MRI imaging versus histologic volumetric estimation of residual injection laryngoplasty material.

Objectives: To examine the degree of agreement between MRI and histologically generated volumetric measurements of residual injection laryngoplasty material. Methods: Following left recurrent laryngeal nerve transection, rabbit vocal cords were injected with jellyfish collagen, Cymetra®, or Restylane®. Laryngeal tissue was harvested 4 or 12 weeks post injection followed by MRI imaging and histologic cross-sectioning. Two raters estimated the volume of remaining injection material in specimens within MRI and histologic axial cross sections. Wilcoxon signed rank tests were employed to detect gross differences between inter-rater measurements and between imaging modalities across time. Agreement between rater measurements and imaging (histology and MRI) was assessed using intra-class correlation coefficients. Results: Data was available from 16 rabbits sacrificed at 4 weeks (n = 8) and 12 weeks (n = 8). Inter-rater testing of MRI imaging revealed no significant differences (p > .05) between rater measurements across time points, and excellent agreement (0.93; 95% confidence interval 0.80-0.98) while histologically estimated volumes demonstrated a significant difference at 4 weeks (p < .05) and overall good agreement (0.89; 95% confidence interval 0.59-0.97). Comparison of MRI and histologically estimated volume measurements revealed significant differences at the 4-week time point (p < .05) but not at 12 weeks (p > .05). Overall, there is only moderate agreement between MRI and histology estimates (0.72; 95% confidence interval 0.22-0.90). Conclusions: MRI imaging demonstrates good reliability and similar estimates of volume to histologically estimated measurements of residual injection laryngoplasty material at time points clinically relevant for future injection laryngoplasty experiments. Level of Evidence: NA.

MRI Study of Jellyfish Collagen, Hyaluronic Acid, and Cadaveric Dermis for Injection Laryngoplasty.

OBJECTIVES/HYPOTHESIS: Test a new jellyfish collagen biomaterial aimed to increase duration of injection medialization laryngoplasty (IL) against two products in clinical practice. STUDY DESIGN: Animal model. METHODS: Left recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received micronized cross-linked jellyfish collagen (MX-JC) and adipose derived stem cells (ADSCs), Group 2, MX-JC alone, Group 3, cross-linked hyaluronic acid (X-HA), and Group 4, micronized acellular dermis (MACD). Animals were sacrificed at 4 and 12 weeks. Major outcomes were MRI tissue volumes and histopathology. RESULTS: After 100 µL IL MRI volumes (means ± STD) at 4 and 12 weeks were: Group 1: 27.2 ± 15.6 and 13.1 ± 5.2 µL, Group 2: 60.8 ± 18 and 27.8 ± 2.47 µL, Group 3: 27.4 ± 12 and 10.6 ± 8 µL, and Group 4: 37.5 ± 11 and 9.85 ± 1 µL. Group 2 volumes were largest and Group 3 were smallest in all comparisons (P < .05). Histologically, low grade inflammatory responses were observed in Group 1, mild histiocytic infiltration in Group 2, widespread muscle fiber loss in Group 3, and plasmocytic infiltration in Group 4. CONCLUSIONS: MX-JC showed the least resorption at 4 and 12 weeks among all groups. T cell inflammatory responses were observed with MX-JC but were reduced by 12 weeks while B cell immune responses, indicative of antibody priming, were predominantly noted with MACD. MX-JC + ADSC showed low grade immunity while the XHA showed greater myocyte loss compared to the other groups. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2452-E2460, 2021.

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae.

A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di-halogenated substrates.

Marine Rhodobacteraceae L-haloacid dehalogenase contains a novel His/Glu dyad that could activate the catalytic water.

The putative L-haloacid dehalogenase gene (DehRhb) from a marine Rhodobacteraceae family was cloned and overexpressed in Escherichia coli. The DehRhb protein was shown to be an L-haloacid dehalogenase with highest activity towards brominated substrates with short carbon chains (≤ C3). The optimal temperature for enzyme activity was 55 °C, and the Vmax and Km were 1.75 µm·min(-1) ·mg(-1) of protein and 6.72 mm, respectively, when using monobromoacetic acid as a substrate. DehRhb showed moderate thermal stability, with a melting temperature of 67 °C. The enzyme demonstrated high tolerance to solvents, as shown by thermal shift experiments and solvent incubation assays. The DehRhb protein was crystallized and structures of the native, reaction intermediate and substrate-bound forms were determined. The active site of DehRhb had significant differences from previously studied L-haloacid dehalogenases. The asparagine and arginine residues shown to be essential for catalytic activity in other L-haloacid dehalogenases are not present in DehRhb. The histidine residue which replaces the asparagine residue in DehRhb was coordinated by a conformationally strained glutamate residue that replaces a conserved glycine. The His/Glu dyad is positioned for deprotonation of the catalytic water which attacks the ester bond in the reaction intermediate. The catalytic water in DehRhb is shifted by ~ 1.5 Å from its position in other L-haloacid dehalogenases. A similar His/Glu or Asp dyad is known to activate the catalytic water in haloalkane dehalogenases. The DehRhb enzyme represents a novel member within the L-haloacid dehalogenase family and it has potential to be used as a commercial biocatalyst.

Development of a high-throughput cell-based assay for identification of IL-17 inhibitors.

Human interleukin 17 (IL-17) is a proinflammatory cytokine derived mainly from activated T cells. Extensive evidence points to a significant role of IL-17 in many autoimmune and infectious diseases, as well as tumorigenesis and transplant rejection, and suggests that targeting IL-17 could be a promising therapeutic strategy. Robust cell-based assays would thus be essential for lead identification and the optimization of therapeutic candidates. Herein, we report a well-characterized two-step assay, consisting of (a) in vitro activation and stimulation of CD4(+) T lymphocytes by a defined complex of antibodies and cytokines, leading to T helper 17 (Th17) cell differentiation and IL-17 production, and (b) IL-17 quantification in cell supernatants using a homogeneous time-resolved fluorescence (HTRF) assay. The system was optimized for and shown to be reliable in high-throughput compatible 96- and 384-well plate formats. The assay is robust (Z' > 0.5) and simple to perform, yields a stable response, and allows for sufficient discrimination of positive (IL-17-producing cells) and negative controls (uninduced cells). The assay was validated by performing dose-response testing of rapamycin and cyclosporine A, which had previously been reported to inhibit IL-17, and determining, for the first time, their in vitro potencies (IC(50)s of 80 ± 23 pM and 223 ± 52 nM, respectively). Also, IKK 16, a selective small-molecule inhibitor of IκB kinase, was found to inhibit IL-17 production, with an IC(50) of 315 ± 79 nM.

Novel anti-infective compounds from marine bacteria.

As a result of the continuous evolution of microbial pathogens towards antibiotic-resistance, there have been demands for the development of new and effective antimicrobial compounds. Since the 1960s, the scientific literature has accumulated many publications about novel pharmaceutical compounds produced by a diverse range of marine bacteria. Indeed, marine micro-organisms continue to be a productive and successful focus for natural products research, with many newly isolated compounds possessing potentially valuable pharmacological activities. In this regard, the marine environment will undoubtedly prove to be an increasingly important source of novel antimicrobial metabolites, and selective or targeted approaches are already enabling the recovery of a significant number of antibiotic-producing micro-organisms. The aim of this review is to consider advances made in the discovery of new secondary metabolites derived from marine bacteria, and in particular those effective against the so called "superbugs", including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), which are largely responsible for the increase in numbers of hospital acquired, i.e., nosocomial, infections.