and pro‐apoptotic (fas ligand, caspase‐3) proteins. Group B compared to control only showed small changes in mRNA levels. Our findings demonstrate that burst endplates, but not equienergetic loading, promotes DD. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:809–816, 2012