Post‐translational modifications of transthyretin affect the triiodonine‐binding potential

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys₁₀), which is frequently affected by oxidative post‐translational modifications. As Cys₁₀ is part of the thyroid hormone‐binding channel within the TTR molecule, PTM of Cys₁₀ may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys₁₀ modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype‐ and site‐specific manner with S‐glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function‐specific patterns of TTR with a substantial decrease in S‐sulphonated, S‐cysteinylglycinated and S‐glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys₁₀ seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.     

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.     

Enhanced Amphiphilic Profile of a Short β-Stranded Peptide Improves Its Antimicrobial Activity

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH₂, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH₂, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state ¹⁹F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by ³¹P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands.     

Spectral Properties of Single Gold Nanoparticles in Close Proximity to Biological Fluorophores Excited by 2-Photon Excitation

Metallic nanoparticles (NPs) are able to modify the excitation and emission rates (plasmonic enhancement) of fluorescent molecules in their close proximity. In this work, we measured the emission spectra of 20 nm Gold Nanoparticles (AuNPs) fixed on a glass surface submerged in a solution of different fluorophores using a spectral camera and 2-photon excitation. While on the glass surface, we observed the presence in the emission at least 3 components: i) second harmonic signal (SHG), ii) a broad emission from AuNPS and iii) fluorescence arising from fluorophores nearby. When on the glass surface, we found that the 3 spectral components have different relative intensities when the incident direction of linear polarization was changed indicating different physical origins for these components. Then we measured by fluctuation correlation spectroscopy (FCS) the scattering and fluorescence signal of the particles alone and in a solution of 100 nM EGFP using the spectral camera or measuring the scattering and fluorescence from the particles. We observed occasional fluorescence bursts when in the suspension we added fluorescent proteins. The spectrum of these burst was devoid of the SHG and of the broad emission in contrast to the signal collected from the gold nanoparticles on the glass surface. Instead we found that the spectrum during the burst corresponded closely to the spectrum of the fluorescent protein. An additional control was obtained by measuring the cross-correlation between the reflection from the particles and the fluorescence arising from EGFP both excited at 488 nm. We found a very weak cross-correlation between the AuNPs and the fluorescence confirming that the burst originate from a few particles with a fluorescence signal.     

Alterations in Peripheral and Central Components of the Auditory Brainstem Response: A Neural Assay of Tinnitus

Chronic tinnitus, or “ringing of the ears”, affects upwards of 15% of the adult population. Identifying a cost-effective and objective measure of tinnitus is needed due to legal concerns and disability issues, as well as for facilitating the effort to assess neural biomarkers. We developed a modified gap-in-noise (GIN) paradigm to assess tinnitus in mice using the auditory brainstem response (ABR). We then compared the commonly used acoustic startle reflex gap-prepulse inhibition (gap-PPI) and the ABR GIN paradigm in young adult CBA/CaJ mice before and after administrating sodium salicylate (SS), which is known to reliably induce a 16 kHz tinnitus percept in rodents. Post-SS, gap-PPI was significantly reduced at 12 and 16 kHz, consistent with previous studies demonstrating a tinnitus-induced gap-PPI reduction in this frequency range. ABR audiograms indicated thresholds were significantly elevated post-SS, also consistent with previous studies. There was a significant increase in the peak 2 (P2) to peak 1 (P1) and peak 4 (P4) to P1 amplitude ratios in the mid-frequency range, along with decreased latency of P4 at higher intensities. For the ABR GIN, peak amplitudes of the response to the second noise burst were calculated as a percentage of the first noise burst response amplitudes to quantify neural gap processing. A significant decrease in this ratio (i.e. recovery) was seen only at 16 kHz for P1, indicating the presence of tinnitus near this frequency. Thus, this study demonstrates that GIN ABRs can be used as an efficient, non-invasive, and objective method of identifying the approximate pitch and presence of tinnitus in a mouse model. This technique has the potential for application in human subjects and also indicates significant, albeit different, deficits in temporal processing in peripheral and brainstem circuits following drug induced tinnitus.     

Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology

Background

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the “gold standard” for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the “CD4% Count Kit-Dry”.

Materials and Methods

Venous blood from 59 adult HIV+ patients (age: 25–58 years; 43 males and 16 females) was collected and stained with the “MiniPOC CD4% Count Kit-Dry”. CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 (“Cytostat tetrachrome kit” for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count).

Results

The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ±5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis.

Conclusion

The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems.     

Some Like It Fat: Comparative Ultrastructure of the Embryo in Two Demosponges of the Genus Mycale (Order Poecilosclerida) from Antarctica and the Caribbean

During embryogenesis, organisms with lecithotrophic indirect development usually accumulate large quantities of energetic reserves in the form of yolk that are necessary for larval survival. Since all sponges have lecithotrophic development, yolk formation is an ineludible step of their embryogenesis. Sponge yolk platelets have a wide range of morphological forms, from entirely lipid or protein platelets to a combined platelet showing both lipids and proteins and even glycogen. So far, there are no comparative studies on the nature and content of yolk in congeneric species of sponges inhabiting contrasting environments, which could have putative effects on the larval adaptation to environmental conditions. Here, we have taken advantage of the worldwide distribution of the sponge genus Mycale, in order to compare the embryogenesis and yolk formation in two species inhabiting contrasting latitudinal areas: M. acerata from Antarctic waters and M. laevis from the Caribbean. We have compared their brooded embryos and larvae using scanning and transmission electron microscopy, and calculated their energetic signatures based on the nature of their yolk. While the general morphological feature of embryos and larvae of both species were very similar, the main difference resided in the yolk nature. The Antarctic species, M. acerata, showed exclusively lipid yolk, whereas the Caribbean species, M. laevis, showed combined platelets of lipids and proteins and less frequently protein yolk platelets. The larvae of M. acerata were estimated to possess a two-fold energetic signature compared to that of M. laevis, which may have important ecological implications for their survival and for maintaining large population densities in the cold waters of the Southern Ocean.