Large-scale, in-cell photocrosslinking at single-residue resolution reveals the molecular basis for glucocorticoid receptor regulation by immunophilins.

The Hsp90 co-chaperones FKBP51 and FKBP52 play key roles in steroid-hormone-receptor regulation, stress-related disorders, and sexual embryonic development. As a prominent target, glucocorticoid receptor (GR) signaling is repressed by FKBP51 and potentiated by FKBP52, but the underlying molecular mechanisms remain poorly understood. Here we present the architecture and functional annotation of FKBP51-, FKBP52-, and p23-containing Hsp90-apo-GR pre-activation complexes, trapped by systematic incorporation of photoreactive amino acids inside human cells. The identified crosslinking sites clustered in characteristic patterns, depended on Hsp90, and were disrupted by GR activation. GR binding to the FKBPFK1, but not the FKBPFK2, domain was modulated by FKBP ligands, explaining the lack of GR derepression by certain classes of FKBP ligands. Our findings show how FKBPs differentially interact with apo-GR, help to explain the differentiated pharmacology of FKBP51 ligands, and provide a structural basis for the development of improved FKBP ligands.

Structural and biochemical insights into FKBP51 as a Hsp90 co-chaperone.

The FK506-binding protein 51 (FKBP51) is a high-molecular-weight immunophilin that emerged as an important drug target for stress-related disorders, chronic pain, and obesity. It has been implicated in a plethora of molecular pathways but remains best characterized as a co-chaperone of Hsp90 in the steroid hormone receptor (SHR) maturation cycle. However, the mechanistic and structural basis for the regulation of SHRs by FKBP51 and the usually antagonistic function compared with its closest homolog FKBP52 remains enigmatic. Here we review recent structural and biochemical studies of FKBPs as regulators in the Hsp90 machinery. These advances provide important insights into the roles of FKBP51 and FKBP52 in SHR regulation.

Differences in Immunogenicity of Three Different Homo- and Heterologous Vaccination Regimens against SARS-CoV-2.

Background: Due to findings on adverse reactions and clinical efficacy of different vaccinations against SARS-CoV-2, the administration of vaccination regimens containing both adenoviral vector vaccines and mRNA-based vaccines has become common. Data are still needed on the direct comparison of immunogenicity for these different regimens. Methods: We compared markers for immunogenicity (anti-S1 IgG/IgA, neutralizing antibodies, and T-cell response) with three different vaccination regimens (homologous ChAdOx1 nCoV-19 (n = 103), or mixture of ChAdOx1 nCoV-19 with mRNA-1273 (n = 116) or BNT162b2 (n = 105)) at two time points: the day of the second vaccination as a baseline and 14 days later. Results: All examined vaccination regimens elicited measurable immune responses that were significantly enhanced after the second dose. Homologous ChAdOx1 nCoV-19 was markedly inferior in immunogenicity to all other examined regimens after administration of the second dose. Between the heterologous regimens, mRNA-1273 as second dose induced greater antibody responses than BNT162b2, with no difference found for neutralizing antibodies and T-cell response. Discussion: While these findings allow no prediction about clinical protection, from an immunological point of view, vaccination against SARS-CoV-2 with an mRNA-based vaccine at one or both time points appears preferable to homologous vaccination with ChAdOx1 nCoV-19. Whether or not the demonstrated differences between the heterologous regimens are of clinical significance will be subject to further research.

B-cell responses to vaccination with BNT162b2 and mRNA-1273 6 months after second dose.

