Kidney Stones in Primary Hyperoxaluria: New Lessons Learnt

To investigate potential differences in stone composition with regard to the type of Primary Hyperoxaluria (PH), and in relation to the patient’s medical therapy (treatment naïve patients versus those on preventive medication) we examined twelve kidney stones from ten PH I and six stones from four PH III patients. Unfortunately, no PH II stones were available for analysis. The study on this set of stones indicates a more diverse composition of PH stones than previously reported and a potential dynamic response of morphology and composition of calculi to treatment with crystallization inhibitors (citrate, magnesium) in PH I. Stones formed by PH I patients under treatment are more compact and consist predominantly of calcium-oxalate monohydrate (COM, whewellite), while calcium-oxalate dihydrate (COD, weddellite) is only rarely present. In contrast, the single stone available from a treatment naïve PH I patient as well as stones from PH III patients prior to and under treatment with alkali citrate contained a wide size range of aggregated COD crystals. No significant effects of the treatment were noted in PH III stones. In disagreement with findings from previous studies, stones from patients with primary hyperoxaluria did not exclusively consist of COM. Progressive replacement of COD by small COM crystals could be caused by prolonged stone growth and residence times in the urinary tract, eventually resulting in complete replacement of calcium-oxalate dihydrate by the monohydrate form. The noted difference to the naïve PH I stone may reflect a reduced growth rate in response to treatment. This pilot study highlights the importance of detailed stone diagnostics and could be of therapeutic relevance in calcium-oxalates urolithiasis, provided that the effects of treatment can be reproduced in subsequent larger studies.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.     

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C₅ acyl side chain of the C₂₄ steroid compound cholate involves the C₂₂ intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C₂₄ steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD⁺ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.     

Subviral Dense Bodies of Human Cytomegalovirus Stimulate Maturation and Activation of Monocyte-Derived Immature Dendritic Cells

Dendritic cells play a central role in the immune control of human cytomegalovirus (HCMV) infection. This work aimed at investigating the impact of noninfectious, subviral dense bodies of HCMV on the maturation and activation of dendritic cells (DC). Treatment of immature DC with dense bodies led to the maturation of these cells and significantly increased their capacity for cytokine release and antigen presentation. Dense body-activated DC may thereby contribute to the development of antiviral immunity.     

Unique wing scale photonics of male Rajah Brooke’s birdwing butterflies

Background

Ultrastructures in butterfly wing scales can take many shapes, resulting in the often striking coloration of many butterflies due to interference of light. The plethora of coloration mechanisms is dazzling, but often only single mechanisms are described for specific animals.

Results

We have here investigated the male Rajah Brooke’s birdwing, Trogonoptera brookiana, a large butterfly from Malaysia, which is marked by striking, colorful wing patterns. The dorsal side is decorated with large, iridescent green patterning, while the ventral side of the wings is primarily brown-black with small white, blue and green patches on the hindwings. Dense arrays of red hairs, creating a distinct collar as well as contrasting areas ventrally around the thorax, enhance the butterfly’s beauty. The remarkable coloration is realized by a diverse number of intricate and complicated nanostructures in the hairs as well as the wing scales. The red collar hairs contain a broad-band absorbing pigment as well as UV-reflecting multilayers resembling the photonic structures of Morpho butterflies; the white wing patches consist of scales with prominent thin film reflectors; the blue patches have scales with ridge multilayers and these scales also have centrally concentrated melanin. The green wing areas consist of strongly curved scales, which possess a uniquely arranged photonic structure consisting of multilayers and melanin baffles that produces highly directional reflections.

Conclusion

Rajah Brooke’s birdwing employs a variety of structural and pigmentary coloration mechanisms to achieve its stunning optical appearance. The intriguing usage of order and disorder in related photonic structures in the butterfly wing scales may inspire novel optical materials as well as investigations into the development of these nanostructures in vivo.

     

The long periodicity phase (LPP) controversy part I: The influence of a natural-like ratio of the CER[EOS] analogue [EOS]-br in a CER[NP]/[AP] based stratum corneum modelling system: A neutron diffraction study

This study used neutron diffraction to investigate a ceramide-[NP] C24/[AP] C24 /[EOS]-br C30/cholesterol/lignoceric acid (0.6: 0.3: 0.1: 0.7: 1) based stratum corneum modelling system. By adding specifically deuterated ceramides-[NP]-D₃, [AP]-D₃, and [EOS]-br-D₃, detailed information on the lamellar and the nanostructure of the system was obtained. For the short periodicity phase a natural-like lamellar repeat distance of 5.47?±?0.02?nm was observed, similar to the [NP]/[AP] base system without the [EOS]-br. Unlike in this system the ceramides here were slightly tilted, hinting towards a slightly less natural arrangement. Due to the deuteration it was possible to observe that the long ceramide chains were overlapping in the lamellar mid-plane. This is considered to be an important feature for the natural stratum corneum. Despite the presence of a ceramide [EOS] analogue – able to form a long phase arrangement – no distinct long periodicity phase was formed, despite a slightly higher than natural ω-acyl ceramide ratio of 10?mol%. The deuterated variant of this ceramide determined that the very long ceramide was integrated into the short periodicity phase, spanning multiple layers instead. The – compared to the base system – unchanged repeat distance highlights the stability of this structure. Furthermore, the localisation of the very long ceramide in the short periodicity phase indicates the possibility of a crosslinking effect and thus a multilayer stabilizing role for the ceramide [EOS]. It can be concluded, that additionally to the mere presence of ceramide-[EOS] more complex conditions have to be met in order to form this long phase. This has to be further investigated in the future.