Mechanistic Evaluation of Bioorthogonal Decaging with trans-Cyclooctene: The Effect of Fluorine Substituents on Aryl Azide Reactivity and Decaging from the 1,2,3-Triazoline.

Bioorthogonal prodrug activation/decaging strategies need to be selective, rapid and release the drug from the masking group upon activation. The rates of the 1,3-dipolar cycloaddition between a trans-cyclooctene (TCO) and a series of fluorine-substituted azido-PABC self-immolative spacers caging two model drugs, and subsequent release from the 1,2,3-triazoline are reported. As the number of fluorine substituents on the PABC linker increases from one to four, the rate of cycloaddition increases by almost one order of magnitude. Using a combination of fluorescence, 1H/19F NMR, and computational experiments, we have been able to determine how substituents on the PABC ring can influence the degradation rates and also the product distribution of the 1,2,3-triazoline. We have also been able to determine how these substituents influence the rate of imine hydrolysis and 1,6-self-immolation decaging rates of the generated anilines. The NMR and computational studies demonstrate that fluorine substituents on the aromatic ring lower the transition state energy required for converting the triazoline to the imine or aziridine intermediates via extrusion of diatomic nitrogen, and that in the case of a tetrafluoro substituted aromatic ring, it is the imine hydrolysis and 1,6-self-immolation that is rate-limiting. This knowledge further enhances the understanding of factors which influence the stability of triazolines, and enables potential applications of fluorinated aromatics, in particular, perfluorinated aromatics, in synthetic chemistry and sustained-release drug delivery systems.

Intra- and Inter-rater Reliability of Navigated Ultrasound in the Assessment of Pelvic Tilt in Symptom-Free Young Adults.

OBJECTIVES: Pelvic tilt is the angle between the anterior pelvic plane and the coronal plane. It affects cup positioning in total hip arthroplasty. The primary objective of this study was to test the intra- and inter-rater reliability of a navigated smart device-based ultrasound system for pelvic tilt assessment. The secondary objective was to test the inter-rater variability of the measurements on a hip phantom. METHODS: A repeated-measures design was used. Two raters measured the pelvic tilt of 12 symptom-free young adults in upright and supine positions. Additionally, pelvic tilt was measured on a hip phantom. Each rater performed 3 measurements in each body position on the participants and 12 measurements on the hip phantom. Intra- and inter-rater reliability were calculated with the use of intraclass correlation coefficients. The variability in measurements on the hip phantom was assessed by a Bland-Altman analysis of agreement. RESULTS: Intraclass correlation coefficient 95% confidence intervals for intra-rater reliability ranged from good to excellent and moderate to excellent for the supine and upright positions respectively. Intraclass correlation coefficient 95% confidence intervals for inter-rater reliability ranged from poor to excellent for both positions. Hip phantom measurements showed no significant average bias (P > .05) and no significant proportional bias (P > .05). The 95% inter-rater limits of agreement were ±1.3° and ±1.7° for the supine and upright positions, respectively. CONCLUSIONS: The intra-rater reliability values achieved were suitable. Intraclass correlation coefficient values for inter-rater reliability remained below an acceptable level. Possible reasons and overcoming strategies were presented. The 95% limits of agreement were good, at less than ±2°.

Oriented Cell Division in the C. elegans Embryo Is Coordinated by G-Protein Signaling Dependent on the Adhesion GPCR LAT-1.

Orientation of spindles and cell division planes during development of many species ensures that correct cell-cell contacts are established, which is vital for proper tissue formation. This is a tightly regulated process involving a complex interplay of various signals. The molecular mechanisms underlying several of these pathways are still incompletely understood. Here, we identify the signaling cascade of the C. elegans latrophilin homolog LAT-1, an essential player in the coordination of anterior-posterior spindle orientation during the fourth round of embryonic cell division. We show that the receptor mediates a G protein-signaling pathway revealing that G-protein signaling in oriented cell division is not solely GPCR-independent. Genetic analyses showed that through the interaction with a Gs protein LAT-1 elevates intracellular cyclic AMP (cAMP) levels in the C. elegans embryo. Stimulation of this G-protein cascade in lat-1 null mutant nematodes is sufficient to orient spindles and cell division planes in the embryo in the correct direction. Finally, we demonstrate that LAT-1 is activated by an intramolecular agonist to trigger this cascade. Our data support a model in which a novel, GPCR-dependent G protein-signaling cascade mediated by LAT-1 controls alignment of cell division planes in an anterior-posterior direction via a metabotropic Gs-protein/adenylyl cyclase pathway by regulating intracellular cAMP levels.