OBJECTIVES: To examine the state of B-cell immunity 6 months after the second vaccination against SARS-CoV-2 in comparison to the state observed 2 weeks after vaccination. METHODS: Sera of 439 participants, whose immune responses to two doses of an mRNA-based vaccine (BNT162b2 or mRNA-1273) were previously characterized, was examined for anti-S1 IgG and IgA, anti-NCP IgG and neutralizing antibodies (nAb), and antinuclear antibodies (ANA). RESULTS: Levels of all examined markers decreased significantly from 2 weeks to 6 months after second vaccination (anti-S1 IgG: 3744 ± 2571.4 vs. 253 ± 144 binding antibody units (BAU)/mL; anti-S1 IgA: 12 ± 0 vs. 1.98 ± 1.75 optical density (OD) ratio; nAb: 100% ± 0% vs. 82% ± 19.3%), the vast majority of participants retaining reactive levels of anti-S1 IgG (436/439) and anti-S1 IgA (334/439) at 6 months. Immune responses were stronger for mRNA-1273 compared with BNT162b2 (anti-S1 IgG: 429 ± 289 vs. 243 ± 143 BAU/mL; anti-S1 IgA: 5.38 ± 3.91 vs. 1.89 ± 1.53 OD ratio; nAb: 90.5% ± 12.6% vs. 81% ± 19.3%). There was no meaningful influence of sex and age on the examined markers. There was a strong correlation between anti-S1 IgG and the surrogate neutralization assay (rho = 0.91, p <0.0001), but not for for IgA and the surrogate neutralization assay (rho = 0.52, p <0.0001). There was a ceiling effect for the association between anti-S1 IgG titres and the inhibition of binding between S1 and ACE2. ANA prevalence was unchanged from 2 weeks to 6 months after the second vaccination (87/498 vs. 77/435), as were the median ANA titres (1:160 vs. 1:160). DISCUSSION: Although the clinical consequences of decreasing anti-SARS-CoV-2 antibody titres cannot be estimated with certainty, a lowered degree of clinical protection against SARS-CoV-2 is possible. Persistently stronger responses to mRNA-1273 suggest that it might confer greater protection than BNT162b2, even 6 months after the second vaccination. Neither examined vaccinations induced ANA within the examined time frame.

Kinetics of the Antibody Response to Boostering With Three Different Vaccines Against SARS-CoV-2.

BACKGROUND: Heterologous vaccinations against SARS-CoV-2 with ChAdOx1 nCoV-19 and a second dose of an mRNA-based vaccine have been shown to be more immunogenic than homologous ChAdOx1 nCoV-19. In the current study, we examined the kinetics of the antibody response to the second dose of three different vaccination regimens (homologous ChAdOx1 nCoV-19 vs. ChAdOx1 nCoV-19 + BNT162b2 or mRNA-1273) against SARS-CoV-2 in a longitudinal manner; whether there are differences in latency or amplitude of the early response and which markers are most suitable to detect these responses. METHODS: We performed assays for anti-S1 IgG and IgA, anti-NCP IgG and a surrogate neutralization assay on serum samples collected from 57 participants on the day of the second vaccination as well as the following seven days. RESULTS: All examined vaccination regimens induced detectable antibody responses within the examined time frame. Both heterologous regimens induced responses earlier and with a higher amplitude than homologous ChAdOx1 nCoV-19. Between the heterologous regimens, amplitudes were somewhat higher for ChAdOx1 nCoV-19 + mRNA-1273. There was no difference in latency between the IgG and IgA responses. Increases in the surrogate neutralization assay were the first changes to be detectable for all regimens and the only significant change seen for homologous ChAdOx1 nCoV-19. DISCUSSION: Both examined heterologous vaccination regimens are superior in immunogenicity, including the latency of the response, to homologous ChAdOx1 nCoV-19. While the IgA response has a shorter latency than the IgG response after the first dose, no such difference was found after the second dose, implying that both responses are driven by separate plasma cell populations. Early and steep increases in surrogate neutralization levels suggest that this might be a more sensitive marker for antibody responses after vaccination against SARS-CoV-2 than absolute levels of anti-S1 IgG.

The temporal course of T- and B-cell responses to vaccination with BNT162b2 and mRNA-1273.

