Biology subjects

Santos, M. S.

Publications and source records attributed to Santos, M. S..

4 recordsLinked to original sources

bioRxiv2025

Population differences of chromosome 22q11.2 duplication structure predisposes differentially to microdeletion and inversion.

The most common genomic disorder, chromosome 22q11.2 microdeletion syndrome (22q11.2DS), is mediated by highly identical and polymorphic segmental duplications (SDs) known as low copy repeats (LCRs; regions A-D) that have been challenging to sequence and characterize. Here, we report the sequence-resolved genomic architecture of 135 chromosome 22q11.2 haplotypes from diverse 1000 Genomes Project samples. We find that more than 90% of the copy number variation is polarized to the most proximal LCR region A (LCRA) where 50 distinct structural configurations are observed ([~]189 kbp to [~]2.15 Mbp or 11-fold length variation). A higher-order SD cassette structure of 105 kbp in length, flanked by 25 kbp long inverted repeats, drives this variation and emerged in the human-chimpanzee ancestral lineage later expanding in humans [~]1.0 [0.8-1.2] million years ago. African LCRA haplotypes are significantly longer (p=0.0047) when compared to non-Africans yet are predicted to be more protected against recurrent microdeletions (p=0.00053) due to a preponderance of flanking SDs in an inverted orientation. Conversely, we identified nine distinct inversion polymorphisms, including five recurrent [~]2.28 Mbp inversions extending across the critical region (LCRA-D) and four smaller inversions (two LCRA-B, one LCRC-D, and one LCRB-D); 7/9 of these events were identified in haplotypes of African and admixed American ancestry. Finally, we sequence and assemble four families and show that LCRA-D deletion breakpoints map to the 105 kbp repeat unit while inversion breakpoints associate with the 25 kbp repeats adjacent to palindromic AT-rich regions. In one family, we observe evidence of more complex unequal crossover events associated with gene conversion and multiple breakpoints. Our findings suggest that specific haplotype configurations are protective and susceptible to chromosome 22q11.2DS while recurrent large-scale inversions help to explain why this syndrome is less prevalent among individuals of African descent.

genomics↗

bioRxiv2024

Engineering the Mechanical Stability of a Therapeutic Affibody/PD-L1 Complex by Anchor Point Selection

Protein-protein complexes can vary in mechanical stability depending on the direction from which force is applied. Here we investigated the anisotropic mechanical stability of a molecular complex between a therapeutic non-immunoglobulin scaffold called Affibody and the extracellular domain of the immune checkpoint protein PD-L1. We used a combination of single-molecule AFM force spectroscopy (AFM-SMFS) with bioorthogonal clickable peptide handles, shear stress bead adhesion assays, molecular modeling, and steered molecular dynamics (SMD) simulations to understand the pulling point dependency of mechanostability of the Affibody:(PD-L1) complex. We observed diverse mechanical responses depending on the anchor point. For example, pulling from residue #22 on Affibody generated an intermediate unfolding event attributed to partial unfolding of PD-L1, while pulling from Affibodys N-terminus generated force-activated catch bond behavior. We found that pulling from residue #22 or #47 on Affibody generated the highest rupture forces, with the complex breaking at up to [~] 190 pN under loading rates of [~]104-105 pN/sec, representing a [~]4-fold increase in mechanostability as compared with low force N-terminal pulling. SMD simulations provided consistent tendencies in rupture forces, and through visualization of force propagation networks provided mechanistic insights. These results demonstrate how mechanostability of therapeutic protein-protein interfaces can be controlled by informed selection of anchor points within molecules, with implications for optimal bioconjugation strategies in drug delivery vehicles.

biophysics↗

bioRxiv2024

Interstitial pneumonia via the oropharyngeal route of infection with Encephalitozoon cuniculi

