Reproducibility of In Vivo Corneal Confocal Microscopy Using an Automated Analysis Program for Detection of Diabetic Sensorimotor Polyneuropathy

Objective

In vivo Corneal Confocal Microscopy (IVCCM) is a validated, non-invasive test for diabetic sensorimotor polyneuropathy (DSP) detection, but its utility is limited by the image analysis time and expertise required. We aimed to determine the inter- and intra-observer reproducibility of a novel automated analysis program compared to manual analysis.

Methods

In a cross-sectional diagnostic study, 20 non-diabetes controls (mean age 41.4±17.3y, HbA1c 5.5±0.4%) and 26 participants with type 1 diabetes (42.8±16.9y, 8.0±1.9%) underwent two separate IVCCM examinations by one observer and a third by an independent observer. Along with nerve density and branch density, corneal nerve fibre length (CNFL) was obtained by manual analysis (CNFL_MANUAL), a protocol in which images were manually selected for automated analysis (CNFL_{SEMI-AUTOMATED}), and one in which selection and analysis were performed electronically (CNFL_{FULLY-AUTOMATED}). Reproducibility of each protocol was determined using intraclass correlation coefficients (ICC) and, as a secondary objective, the method of Bland and Altman was used to explore agreement between protocols.

Results

Mean CNFL_Manual was 16.7±4.0, 13.9±4.2 mm/mm² for non-diabetes controls and diabetes participants, while CNFL_{Semi-Automated} was 10.2±3.3, 8.6±3.0 mm/mm² and CNFL_{Fully-Automated} was 12.5±2.8, 10.9 ± 2.9 mm/mm². Inter-observer ICC and 95% confidence intervals (95%CI) were 0.73(0.56, 0.84), 0.75(0.59, 0.85), and 0.78(0.63, 0.87), respectively (p = NS for all comparisons). Intra-observer ICC and 95%CI were 0.72(0.55, 0.83), 0.74(0.57, 0.85), and 0.84(0.73, 0.91), respectively (p<0.05 for CNFL_{Fully-Automated} compared to others). The other IVCCM parameters had substantially lower ICC compared to those for CNFL. CNFL_{Semi-Automated} and CNFL_{Fully-Automated} underestimated CNFL_Manual by mean and 95%CI of 35.1(-4.5, 67.5)% and 21.0(-21.6, 46.1)%, respectively.

Conclusions

Despite an apparent measurement (underestimation) bias in comparison to the manual strategy of image analysis, fully-automated analysis preserves CNFL reproducibility. Future work must determine the diagnostic thresholds specific to the fully-automated measure of CNFL.     

Potential pathogenic bacteria in metalworking fluids and aerosols from a machining facility

The metalworking and machining industry utilizes recirculating metalworking fluids for integral aspects of the fabrication process. Despite the use of biocides, these fluids sustain substantial biological growth. Subsequently, the high-shear forces incurred during metalworking processing aerosolize bacterial cells and may cause dermatologic and respiratory effects in exposed workers. We quantified and identified the bacterial load for metalworking fluid and aerosol samples of a machining facility in the US Midwest during two seasons. To investigate the presence of potentially pathogenic bacteria in fluid and air, we performed 16S rRNA gene surveys. The concentration of total bacterial cells (including culturable and nonculturable cells) was relatively constant throughout the study, averaging 5.1 × 10? cells mL?1 in the fluids and 4.8 × 10? cells m?3 in the aerosols. We observed bacteria of potential epidemiologic significance from several different bacterial phyla in both fluids and aerosols. Most notably, Alcaligenes faecalis was identified through both direct sequencing and culturing in every sample collected. Elucidating the bacterial community with gene surveys showed that metalworking fluids were the source of the aerosolized bacteria in this facility.     

Spinophilin facilitates dephosphorylation of doublecortin by PP1 to mediate microtubule bundling at the axonal wrist

The axonal shafts of neurons contain bundled microtubules, whereas extending growth cones contain unbundled microtubule filaments, suggesting that localized activation of microtubule-associated proteins (MAP) at the transition zone may bundle these filaments during axonal growth. Dephosphorylation is thought to lead to MAP activation, but specific molecular pathways have remained elusive. We find that Spinophilin, a Protein-phosphatase 1 (PP1) targeting protein, is responsible for the dephosphorylation of the MAP Doublecortin (Dcx) Ser 297 selectively at the "wrist" of growing axons, leading to activation. Loss of activity at the "wrist" is evident as an impaired microtubule cytoskeleton along the shaft. These findings suggest that spatially restricted adaptor-specific MAP reactivation through dephosphorylation is important in organization of the neuronal cytoskeleton.     

Characterisation of recombinant Hevea brasiliensis allene oxide synthase: effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity.

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His(6)-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a K(m) value of 4.02+/-0.64 microM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His(6)-HbAOS enzyme activity. Although JA had no effect on His(6)-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His(6)-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.     

