Prediction of 2-year clinical outcome trajectories in patients undergoing anterior cervical discectomy and fusion for spondylotic radiculopathy.

OBJECTIVE: Anterior cervical discectomy and fusion (ACDF) is often described as the gold standard surgical technique for cervical spondylotic radiculopathy. Although outcomes are considered favorable, there is little prognostic evidence to guide patient selection for ACDF. This study aimed to 1) describe the 24-month postoperative trajectories of arm pain, neck pain, and pain-related disability; and 2) identify perioperative prognostic factors that predict trajectories representing poor clinical outcomes. METHODS: In this retrospective cohort study, patients with cervical spondylotic radiculopathy who underwent ACDF at 1 of 12 orthopedic or neurological surgery centers were recruited. Potential outcome predictors included demographic, health, clinical, and surgery-related prognostic factors. Surgical outcomes were classified by trajectories of arm pain intensity, neck pain intensity (numeric pain rating scales), and pain-related disability (Neck Disability Index) from before surgery to 24 months postsurgery. Trajectories of postoperative pain and disability were estimated with latent class growth analysis, and prognostic factors associated with poor outcome trajectory were identified with robust Poisson models. RESULTS: The authors included data from 352 patients (mean age 50.9 [SD 9.5] years; 43.8% female). The models estimated that 15.5%-23.5% of patients followed a trajectory consistent with a poor clinical outcome. Lower physical and mental health-related quality of life, moderate to severe risk of depression, and longer surgical wait time and procedure time predicted poor postoperative trajectories for all outcomes. Receiving compensation and smoking additionally predicted a poor neck pain outcome. Regular exercise, physiotherapy, and spinal injections before surgery were associated with a lower risk of poor disability outcome. Patients who used daily opioids, those with worse general health, or those who reported predominant neck pain or a history of depression were at greater risk of poor disability outcome. CONCLUSIONS: Patients who undergo ACDF for cervical spondylotic radiculopathy experience heterogeneous postoperative trajectories of pain and disability, with 15.5%-23.5% of patients experiencing poor outcomes. Demographic, health, clinical, and surgery-related prognostic factors can predict ACDF outcomes. This information may further assist surgeons with patient selection and with setting realistic expectations. Future studies are needed to replicate and validate these findings prior to confident clinical implementation.

The relationships between physical activity, lumbar multifidus muscle morphology, and low back pain from childhood to early adulthood: a 12-year longitudinal study.

We investigated the longitudinal associations between physical activity (PA), lumbar multifidus morphology, and impactful low back pain (LBP) in young people. Nine-year-old children were recruited from 25 primary schools and followed up at age 13, 16, and 21 years. We measured PA with accelerometers at age 9, 13, and 16; quantified patterns of lumbar multifidus intramuscular adipose tissue (IMAT) change from 13 to 16 years using magnetic resonance imaging; and recorded LBP and its impact with standardised questionnaires and interviews. Associations were examined with crude and adjusted logistic or multinomial models and reported with odds ratios (OR) or relative risk ratios (RRR). We included data from 364 children (mean[SD] age = 9.7[.4] years). PA behaviour was not associated with LBP. Having persistently high IMAT levels at age 13 and 16 was associated with greater odds of LBP (OR[95% CI] = 2.98[1.17 to 7.58]). Increased time in moderate and vigorous intensity PA was associated with a lower risk of higher IMAT patterns (RRR[95% CI] = .67[.46 to .96] to .74[.55 to 1.00]). All associations became non-significant after adjusting for sex and body mass index (BMI). Future studies investigating the relationships between PA behaviour, lumbar multifidus IMAT, and impactful LBP should account for potential confounding by sex and BMI.

Decreased white matter fractional anisotropy is associated with poorer functional motor skills following spinal cord injury: a pilot study.

