Copper homeostasis in Enterococcus hirae

Copper is an essential component of life because of its convenient redox potential of 200-800 mV when bound to protein. Extensive insight into copper homeostasis has only emerged in the last decade and Enterococcus hirae has served as a paradigm for many aspects of the process. The cop operon of E. hirae regulates copper uptake, availability, and export. It consists of four genes that encode a repressor, CopY, a copper chaperone, CopZ, and two CPx-type copper ATPases, CopA and CopB. Most of these components have been conserved across the three evolutionary kingdoms. The four Cop proteins have been studied in vivo as well as in vitro and their function is understood in some detai

BMP-2 and TGF-β3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossification of the annulus fibrosus

Introduction: Different approaches for disc regeneration are currently under investigation. Beside gene therapy and tissue engineering techniques, the application of growth and differentiation factors own promising potential. Studies using reduced intervertebral disc models, such as cell or tissue fragment cultures, have limited validity and show controversial results depending on the employed experimental model. Therefore, the goal of the current study was to investigate the effect of BMP-2 and TGF-β3 on intervertebral disc degeneration using an in vitro full-organ disc/endplate culture system. Materials and methods: Intervertebral rabbit disc explants were cultured in the presence of 1μg/ml BMP-2 or TGF-β3 for 21days in DMEM/F12 media. Nucleus and annulus were analyzed for gene expression of collagen type I and II (Col I/II), aggrecan, collagenases (MMP-1/MMP-13) with RT-qPCR, histological changes with bone and proteoglycan-specific staining (von Kossa, toluidine blue) and differences in cellularity (DNA) and proteoglycan content (alcian blue binding assay). Results: The results demonstrate that disc proteoglycan concentration decreased with time in the TGF-β3 and BMP-2 groups. In the annulus fibrosus (AF), TGF-β3 and BMP-2 resulted in an up-regulation of Col I and type II, and of aggrecan gene expression. In contrast, MMP genes were inhibited. In the nucleus, the growth factors decreased gene expression of aggrecan and spontaneous Col I up-regulation was inhibited by TGF-β3, whereas expression of Col II was decreased with BMP-2. There was no effect on expression of MMP-1 and MMP-13 for most sampling points. However, TGF-β3 and BMP-2 induced ossification of the AF was demonstrated by histology. Conclusion: It can be concluded that both growth factors, at the tested concentrations, may not be suitable to regenerate the whole intervertebral disc organ but they are interesting candidates for being injected alone or in combination into a painful intervertebral disc to induce osseous fusion (spondylodesis

The copper-inducible ComR (YcfQ) repressor regulates expression of ComC (YcfR), which affects copper permeability of the outer membrane of Escherichia coli

The pathway of copper entry into Escherichia coli is still unknown. In an attempt to shed light on this process, a lux-based biosensor was utilized to monitor intracellular copper levels in situ. From a transposon-mutagenized library, strains were selected in which copper entry into cells was reduced, apparent as clones with reduced luminescence when grown in the presence of copper (low-glowers). One low-glower had a transposon insertion in the comR gene, which encodes a TetR-like transcriptional regulator. The mutant strain could be complemented by the comR gene on a plasmid, restoring luminescence to wild-type levels. ComR did not regulate its own expression, but was required for copper-induction of the neighboring, divergently transcribed comC gene, as shown by real-time quantitative PCR and with a promoter-lux fusion. The purified ComR regulator bound to the promoter region of the comC gene in vitro and was released by copper. By membrane fractionation, ComC was shown to be localized in the outer membrane. When grown in the presence of copper, ∆comC cells had higher periplasmic and cytoplasmic copper levels, compared to the wild-type, as assessed by the activation of the periplasmic CusRS sensor and the cytoplasmic CueR sensor, respectively. Thus, ComC is an outer membrane protein which lowers the permeability of the outer membrane to copper. The expression of ComC is controlled by ComR, a novel, TetR-like copper-responsive represso

Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

There is a major controversy whether spinal trauma with vertebral endplate fractures can result in post-traumatic disc degeneration. Intervertebral discs, which are adjacent to burst endplates, are frequently removed and an intercorporal spondylodesis is performed. In any case, the biological effects within the discs following endplate factures are poorly elucidated to date. The aim of our investigations was therefore to establish a novel disc/endplate trauma culture model to reproducibly induce endplate fractures and investigate concurrent disc changes in vitro. This model is based on a full-organ disc/endplate culture system, which has been validated by the authors before. Intervertebral disc/endplate specimens were isolated from Burgundy rabbits and cultured in standard media (DMEM/F12, 10%FCS). Burst endplate fractures were induced in half of the specimens with a custom-made fracture device and subsequently cultured for 9days. The biological effects such as necrotic or apoptotic cell death and the expression of pro-apoptotic genes and other genes involved in organ degeneration, e.g. matrix metalloproteinases (MMPs) were analyzed. Cell damage was assessed by quantification of the lactate dehydrogenase (LDH) activity in the supernatant. The expression of genes involved in the cellular apoptotic pathway (caspase3) and the pro-apoptotic proteins FasL and TNF-α were monitored. The results demonstrate that LDH levels increased significantly post trauma compared to the control and remained elevated for 3days. Furthermore, a constant up-regulation of the caspase3 gene in both disc compartments was present. The pro-apoptotic proteins FasL and TNF-α were up regulated predominantly in the nucleus whereas the MMP-1 and -13 transcripts (collagenases) were increased in both disc structures. From this study we can conclude that endplate burst fractures result in both necrotic and apoptotic cell death in nucleus and annulus tissue. Moreover, FasL and TNF-α expression by nucleus cells may lead to continued apoptosis induced by Fas- and TNF-α receptor bearing cells. In addition TNF-α over-expression has potentially deleterious effects on disc metabolism such as over-expression of matrix proteinases. Taken together, the short term biological response of the disc following endplate fracture exhibits characteristics, which may initiate the degeneration of the orga

Immunomodulation for primary prevention of urinary tract infections in patients with spinal cord injury during primary rehabilitation: protocol for a randomized placebo-controlled pilot trial (UROVAXOM-pilot).

BACKGROUND Urinary tract infections (UTIs) are common in individuals with neurogenic lower urinary tract dysfunction (NLUTD) following spinal cord injury (SCI). They are not only a great burden for affected individuals, but also cause considerable health costs. Furthermore, recurrent antibiotic treatments of UTIs contribute to the growing problem of bacterial resistance to antimicrobial compounds. Even though there is a multitude of different measures to prevent UTIs in individuals with NLUTD, no clear evidence exists for any of these. Oral immunomodulation with UTI-relevant Escherichia coli lysate may be a promising preventative measure with a good safety profile in individuals with NLUTD. However, currently available data are sparse. METHODS This is a randomized, quasi-blinded, placebo-controlled, mono-centric pilot trial investigating the feasibility of a main trial regarding the effects of a lyophilized lysate of E. coli strains for oral application (Uro-Vaxom®, OM Pharma SA, Meyrin, Switzerland). There will be two parallel groups of 12 participants each. Individuals with acute SCI (duration SCI ≤ 56 days) from 18 to 70 years of age admitted for primary rehabilitation will be eligible. Blood and urine samples will be taken prior to intervention start, at the end of the intervention, and 3 months after intervention termination. The trial intervention will last 90 days. The participants will not be informed regarding the treatment allocation (quasi-blinded). The nursing staff will prepare the daily dose of the allocated treatment from the original packaging. The trial personnel and the biostatistician will be blinded. Feasibility (e.g., recruitment rate, patient attrition), clinical (e.g., number of symptomatic UTIs), and laboratory parameters (e.g., urinary culture, urinary proteo- and microbiome, blood cell counts) as well as adverse events will be collected. DISCUSSION Effective and efficient measures for the prevention of UTIs in individuals with NLUTD are urgently needed. If the conclusion of this pilot is positive regarding feasibility, the effects of oral immunomodulation with a E. coli lysate will be investigated in a larger, sufficiently powered, multi-center trial. TRIAL REGISTRATION ClinicalTrials.gov NCT04049994 . Registered on 8 August 2019

Assessing the Feasibility of a Multimodal Approach to Pain Evaluation in Early Stages after Spinal Cord Injury.

This research evaluates the feasibility of a multimodal pain assessment protocol during rehabilitation following spinal cord injury (SCI). The protocol amalgamates clinical workup (CW), quantitative sensory testing (QST), and psychosocial factors (PSF) administered at 4 (T1), 12 (T2), and 24 (T3) weeks post injury and at discharge (T4). Molecular blood biomarkers (BB) were evaluated via gene expression and proteomic assays at T1 and T4. Different pain trajectories and temporal changes were identified using QST, with inflammation and pain-related biomarkers recorded. Higher concentrations of osteopontin and cystatin-C were found in SCI patients compared to healthy controls, indicating their potential as biomarkers. We observed altered inflammatory responses and a slight increase in ICAM-1 and CCL3 were noted, pointing towards changes in cellular adhesion linked with spinal injury and a possible connection with neuropathic pain. Despite a small patient sample hindering the correlation of feasibility data, descriptive statistical analyses were conducted on stress, depression, anxiety, quality of life, and pain interferences. The SCI Pain Instrument (SCIPI) was efficient in distinguishing between nociceptive and neuropathic pain, showing a progressive increase in severity over time. The findings emphasize the need for the careful consideration of recruitment setting and protocol adjustments to enhance the feasibility of multimodal pain evaluation studies post SCI. They also shed light on potential early adaptive mechanisms in SCI pathophysiology, warranting the further exploration of prognostic and preventive strategies for chronic pain in the SCI population

Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae

Abstract : In Enterococcus hirae, copper homeostasis is controlled by the cop operon, which encodes the copper-responsive repressor CopY, the copper chaperone CopZ, and two copper ATPases, CopA and CopB. The four genes are under control of CopY, which is a homodimeric zinc protein, [Zn(II)CopY]2. It acts as a copper-responsive repressor: when media copper is raised, CopY is released from the DNA, allowing transcription to proceed. This involves the conversion of [Zn(II)CopY]2 to [Cu(I)2CopY]2, which is no longer able to bind to the promoter. Binding analysis of [Zn(II)CopY]2 to orthologous promoters and to control DNA by surface plasmon resonance analysis defined the consensus sequence TACAnnTGTA as the repressor binding element, or "cop box”, of Gram-positive bacteria. Association and dissociation rates for the CopY-DNA interaction in the absence and presence of added copper were determined. The dissociation rate of [Zn(II)CopY]2 from the promoter was 7.3×10-6s-1 and was increased to 5×10-5s-1 in the presence of copper. This copper-induced change may be the underlying mechanism of copper induction. Induction of the cop operon was also assessed in vivo with a biosensor containing a lux reporter system under the control of the E. hirae cop promoter. Half-maximal induction of this biosensor was observed at 5μM media copper, which delineates the ambient copper concentration to which the cop operon responds in viv

Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells

Introduction: Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells. Materials and methods: Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrow-derived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cell-scaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry. Results: MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower. Conclusions: Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cell

Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro

The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the inflammatory processes in the IVD remains unclear. We found that tissue obtained by discectomy of degenerated and post-traumatic IVD contains significant amounts of IgG antibodies, a sign of lymphocyte infiltration. Further we investigated whether MSCs in vitro, which were characterized for their multilineage differentiation potential and may have immunomodulatory effects on IVD fragments. IVD fragments were co-cultured in contact with peripheral blood lymphocytes (PBLs) and MSCs, and as functional controls we used contact co-cultures of PBLs stimulated with pokeweed mitogen (2.5μg/mL) and MSCs. The time course of lymphocyte proliferation (Alamar Blue), IgG (ELISA) and gene expression (RT-PCR) of anti-inflammatory cytokines (TGF-β1, IL-10) by MSCs and pro-inflammatory molecules (IL-1α, IL-1β and TNF-α) by the IVD fragments were analyzed. Depending on the response to the presence of MSCs, the IVD fragments (n=13) were divided in two groups: responders (n=9), where inflammation was inhibited by MSCs and non-responders (n=4), where MSCs did not decrease inflammation. At 1week in co-culture, MSCs reduced significantly the IgG production in the IVD responders group to 69% and PBLs proliferation to 57% of the control. MSCs expression of the anti-inflammatory TGF-β1 increased with time, while IL-10 was expressed only at day 1. IVD gene expression of TNF-α decreased constantly, whereas IL-1α and IL-1β expression increased. In conclusion, these data suggest that MSCs may modulate disc-specific inflammatory and pain status and aid regeneration of the host tissu

Energy Availability and Nutritional Intake during Different Training Phases of Wheelchair Athletes.

Optimizing nutritional intake and timing helps athletes to improve performance and long-term health. Different training phases can require varying nutritional needs. In this study, we conducted a descriptive assessment of dietary intake, energy availability (EA), and blood biochemical parameters in elite wheelchair athletes during distinct training phases. Data analyzed in this study were collected as part of a randomized controlled crossover trial exploring the feasibility of probiotics and prebiotic supplementation. Data were obtained from consecutive three-day diaries and blood samples, both collected at four different time points across four consecutive months. We included 14 athletes (mean (standard deviation) age 34 (9) years, eight females, and six males) active in different wheelchair sports. The mean daily nutritional intake (g/kg body mass) for females and males was 2.7 (0.9) and 4.0 (0.7) for carbohydrates, 1.1 (0.3) and 1.5 (0.3) for protein, and 0.8 (0.3) and 1.4 (0.2) for fat. EA did not change across the four time points in either female (p = 0.30) or male (p = 0.05) athletes. The mean EA was lower in female athletes compared to male athletes (p = 0.03). Low EA (≤30 kcal/ kg fat-free mass/day) was observed in female (58 (29) % of days) and male (34 (23) % of days) athletes. Iron deficiency with anemia was observed in two female athletes. Mean vitamin D levels were insufficient (<75 nmol/L). Macronutrient intake, EA, and blood biochemical parameters were suboptimal in this cohort of elite wheelchair athletes, especially in female athletes