OBJECTIVES: To investigate the response of the immune system (and its influencing factors) to vaccination with BNT162b2 or mRNA-1273. METHODS: 531 vaccinees, recruited from healthcare professionals, donated samples before, in between, and after the administration of the two doses of the vaccine. T- and B-cell responses were examined via interferon-γ (IFN-γ) release assay, and antibodies against different epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (S1 and NCP) were detected via ELISA and surrogate neutralization assay. Results were correlated with influencing factors such as age, sex, prior infection, vaccine received (BNT162b2 or mRNA-1273), and immunosuppression. Furthermore, antinuclear antibodies (ANAs) were measured to screen for autoimmune responses following vaccination with an mRNA vaccine. RESULTS: No markers of immunity against SARS-CoV-2 were found before the first vaccination. Two weeks after it, specific responses against SARS-CoV-2 were already measurable (median ± median absolute deviation (MAD): anti-S1 IgG 195.5 ± 172.7 BAU/mL; IgA 6.7 ± 4.9 OD; surrogate neutralization 39 ± 23.7%), and were significantly increased two weeks after the second dose (anti-S1 IgG 3744 ± 2571.4 BAU/mL; IgA 12 ± 0 OD; surrogate neutralization 100 ± 0%, IFN-γ 1897.2 ± 886.7 mIU/mL). Responses were stronger for younger participants (this difference decreasing after the second dose). Further influences were previous infection with SARS-CoV-2 (causing significantly stronger responses after the first dose compared to unexposed individuals (p ≤ 0.0001)) and the vaccine received (significantly stronger reactions for recipients of mRNA-1273 after both doses, p < 0.05-0.0001). Some forms of immunosuppression significantly impeded the immune response to the vaccination (with no observable immune response in three immunosuppressed participants). There was no significant induction of ANAs by the vaccination (no change in qualitative ANA results (p 0.2592) nor ANA titres (p 0.08) from pre-to post-vaccination. CONCLUSIONS: Both vaccines elicit strong and specific immune responses against SARS-CoV-2 which become detectable one week (T-cell response) or two weeks (B-cell response) after the first dose.

Effects of Acoustic Paired Associative Stimulation on Late Auditory Evoked Potentials.

Paired associative stimulation (PAS), a form of non-invasive cortical stimulation pairing transcranial magnetic stimulation (TMS) with a peripheral sensory stimulus, has been shown to induce neuroplastic effects in the human motor, somatosensory and auditory cortex. The current study investigated the effects of acoustic PAS on late auditory evoked potentials (LAEP) and the influence of tone duration and placebo stimulation. In two experiments, 18 participants underwent a PAS with a 4 kHz paired tone of 400 ms duration using 200 pairs of stimuli (TMS-pulse over the left auditory cortex 45 ms after tone-onset) presented at 0.1 Hz. In Experiment 1 this protocol was contrasted with a protocol using a short paired tone of 23 ms duration (PAS-23 ms vs. PAS-400 ms). In Experiment 2 this PAS protocol was contrasted with sham stimulation (PAS-400 ms-sham vs. PAS-400 ms). Before and after PAS, LAEP were recorded for tones of 4 kHz (same carrier frequency as the paired tone) and 1 kHz as control tone. In Experiment 1, there was a significant difference between LAEP amplitudes of the 4 kHz tone after PAS-23 ms and PAS-400 ms with higher LAEP amplitudes after PAS-23 ms. Before both conditions, no difference could be detected. In Experiment 2 we observed a significant overall decrease in LAEP amplitudes pre to post PAS. Unspecific decreases of LAEP following PAS with a long paired tone (PAS-400 ms) might be related to habituation effects due to repeated presentation of sound stimuli which are not evident for PAS with a short paired tone (PAS-23 ms). Interpreting this result using the concept of temporal integration time allows us to discuss it in the context of spike-timing dependent plasticity.

New Stereoselective Biocatalysts for Carboligation and Retro-Aldol Cleavage Reactions Derived from d-Fructose 6-Phosphate Aldolase.

d-Fructose 6-phosphate aldolase (FSA) catalyzes the asymmetric cross-aldol addition of phenylacetaldehyde and hydroxyacetone. We conducted structure-guided saturation mutagenesis of noncatalytic active-site residues to produce new FSA variants, with the goal of widening the substrate scope of the wild-type enzyme toward a range of para- and meta-substituted arylated aldehydes. After a single generation of mutagenesis and selection, enzymes with diverse substrate selectivity scopes were identified. The kinetic parameters and stereoselectivities for a subset of enzyme/substrate combinations were determined for the reactions in both the aldol addition and cleavage reaction directions. The achieved collection of new aldolase enzymes provides new tools for controlled asymmetric synthesis of substituted aldols.

Tip of the Iceberg: Forme Fruste Tuberous Sclerosis in a Child.