Adhesion GPCRs Govern Polarity of Epithelia and Cell Migration.

In multicellular organisms cells spatially arrange in a highly coordinated manner to form tissues and organs, which is essential for the function of an organism. The component cells and resulting structures are often polarised in one or more axes, and how such polarity is established and maintained correctly has been one of the major biological questions for many decades. Research progress has shown that many adhesion GPCRs (aGPCRs) are involved in several types of polarity. Members of the two evolutionarily oldest groups, Flamingo/Celsr and Latrophilins, are key molecules in planar cell polarity of epithelia or the propagation of cellular polarity in the early embryo, respectively. Other adhesion GPCRs play essential roles in cell migration, indicating that this receptor class includes essential molecules for the control of various levels of cellular organisation.

Surgically modifiable factors measured by computer-navigation together with patient-specific factors predict knee society score after total knee arthroplasty.

BACKGROUND: The purpose was to investigate whether patient-specific factors (PSF) and surgically modifiable factors (SMF), measured by means of a computer-assisted navigation system, can predict the Knee Society Scores (KSS) after total knee arthroplasty (TKA). METHODS: Data from 99 patients collected during a randomized clinical trial were used for this secondary data analysis. The KSS scores of the patients were measured preoperatively and at 4-years follow-up. Multiple regression analyses were performed to investigate which combination of variables would be the best to predict the 4-years KSS scores. RESULTS: When considering SMF alone the combination of four of them significantly predicted the 4-years KSS-F score (p = 0.009), explaining 18 % of its variation. When considering only PSF the combination of age and body weight significantly predicted the 4-years KSS-F (p = 0.008), explaining 11 % of its variation. When considering both groups of predictors simultaneously the combination of three PSF and two SMF significantly predicted the 4-years KSS-F (p = 0.007), explaining 20 % of its variation. CONCLUSIONS: Younger age, better preoperative KSS-F scores and lower BMI before surgery, a positive tibial component slope and small changes in femoral offset were predictors of better KSS-F scores at 4-years.

Patient-specific and intra-operatively modifiable factors assessed by computer navigation predict maximal knee flexion one year after TKA.

PURPOSE: There are multiple factors affecting maximal knee flexion (MKF) after total knee arthroplasty (TKA). The aim of the study was to investigate whether patient-specific factors (PSF) and surgically modifiable factors (SMF), measured by means of a computer-assisted navigation system, can predict the MKF after TKA. METHODS: Data from 99 patients collected during a randomized clinical trial were used for this secondary data analysis. The MKF of the patients was measured preoperatively and 1-year post-surgery. Multiple regression analyses were performed to investigate which combination of variables would be the best to predict the 1-year MKF. RESULTS: When considering SMF alone, the combination of three factors significantly predicted the 1-year MKF (p = 0.001), explaining 22 % of its variation. When considering only PSF, the combination of pre-op MKF and BMI significantly predicted the 1-year MKF (p < 0.001), explaining 23 % of its variation. When considering both groups of potential predictors simultaneously, the combination of five SMF with two PSF significantly predicted the 1-year MKF (p = 0.001), explaining 32 % of its variation. CONCLUSIONS: Computer navigation variables alone could explain 22 % of the variance in the 1-year MKF. The larger proportion (32 %) of the 1-year MKF variation could be explained with a combination of SMF and PSF. The results of studies in this area could be used to identify patients at risk of poor outcomes. LEVEL OF EVIDENCE: Level II, Prognostic study.

The N terminus-only (trans) function of the Adhesion GPCR Latrophilin-1 controls multiple processes in reproduction of C. elegans.