STUDY DESIGN: Prospective cross-sectional study OBJECTIVES: The objective of this study was to assess associations between white matter changes and functional motor markers including grip strength and prehension in the upper limb. SETTING: Single Center Imaging Study, in Vancouver Canada. METHODS: Diffusion tensor imaging produced FA (Fractional Anisotropy) maps of the brain for participants with SCI (n = 7) and controls (n = 6). These FA maps were analyzed using tract-based spatial statistics. Correlations between the FA values (of the genu of the corpus callosum, the left superior longitudinal fasciculus and the right anterior thalamic radiation) of the SCI group and functional outcomes (grip strength, Graded Redefined Assessment of Strength, Sensibility and Prehension (GRASSP)) were assessed. RESULTS: Significant differences (p < 0.05) were found between the FA values of the controls and the SCI group in two white matter clusters, with lower values in the SCI group. Strong correlations were found between the FA values of the identified clusters and the age of SCI participant, and the right GRASSP Quantitative Prehension and right total GRASSP score. CONCLUSIONS: This preliminary data suggests that decreased FA in the genu of the corpus callosum may be a biomarker for functional motor ability of the upper limb with higher FA indicating better ability. Further research needs to be done to determine if other white matter tracts are also associated with strength and use of the hand following SCI. SPONSORSHIP: The International Collaboration on Repair Discoveries (operating grant) and Canada Research Chair Program (for JJE) provided support for this research.

Mesenchymal Stromal Cells Modulate Macrophages in Clinically Relevant Lung Injury Models by Extracellular Vesicle Mitochondrial Transfer.

RATIONALE: Acute respiratory distress syndrome (ARDS) remains a major cause of respiratory failure in critically ill patients. Mesenchymal stromal cells (MSCs) are a promising candidate for a cell-based therapy. However, the mechanisms of MSCs' effects in ARDS are not well understood. In this study, we focused on the paracrine effect of MSCs on macrophage polarization and the role of extracellular vesicle (EV)-mediated mitochondrial transfer. OBJECTIVES: To determine the effects of human MSCs on macrophage function in the ARDS environment and to elucidate the mechanisms of these effects. METHODS: Human monocyte-derived macrophages (MDMs) were studied in noncontact coculture with human MSCs when stimulated with LPS or bronchoalveolar lavage fluid (BALF) from patients with ARDS. Murine alveolar macrophages (AMs) were cultured ex vivo with/without human MSC-derived EVs before adoptive transfer to LPS-injured mice. MEASUREMENTS AND MAIN RESULTS: MSCs suppressed cytokine production, increased M2 macrophage marker expression, and augmented phagocytic capacity of human MDMs stimulated with LPS or ARDS BALF. These effects were partially mediated by CD44-expressing EVs. Adoptive transfer of AMs pretreated with MSC-derived EVs reduced inflammation and lung injury in LPS-injured mice. Inhibition of oxidative phosphorylation in MDMs prevented the modulatory effects of MSCs. Generating dysfunctional mitochondria in MSCs using rhodamine 6G pretreatment also abrogated these effects. CONCLUSIONS: In the ARDS environment, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype through EV-mediated mitochondrial transfer. MSC-induced changes in macrophage phenotype critically depend on enhancement of macrophage oxidative phosphorylation. AMs treated with MSC-derived EVs ameliorate lung injury in vivo.

Clustering of Ca2+ transients in interstitial cells of Cajal defines slow wave duration.

Interstitial cells of Cajal (ICC) in the myenteric plexus region (ICC-MY) of the small intestine are pacemakers that generate rhythmic depolarizations known as slow waves. Slow waves depend on activation of Ca2+-activated Cl- channels (ANO1) in ICC, propagate actively within networks of ICC-MY, and conduct to smooth muscle cells where they generate action potentials and phasic contractions. Thus, mechanisms of Ca2+ regulation in ICC are fundamental to the motor patterns of the bowel. Here, we characterize the nature of Ca2+ transients in ICC-MY within intact muscles, using mice expressing a genetically encoded Ca2+ sensor, GCaMP3, in ICC. Ca2+ transients in ICC-MY display a complex firing pattern caused by localized Ca2+ release events arising from multiple sites in cell somata and processes. Ca2+ transients are clustered within the time course of slow waves but fire asynchronously during these clusters. The durations of Ca2+ transient clusters (CTCs) correspond to slow wave durations (plateau phase). Simultaneous imaging and intracellular electrical recordings revealed that the upstroke depolarization of slow waves precedes clusters of Ca2+ transients. Summation of CTCs results in relatively uniform Ca2+ responses from one slow wave to another. These Ca2+ transients are caused by Ca2+ release from intracellular stores and depend on ryanodine receptors as well as amplification from IP3 receptors. Reduced extracellular Ca2+ concentrations and T-type Ca2+ channel blockers decreased the number of firing sites and firing probability of Ca2+ transients. In summary, the fundamental electrical events of small intestinal muscles generated by ICC-MY depend on asynchronous firing of Ca2+ transients from multiple intracellular release sites. These events are organized into clusters by Ca2+ influx through T-type Ca2+ channels to sustain activation of ANO1 channels and generate the plateau phase of slow waves.