Synthesis and radiolabeling of selective high-affinity ligands designed to target non-Hodgkin's lymphoma and leukemia

Selective high-affinity ligands (SHALs) were synthesized as molecular targeting agents for HLA-DR10, a cell surface receptor upregulated on malignant B-cell lymphocytes in non-Hodgkin's lymphoma and leukemia. SHALs are designed to mimic the affinity and selectivity of Lym-1, an antibody that binds to the beta-subunit of HLA-DR10. To bind selectively to HLA-DR10, SHALs were constructed to bind to two adjacent pockets on the surface of the beta-subunit of HLA-DR10 located within an epitope recognized by the Lym-1 antibody. A series of multivalent SHALs with molecular masses of 1500-3000 Da were synthesized using solid/polymer-supported synthesis on chlorotrityl chloride resin in 50-80% yield. To enable their use as radionuclide carriers in mouse studies, SHALs were conjugated to DOTA in a solution-phase reaction with 70-100% yield. 57Co/CoCl2 titrations revealed that 50-60% of the DOTA in the DOTA-conjugated SHALs was available for radiometal chelation. These DOTA-SHALs were labeled with 111In and used to carry out pharmacokinetic studies in mice. Radiolabeling reactions of DOTA-SHALs, with exactly one DOTA entity per targeting SHAL molecule, yielded products with greater than 90% radiochemical purity and specific activities ranging from 97 to 150 muCi/mug.     

Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.     

A global role for zebrafish klf4 in embryonic erythropoiesis

There are two waves of erythropoiesis, known as primitive and definitive waves in mammals and lower vertebrates including zebrafish. The founding member of the Kruppel-like factor (KLF) family of CACCC-box binding proteins, EKLF/Klf1, is essential for definitive erythropoiesis in mammals but only plays a minor role in primitive erythropoiesis. Morpholino knockdown experiments have shown a role for zebrafish klf4 in primitive erythropoiesis and hatching gland formation. In order to generate a global understanding of how klf4 might influence gene expression and differentiation, we have performed expression profiling of klf4 morphants, and then performed validation of many putative target genes by qRT-PCR and whole mount in situ hybridization. We found a critical role for klf4 in embryonic globin, heme synthesis and hatching gland gene expression. In contrast, there was an increase in expression of definitive hematopoietic specific genes such as larval globin genes, runx1 and c-myb from 24 hpf, suggesting a selective role for klf4 in primitive rather than definitive erythropoiesis. In addition, we show klf4 preferentially binds CACCC box elements in the primitive zebrafish beta-like globin gene promoters. These results have global implications for primitive erythroid gene regulation by KLF-CACCC box interactions.     

Bordetella pertussis adenylate cyclase toxin (ACT) induces cyclooxygenase-2 (COX-2) in murine macrophages and is facilitated by ACT interaction with CD11b/CD18 (Mac-1)

Bordetella pertussis causes a profound inflammatory response in lungs of infected individuals. The adenylate cyclase toxin (ACT) of B. pertussis is a potent enzyme that converts cytosolic ATP into cAMP, and is required for virulence in vivo. During infection, secreted ACT binds to macrophages utilizing the beta2 integrin, Mac-1 (CR3, CD11b/CD18), and subsequent intoxication by ACT inhibits essential antibacterial activities of macrophages. Additionally, Mac-1 has been reported to be a co-receptor for TLR4 required for the full induction of some LPS-responsive genes, including pro-inflammatory cyclooxygenase 2 (COX-2). We have examined the effect of ACT on COX-2 expression in HEK293T cells expressing Mac-1 and in murine macrophages. We report that ACT induces COX-2 in a manner that absolutely requires the catalytic activity of this enzyme and Mac-1 expression dramatically enhanced the sensitivity of cells to ACT-dependent COX-2 induction. The mechanism of COX-2 induction by ACT utilizes the cAMP-PKA-CREB-dependent pathway. Finally, ACT and TLR2 or TLR4 act synergistically to increase COX-2 expression. These data suggest that ACT contributes significantly to the inflammatory response induced by B. pertussis infection by augmenting COX-2 expression and provides evidence against the concept that ACT functions exclusively via its inhibitory effects on phagocytic leucocytes.     

Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis

In addition to their role in cellular bioenergetics, mitochondria also initiate common forms of programmed cell death (apoptosis) through the release of proteins such as cytochrome c from the intermembrane and intracristal spaces. The release of these proteins is studied in populations of cells by western blotting mitochondrial and cytoplasmic fractions of cellular extracts, and in single cells by fluorescence microscopy using fluorescent indicators and fusion proteins. However, studying the changes in ultrastructure associated with release of proteins requires the higher resolution provided by transmission electron microscopy. Here, we have used fluorescence microscopy to characterize the state of apoptosis in HeLa cells treated with etoposide followed by electron microscopy and three-dimensional electron microscope tomography of the identical cells to study the sequence of structural changes. We have identified a remodelling of the inner mitochondrial membrane into many separate vesicular matrix compartments that accompanies release of proteins; however, this remodelling is not required for efficient release of cytochrome c. Swelling occurs only late in apoptosis after release of cytochrome c and loss of the mitochondrial membrane potential.