Microsporidia causes opportunistic infections in immunosuppressed individuals. Mammals shed these spores of fungi in feces, urine, or respiratory secretions, which could contaminate water and food, thereby reaching the human body and causing infection. The oral route is the most common route of infection, although experiments have demonstrated that intraperitoneal and intravenous routes may also spread infection. Respiratory tract infection, although considered to be possible, has not been reported to date. The present study, therefore, aimed to demonstrate infection with the microsporidia of Encephalitozoon cuniculi via the oropharyngeal route as a model for opportunistic pneumonia. The objectives were the study of opportunistic pneumonia in general while also confirming transmission via the respiratory route to expand the understanding of the epidemiology of zoonoses. C57BL mice, both male and female, up to 12 weeks of age, and free of specific pathogens (SPF) were inoculated (Infected group) or not infected (Non-Infected group) with 1 x 107 spores of E. cuniculi via the oropharyngeal route. The animals immunosuppressed with cyclophosphamide (Cy) were infected (Cy-Infected group) or not infected (Cy-Uninfected group), and then assessed for the influence of immunosuppression on infection. In both groups, animals inoculated with 0.9% saline solution via the oropharyngeal route served as controls (Sham and Cy-Sham groups, respectively). After 14 days of infection, the lungs of all animals were retrieved for histopathological analysis, phenotyping of lung inflammatory cells using flow cytometry, measurement of fungal load using qPCR, and measurement of the serum levels of Th1, Th2, and Th17 cytokines. The results revealed that the infected animals developed interstitial pneumonia characterized by perialveolar inflammatory infiltrative lesions with a predominance of lymphocytes and plasma cells. However, the fungal load and the extent of the inflammatory infiltrate were relatively lower in the Cy-Infected group, with a predominance of the CD8+ T lymphocyte population in the lungs compared to Infected group. In the Infected group not treated with Cy, an increase in the population of alveolar macrophages (F4/80+CD11b-SiglecF+) was noted, along with higher fungal load and inflammatory infiltrate in the lungs, indicating a further pronounced Encephalitozoon pneumonia compared to the immunosuppressed animals. The infected groups presented Th1 cytokine profiles, with the Cy-Infected group exhibiting increased Th17 levels. Collectively, these results demonstrated that oropharyngeal infection promoted pneumonia caused by E. cuniculi in mice treated or not with cyclophosphamide, with greater severity occurring in the non-immunosuppressed mice, thereby establishing this model as a suitable one for interstitial pneumonia via aspiration. Author SummaryOpportunistic fungi infection in immunosuppressed individuals may lead to pneumonia that is lethal. The airways are the most common routes for these infections. The opportunistic fungal species Encephalitozoon cuniculi is associated with pneumonia, although this species has not been reported to cause airway infection to date. The present study aimed to demonstrate that the oropharyngeal route, which is a frequent route of infection in patients with reflux, could lead to severe pneumonia in the mice without immunosuppression associated with a low CD8+ T lymphocyte response.

pathology↗

bioRxiv2023

Cell-derived ECM loaded electrospun Polycaprolactone/Chitosan nanofibrous scaffolds for periodontal regeneration

Periodontitis is an inflammatory infection caused by bacterial plaque accumulation that affects the periodontium, a complex structure of different tissues (cementum, periodontal ligament and alveolar bone) that surrounds and supports the teeth. Current treatments lack bioactive signals to induce tissue repair and coordinated regeneration of the periodontium, thus alternative strategies are needed to improve clinical outcomes. Cell-derived extracellular matrix (ECM) has been combined with biomaterials to enhance their biofunctionality for various tissue engineering (TE) applications. In this work, bioactive cell-derived ECM loaded electrospun polycaprolactone/chitosan (PCL/CTS) nanofibrous scaffolds were developed combining polymer solutions with lyophilized decellularized ECM (dECM) derived from human Periodontal Ligament Stem/Stromal Cells (PDLSCs). The works aims were to fabricate and characterize cell-derived ECM electrospun PCL/CTS scaffolds in terms of morphology, physico-chemical, thermal and mechanical properties and assess their ability to enhance the osteogenic differentiation of PDLSCs, envisaging periodontal TE applications. PDLSCs were cultured and used for dECM production. PDLSCs-derived dECM was characterized regarding morphology, protein expression, DNA removal efficiency, and glycosaminoglycans and collagen contents. Osteogenic differentiation of PDLSCs was performed on PCL, PCL/CTS and PCL/CTS/ECM electrospun scaffolds for 21 days. The obtained results demonstrate that PCL/CTS/ECM scaffolds promoted cell proliferation compared to PCL and PCL/CTS scaffolds, while maintaining similar physical and mechanical properties of PCL/CTS scaffolds. PCL/CTS/ECM scaffolds enhanced the osteogenic differentiation of PDLSCs, confirmed by increased alkaline phosphatase activity, calcium deposition, and bone-specific marker genes expression. Moreover, PCL/CTS scaffolds showed higher levels of cell mineralization than PCL scaffolds. Overall, this work describes the first use of lyophilized cell-derived ECM loaded electrospun scaffolds for periodontal TE applications and highlights its potential as a promising therapeutic strategy for periodontitis treatment.

bioengineering↗