Unraveling the early steps of prokaryotic replication.

In prokaryotes, many of the physical mechanisms governing the process of initiating DNA replication are now emerging. For example, certain organizational features of origins, such as the use of repetitive sequence elements for initiator-binding sites, are found throughout bacteria and many archaea. Common themes in the regulation of initiation, including origin sequestration by trans-acting factors, titration of initiator levels by cis- and trans-acting factors, and control of initiator function by ATP, also exist. Recent studies have shown that prokaryotic initiator structures are both modular and conserved, and have begun to reveal how these proteins specifically recognize target DNA sequences. These properties probably control initiator self-assembly and DNA remodeling to properly fire replication origins.

The folding landscape of Streptomyces griseus protease B reveals the energetic costs and benefits associated with evolving kinetic stability.

Like most extracellular bacterial proteases, Streptomyces griseus protease B (SGPB) and alpha-lytic protease (alphaLP) are synthesized with covalently attached pro regions necessary for their folding. In this article, we characterize the folding free energy landscape of SGPB and compare it to the folding landscapes of alphaLP and trypsin, a mammalian homolog that folds independently of its zymogen peptide. In contrast to the thermodynamically stable native state of trypsin, SGPB and alphaLP fold to native states that are thermodynamically marginally stable or unstable, respectively. Instead, their apparent stability arises kinetically, from unfolding free energy barriers that are both large and highly cooperative. The unique unfolding transitions of SGPB and alphaLP extend their functional lifetimes under highly degradatory conditions beyond that seen for trypsin; however, the penalty for evolving kinetic stability is remarkably large in that each factor of 2.4-8 in protease resistance is accompanied by a cost of ~10(5) in the spontaneous folding rate and ~5-9 kcal/mole in thermodynamic stability. These penalties have been overcome by the coevolution of increasingly effective pro regions to facilitate folding. Despite these costs, kinetic stability appears to be a potent mechanism for developing native-state properties that maximize protease longevity.

Disabling the folding catalyst is the last critical step in alpha-lytic protease folding.

Alpha-Lytic protease (alphaLP) is an extracellular bacterial pro-protease marked by extraordinary conformational rigidity and a highly cooperative barrier to unfolding. Although these properties successfully limit its proteolytic destruction, thereby extending the functional lifetime of the protease, they come at the expense of foldability (t(1/2) = 1800 yr) and thermodynamic stability (native alphaLP is less stable than the unfolded species). Efficient folding has required the coevolution of a large N-terminal pro region (Pro) that rapidly catalyzes alphaLP folding (t(1/2) = 23 sec) and shifts the thermodynamic equilibrium in favor of folded protease through tight native-state binding. Release of active alphaLP from this stabilizing, but strongly inhibitory, complex requires the proteolytic destruction of Pro. alphaLP is capable of initiating Pro degradation via cleavage of a flexible loop within the Pro C-terminal domain. This single cleavage event abolishes Pro catalysis while maintaining strong native-state binding. Thus, the loop acts as an Achilles' heel by which the Pro foldase machinery can be safely dismantled, preventing Pro-catalyzed unfolding, without compromising alphaLP native-state stability. Once the loop is cleaved, Pro is rapidly degraded, releasing active alphaLP.

Interdependent folding of the N- and C-terminal domains defines the cooperative folding of alpha-lytic protease.

