Biology subjects

Wrobel, A.

Publications and source records attributed to Wrobel, A..

5 recordsLinked to original sources

bioRxiv2025

Fresh Paraformaldehyde Preserves Thrombus Biochemistry - An ATR-FTIR and PCA Investigation of Pulmonary Artery Thrombi

BackgroundBiochemical analyses of pulmonary embolism derived thrombi depend critically on fixative quality. ObjectiveTo quantify the impact of paraformaldehyde (PFA) shelf life on thrombus molecular integrity using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and time-of-flight secondary ion mass spectroscopy (SIMS) combined with principal component analysis. MethodsTen pulmonary artery thrombi were fixed in either aged PFA (6 months; n = 6) or freshly prepared 4% PFA (n = 4); an in-vitro plasma clot control received the same fresh PFA. Triplicate ATR-FTIR spectra (400-4000 cm-1) were collected and vector-normalized to the Amide I band. ToF-SIMS measurements were performed using a 30 keV Bi primary ion beam Fixative chemistry was assessed by the carbonyl absorbance at 1700 cm-1. ResultsFreshly fixed thrombi displayed narrow Amide I (~1650 cm-1) and Amide II (~1540 cm-1) bands with a higher signal-to-noise ratio (SNR; median 41.8) than aged-PFA samples (18.2; P < .01). Carbonyl absorbance at 1700 cm-1 was markedly higher in fresh PFA. Aged PFA introduces chemical variances of biological samples in low-mass molecule fragments. PCA showed clear separation of fresh versus aged specimens and alignment of fresh thrombi with plasma controls across spectral windows. ConclusionsPFA solutions older than three months markedly deteriorate thrombus biochemical fidelity. ATR-FTIR and ToF-SIMS offer a rapid quality-control assay prior to molecular analyses. Key pointsO_LIPFA stored [≥]3 months loses >70% of reactive aldehydes, yielding broad Amide envelopes, attenuated lipid bands and a two fold SNR drop. C_LIO_LIAged PFA introduces chemical variances of biological samples in low-mass molecule fragments. C_LIO_LIPCA of fingerprint, protein or lipid windows cleanly separates aged from fresh fixations (PC1 up to 80% variance). C_LIO_LIA rapid QC workflow (carbonyl absorbance [≥]0.25 a.u. plus PCA verification) safeguards molecular integrity irrespective of patient age, DVT history or clinical severity. C_LIO_LIAdopting this workflow will harmonise multi centre clot biorepositories and enhance the reproducibility of proteomic, lipidomic and imaging studies. C_LI

biophysics↗

bioRxiv2025

p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) internalizes cell-surface receptors via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to these sites by the adaptor protein complex AP2. AP2 cycles between a closed cytoplasmic conformation and an open membrane-bound state, and efficient CME requires both conformations and their dynamic interconversion. The mechanisms regulating these conformational changes, which include post-translational modifications of the AP2, remain incompletely understood. Here, we report that p70S6 kinase phosphorylates the {micro}2 subunit of the AP2 and that the phosphorylation of serine 45 (S45) depends on p70S6K activity. Loss of S45-{micro}2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. In Caenorhabditis elegans, lack of S45-{micro}2 phosphorylation produces directionally similar but markedly weaker phenotypes than AP2 loss of function. Live imaging and in silico dynamic modelling suggest that S45-2 phosphorylation has impact on the conformational changes of the AP2 complex. These findings identify a p70S6K-dependent mechanism that modulates AP2 function and further strengthen the importance of post-translational regulation in controlling CME. Summary statementClathrin-mediated endocytosis is essential for receptor internalization and cell signaling. This study identifies a novel regulatory mechanism in which p70S6K-mediated phosphorylation of the AP2 subunit 2 at Ser45 modulates AP2 conformational dynamics and facilitates receptor endocytosis.

cell biology↗

bioRxiv2024

The bat Influenza A virus subtype H18N11 induces nanoscale MHCII clustering upon host cell attachment

Prior to the discovery of the bat influenza A virus (IAV) subtypes H17N10 and H18N11, it was believed that all IAVs bind via the viral hemagglutinin (HA) to sialic acid residues to mediate attachment and subsequent viral entry. Both HA subtypes, H17 and H18, however, do not bind to sialic acids but instead engage a proteinaceous receptor: major histocompatibility complex class II (MHCII). The mechanistic details of this hitherto unknown protein-mediated entry are not understood. Since conventional IAVs require attachment to clusters of sialylated glycans to overcome the low affinity of the HA-sialic acid interaction, we hypothesized that bat HA would likewise interact with multiple MHCII molecules. Here, we used photoactivated localization microscopy (PALM) on fixed and live cells expressing MHCII fused to appropriate fluorescent reporters. We show that bat IAV particles attach to pre-existing MHCII clusters present on susceptible cells and that the local HA-MHCII interaction results in an increased cluster size. To measure the impact of viral attachment on the dynamics of MHCII, we utilized an experimental setup designated "inverse infection" where intact viral particles were immobilized on coverslips before live MHCII-expressing cells were seeded on top. Recording the trajectories of single MHCII molecules, this approach revealed that the mobility of MHCII was indeed slowed down in viral proximity leading to a local enrichment of MHCII molecules beneath the viral particle. Taken together, these data suggest that attachment of viral particles leads to clustering of MHCII, a process similar to the MHCII dynamics observed during the formation of an immunological synapse.

microbiology↗

bioRxiv2024

Local variation in brain temperature explains gender-specificity of working memory performance

Exploring gender differences in cognitive abilities offers vital insights into human brain functioning. Our study utilized advanced techniques like magnetic resonance thermometry, standard working memory n-back tasks, and functional MRI to investigate if gender-based variations in brain temperature correlate with distinct neuronal responses and working memory capabilities. Interestingly, our findings revealed no gender disparity in working memory performance. However, we observed a significant decrease in average brain temperature in males during working memory tasks, a phenomenon not seen in females. Although changes in female brain temperature were not statistically significant, we found an inverse relationship between the absolute temperature change (ATC) and cognitive performance, alongside a correlation with blood oxygen level dependent (BOLD) neuronal responses. This suggests that in females, ATC is a crucial determinant for the link between cognitive performance and BOLD responses, a linkage not evident in males. Our results also suggest that females compensate for their brains heightened temperature sensitivity by activating additional neuronal networks to support working memory. This study not only underscores the complexity of gender differences in cognitive processing but also opens new avenues for understanding how temperature fluctuations influence brain functionality. SignificanceSex/gender differences in cognition are of high scientific and social interest. Yet, those differences (if any) remain elusive. Here we used magnetic resonance thermometry and functional MRI to examine, whether gender differences in working memory performance (WMP) are determined by subtle, yet detectable between-sex differences in local brain temperature fluctuations mediated by blood oxygen level-dependent (BOLD) neuronal responses. We found that WMP did not differ between genders. Yet, a females WMP was more sensitive to brain temperature variation compared to males. Furthermore, the negative impact of temperature on female cognitive functions was compensated by higher BOLD activity in other task-specific brain areas. This compensation may account for equivocal results of studies on the between-sex differences in cognitive performance.

neuroscience↗

bioRxiv2020

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans

Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections1. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a recent zoonotic infection that has quickly reached pandemic proportions2,3. Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 spike (S) glycoprotein, we demonstrate the presence of pre-existing humoral immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. These were predominantly of the IgG class and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, as well as concomitant IgM and IgA antibodies, lasting throughout the observation period of 6 weeks since symptoms onset. SARS-CoV-2-uninfected donor sera also variably reacted with SARS-CoV-2 S and nucleoprotein (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralising activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

immunology↗