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder characterized by dermatologic manifestations and growth of multiple benign tumors often involving the brain, skin, kidneys, heart, lungs, and liver. It exhibits wide phenotypic variation, ranging from the most severe cases with intellectual disability and intractable epilepsy to the mildest, clinically silent forms of the disease. The incidence of TSC is reported to be 1/6000; however, this does not account for those with milder forms of the disease, of which forme fruste is the mildest. Forme fruste is a French term for a "crude or unfinished form." In medicine, it refers to an atypical or attenuated manifestation of a clinical condition and implies an incomplete, partial, or an aborted disease state. Here, we describe a rare case of forme fruste TSC incidentally diagnosed in an otherwise healthy child, highlighting the implications of the diagnosis for treatment and screening in similarly affected pediatric patients.

Redox-gradient driven electron transport in a mixed community anodic biofilm.

Here, we describe the long-distance (multi-cell-length) extracellular electron transport (LD-EET) that occurs in an anode-grown mixed community biofilm (MCB) enriched from river sediment that contains 3%-45% Geobacter spp. High signal-to-noise temperature-dependent electrochemical gating measurements (EGM) using interdigitated microelectrode arrays reveal a peak-shaped electrical conductivity vs. potential dependency, indicating MCB acts as a redox conductor, similar to pure culture anode-grown Geobacter sulfurreducens biofilms (GSB). EGM also reveal that the maximum sustained rate of LD-EET in MCB is comparable to GSB, and the same whether under acetate-oxidizing or acetate-free conditions. Voltammetry indicated that MCB possesses 3- to 5-fold less electrode-accessible redox cofactors than GSB, suggesting that MCB may be more efficiently organized than GSB for LD-EET or that a small portion of electrode accessible redox cofactors of GSB are involved in LD-EET. The activation energy for LD-EET (0.11 ± 0.01 eV) was comparable to GSB, consistent with the possible role of c-type cytochromes as LD-EET cofactors, detected in abundance by confocal resonance Raman microscopy. Taken together, the results demonstrate LD-EET for a mixed community anode-grown microbial biofilm that is remarkably similar to GSB even though it contains many different types of microorganisms and appears to utilize far fewer EET redox cofactors.

Paired Associative Stimulation of the Temporal Cortex: Effects on the Auditory Steady-State Response.

BACKGROUND: Paired associative stimulation (PAS) is the repeated combination of a sensory stimulus with transcranial magnetic stimulation (TMS) in close temporal association. Recently, a study demonstrated that PAS of an auditory stimulus together with TMS of the temporal cortex is capable of changing the amplitude of auditory evoked potentials (AEP). OBJECTIVE: This study examined the influence of tone duration and habituation in temporal cortex PAS as elicited by 40 and 20 Hz amplitude modulated auditory steady-state responses (aSSR). METHODS: Eighteen subjects participated in two experiments, including two PAS protocols each, which consisted of 200 auditory stimuli (4 kHz) paired with temporal cortex TMS with an interstimulus interval (ISI) of 45 ms between tone onset and TMS pulse, delivered at 0.1 Hz. Experiment 1 compared auditory stimuli with different lengths [PAS (23 ms) vs. PAS (400 ms)]. Experiment 2 investigated verum vs. sham PAS. aSSR for the paired tone (4 kHz) and a control tone (1 kHz) were measured pre- and post-interventional-using 40 Hz aSSR in experiment 1 and both 20 and 40 Hz aSSR in experiment 2. RESULTS: A statistically significant, sham-controlled decrease in amplitude was observed for the 20 Hz aSSR using the 4 kHz PAS carrier frequency in experiment 2. CONCLUSION: Frequency-specific effects for the 20 Hz aSSR confirm the feasibility of auditory PAS and highlight the secondary auditory cortex as its target site, introducing new possible treatment protocols for patients suffering from tinnitus. The amplitude decrease can be explained by principles of spike timing-dependent plasticity and the superposition model of aSSR.

Upregulation of the zebrafish Nogo-A homologue, Rtn4b, in retinal ganglion cells is functionally involved in axon regeneration.

BACKGROUND: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals. RESULTS: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 µM MO1) and 74% (140 µM MO1), respectively, with MO2: 59% (70 µM MO2) and 73% (140 µM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO. CONCLUSIONS: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.