Alpha-lytic protease (alphaLP) serves as an important model in achieving a quantitative and physical understanding of protein folding reactions. Synthesized as a pro-protease, alphaLP belongs to an interesting class of proteins that require pro regions to facilitate their proper folding. alphaLP's pro region (Pro) acts as a potent folding catalyst for the protease, accelerating alphaLP folding to its native conformation nearly 10(10)-fold. Structural and mutational studies suggested that Pro's considerable foldase activity is directed toward structuring the alphaLP C-terminal domain (CalphaLP), a seemingly folding-impaired domain, which is believed to contribute significantly to the high-energy folding and unfolding transition states of alphaLP. Pro-mediated nucleation of alphaLP folding within CalphaLP was hypothesized to subsequently enable the alphaLP N-terminal domain (NalphaLP) to dock and fold, completing the formation of native protease. In this paper, we find that ternary folding reactions of Pro and noncovalent NalphaLP and CalphaLP domains are unaffected by the order in which the components are added or by the relative concentrations of the alphaLP domains, indicating that neither discrete CalphaLP structuring nor docking of the two alphaLP domains is involved in the folding transition state. Instead, the rate-limiting step of these folding reactions appears to be a slow and concerted rearrangement of the NalphaLP and CalphaLP domains to form active protease. This cooperative and interdependent folding of both protease domains defines the large alphaLP folding barrier and is an apparent extension of the highly cooperative alphaLP unfolding transition that imparts the protease with remarkable kinetic stability and functional longevity.

Detection of hypoxia in human brain tumor xenografts using a modified comet assay.

We used the standard comet assay successfully to generate in vitro dose-response curves under oxic and hypoxic conditions. We then made mixtures of cells that had been irradiated with 3 and 9 Gy of X-rays to simulate two subpopulations in a tumor, but efforts to accurately detect and quantify the subpopulations using the standard comet assay were unsuccessful. Therefore, we investigated a modified comet assay to determine whether it could be used for measuring hypoxia in our model systems. U251 MG cells were grown as subcutaneous tumors in athymic mice; U251 MG and U87 MG cells were grown as intracerebral (i.c.) tumors in athymic rats. Animals were injected with RSU 1069, irradiated, and euthanized. Tumors and normal brains were removed, and the cells were analyzed using a modified comet assay. Differences in comet tail moment distributions between tumor and contralateral normal brain, using tail moments at either the 25th or 50th percentile in each distribution, were taken as measures of the degree of tumor hypoxia. For U251 MG tumors, there was a positive relationship between tumor size and the degree of hypoxia, whereas preliminary data from U87 MG i.c. tumors showed less hypoxia and no apparent relationship between tumor size and hypoxia.

The pro region N-terminal domain provides specific interactions required for catalysis of alpha-lytic protease folding.

The extracellular bacterial protease, alpha-lytic protease (alphaLP), is synthesized with a large, two-domain pro region (Pro) that catalyzes the folding of the protease to its native conformation. In the absence of its Pro folding catalyst, alphaLP encounters a very large folding barrier (DeltaG = 30 kcal mol(-1)) that effectively prevents the protease from folding (t(1/2) of folding = 1800 years). Although homology data, mutational studies, and structural analysis of the Pro.alphaLP complex suggested that the Pro C-terminal domain (Pro C-domain) serves as the minimum "foldase" unit responsible for folding catalysis, we find that the Pro N-terminal domain (Pro N-domain) is absolutely required for alphaLP folding. Detailed kinetic analysis of Pro N-domain point mutants and a complete N-domain deletion reveal that the Pro N-domain both provides direct interactions with alphaLP that stabilize the folding transition state and confers stability to the Pro C-domain. The Pro N- and C-domains make conflicting demands upon native alphaLP binding that are alleviated in the optimized interface of the folding transition state complex. From these studies, it appears that the extremely high alphaLP folding barrier necessitates the presence of both the Pro domains; however, alphaLP homologues with less demanding folding barriers may not require both domains, thus possibly explaining the wide variation in the pro region size of related pro-proteases.