Adhesion G protein-coupled receptors (aGPCR) are unique molecules. They are able to transmit classical signals via G-protein activation as well as mediate functions solely through their extracellular N termini, completely independently of the seven transmembrane helices domain (7TM) and the C terminus. This dual mode of action is highly unusual for GPCRs and allows for a plethora of possible cellular consequences. However, the physiological implications and molecular details of this N terminus-mediated signaling are poorly understood. Here, we show that several distinct 7TM-independent/trans functions of the aGPCR Latrophilin homolog LAT-1 in the nematode Caenorhabditis elegans together regulate reproduction: sperm guidance, ovulation, and germ cell apoptosis. In these contexts, the receptor elicits its functions in a non-cell autonomous manner. The functions might be realized through alternative splicing of the receptor specifically generating N terminus-only variants. Thus, our findings shed light on the versatility of 7TM-independent/N terminus-only/trans functions of aGPCR and discuss possible molecular details.

Deciphering and modulating G protein signalling in C. elegans using the DREADD technology.

G-protein signalling is an evolutionary conserved concept highlighting its fundamental impact on developmental and functional processes. Studies on the effects of G protein signals on tissues as well as an entire organism are often conducted in Caenorhabditis elegans. To understand and control dynamics and kinetics of the processes involved, pharmacological modulation of specific G protein pathways would be advantageous, but is difficult due to a lack in accessibility and regulation. To provide this option, we designed G protein-coupled receptor-based designer receptors (DREADDs) for C. elegans. Initially described in mammalian systems, these modified muscarinic acetylcholine receptors are activated by the inert drug clozapine-N-oxide, but not by their endogenous agonists. We report a novel C. elegans-specific DREADD, functionally expressed and specifically activating Gq-protein signalling in vitro and in vivo which we used for modulating mating behaviour. Therefore, this novel designer receptor demonstrates the possibility to pharmacologically control physiological functions in C. elegans.

Do Well-Balanced Primary TKA Patients Achieve Better Outcomes Within the First Year After Surgery?

Some surgically modifiable factors are related to soft tissue balance. With computer-assisted surgery, it is possible to access these variables quantitatively. The aim of this analysis was to study the influence of gap balance on clinical outcomes within the first year after computer-navigated total knee arthroplasty (TKA). Based on navigation data, 3 independent variables reflecting gap balance were used to split the patients in 2 groups. The Knee Society Scores (Function [KSS-F] and Knee [KSS-K]) and the maximal knee flexion (MKF) measured preoperatively and at 3, 6 and 12 months were compared using analyses of variance (2×4 design) for repeated measures. Higher flexion-extension gap equality led to statistically higher KSS-F and KSS-K scores at 1 year (P=.02). Higher medial-lateral flexion gap equality led to superior mean MKF at all measurement points; however the differences were statistically only significant at 3 months (P=.01). The coefficients of variation of the variables used to select the patients were overall very low. With computer-assisted navigation, it is possible to access quantitatively the size of the medial and lateral flexion and extension gaps. Higher flexion-extension gap equality values led to statistically significant better KSS-F and KSS-K scores at 1 year. Higher medial-lateral flexion gap equality values led to better MKF values; however the differences were only statistically significant at 3 months. [Orthopedics. 2016; 39(3):S6-S12.].

Bioorthogonal prodrug activation driven by a strain-promoted 1,3-dipolar cycloaddition.

Due to the formation of hydrolysis-susceptible adducts, the 1,3-dipolar cycloaddition between an azide and strained trans-cyclooctene (TCO) has been disregarded in the field of bioorthogonal chemistry. We report a method which uses the instability of the adducts to our advantage in a prodrug activation strategy. The reaction of trans-cyclooctenol (TCO-OH) with a model prodrug resulted in a rapid 1,3-dipolar cycloaddition with second-order rates of 0.017 M-1 s-1 and 0.027 M-1 s-1 for the equatorial and axial isomers, respectively, resulting in release of the active compound. 1H NMR studies showed that activation proceeded via a triazoline and imine, both of which are rapidly hydrolyzed to release the model drug. Cytotoxicity of a doxorubicin prodrug was restored in vitro upon activation with TCO-OH, while with cis-cyclooctenol (CCO-OH) no activation was observed. The data also demonstrates the potential of this reaction in organic synthesis as a mild orthogonal protecting group strategy for amino and hydroxyl groups.