The role of osmolarity adjusting agents in the regulation of encapsulated cell behavior to provide a safer and more predictable delivery of therapeutics

Transplantation of cells within alginate microspheres has been extensively studied for sustained drug delivery. However, the lack of control over cell behavior represents a major concern regarding the efficacy and the safety of the therapy. Here, we demonstrated that when formulating the biosystem, an adequate selection of osmolarity adjusting agents significantly contributes to the regulation of cell responses. Our data showed that these agents interact in the capsule formation process, influencing the alginate crosslinking degree. Therefore, when selecting inert or electrolyte-based osmolarity adjusting agents to encapsulate D1 multipotent mesenchymal stromal cells (MSCs), alginate microcapsules with differing mechanical properties were obtained. Since mechanical forces acting on cells influence their behavior, contrasting cell responses were observed both, in vitro and in vivo. When employing mannitol as an inert osmolarity adjusting agent, microcapsules presented a more permissive matrix, allowing a tumoral-like behavior. This resulted in the formation of enormous cell-aggregates that presented necrotic cores and protruding peripheral cells, rendering the therapy unpredictable, dysfunctional, and unsafe. Conversely, the use of electrolyte osmolarity adjusting agents, including calcium or sodium, provided the capsule with a suitable crosslinking degree that established a tight control over cell proliferation and enabled an adequate therapeutic regimen in vivo. The crucial impact of these agents was confirmed when gene expression studies reported pivotal divergences not only in proliferative pathways, but also in genes involved in survival, migration, and differentiation. Altogether, our results prove osmolarity adjusting agents as an effective tool to regulate cell behavior and obtain safer and more predictable therapies.     

Disconnecting bones within the jaw‐otic network modules underlies mammalian middle ear evolution

The origin of the mammalian middle ear ossicles from the craniomandibular articulation of their synapsid ancestors is a key event in the evolution of vertebrates. The richness of the fossil record and the multitude of developmental studies have provided a stepwise reconstruction of this evolutionary innovation, highlighting the homology between the quadrate, articular, pre‐articular and angular bones of early synapsids with the incus, malleus, gonial and ectotympanic bones of derived mammals, respectively. There are several aspects involved in this functional exaptation: (i) an increase of the masticatory musculature; (ii) the separation of the quadrate bone from the cranium; and (iii) the disconnection of the post‐dentary bones from the dentary. Here, we compared the jaw‐otic complex for 43 synapsid taxa using anatomical network analysis, showing that the disconnection of mandibular bones was a key step in the mammalian middle ear evolution, changing the skull anatomical modularity concomitant to the acquisition of new functions. Furthermore, our analysis allows the identification of three types of anatomical modules evolving through five evolutionary stages during the anatomical transformation of the jawbones into middle ear bones, with the ossification and degradation of Meckel's cartilage in mammals as the key ontogenetic event leading the change of anatomical modularity.     

Prognostic significance of performing universal HER2 testing in cases of advanced gastric cancer

Background

Trastuzumab significantly improves overall survival (OS) when added to cisplatin and fluoropyrimidine as a treatment for HER2-positive advanced gastric cancers (AGC). The aim of this study was to evaluate the impact of the gradual implementation of HER2 testing on patient prognosis in a national registry of AGC.

Methods

This Spanish National Cancer Registry includes cases who were consecutively recruited at 28 centers from January 2008 to January 2016. The effect of missing HER2 status was assessed using stratified Cox proportional hazards (PH) regression.

Results

The rate of HER2 testing increased steadily over time, from 58.3 % in 2008 to 92.9 % in 2016. HER2 was positive in 194 tumors (21.3 %). In the stratified Cox PH regression, each 1 % increase in patients who were not tested for HER2 at the institutions was associated with an approximately 0.3 % increase in the risk of death: hazard ratio, 1.0035 (CI 95 %, 1.001–1.005), P = 0.0019. Median OS was significantly lower at institutions with the highest proportions of patients who were not tested for HER2.

Conclusion

Patients treated at centers that took longer to implement HER2 testing exhibited worse clinical outcomes. The speed of implementation behaves as a quality-of-care indicator. Reviewed guidelines on HER2 testing should be used to achieve this goal in a timely manner.

     

White matter involvement on DTI-MRI in Cushing’s syndrome relates to mood disturbances and processing speed: a case-control study

Purpose

Cushing’s syndrome (CS) is an endocrine disorder due to prolonged exposure to cortisol. Recently, microstructural white matter (WM) alterations detected by diffusion tensor imaging (DTI) have been reported in CS patients, and related to depression, but other functional significances. remain otherwise unclear. We aimed at investigating in more depth mood symptoms in CS patients, and how these relate to cognition (information processing speed), and to WM alterations on DTI.

Methods

The sample comprised 35 CS patients and 35 healthy controls. Beck Depression Inventory-II (BDI-II) was used to measure depressive symptoms, State-Trait Anxiety Inventory (STAI) to assess anxiety, and processing speed was measured by the Symbol Digit Modalities Test (SDMT). DTI studies were acquired using a 3-Tesla Philips-Achieva MR-facility. Voxelwise statistical analysis of fractional anisotropy (FA), mean, axial and radial diffusivities (MD, AD, RD) data were performed using FMRIB Software Library. Correlation analysis were obtained between mood and processing speed variables, and FA, MD, AD and RD values, taking both CS patients and healthy controls.

Results

Active, controlled and cured CS patients showed greater depression (F?=?12.4, p?<?0.001), anxious state (F?=?4.8, p?=?0.005) and anxious trait (F?=?9.6, p?<?0.001) scores, than controls. Using the entire sample, depression scores correlated negatively to FA and positively to RD values. Although there were no differences in processing speed between groups, SDMT scores correlated positively to both FA and AD values.

Conclusions

There were greater depressive and anxious symptoms in CS patients than in healthy controls, but no difference in processing speed. However, DTI is related to depression and information processing speed in CS.

     

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species

α-synuclein aggregation is present in Parkinson’s disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

Aberrant aggregation of α-synuclein (α-syn) into amyloid fibrils and subsequent accumulation into intracellular inclusions is a hallmark of neurodegenerative disorders such as Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy (1–3). In these diseases, soluble α-syn monomers misfold and self-assemble, forming small oligomeric species that retain the highly disordered structure of the monomeric state (4). These species are rather unstable and can undergo structural rearrangements, including a gain in β-sheet structure that generates more stable species (4, 5). β-structured oligomers can grow further through monomer addition or self-association, finally giving rise to well-defined amyloid fibrils (4–6). Despite the controversial evidence about the relationship between the different species that populate the aggregation process and cellular toxicity, the prevalent view is that both intermediate oligomers and small fibrils are neurotoxic (7). Due to their abnormal interactions with cellular components, certain types of oligomers are key pathogenic agents in the development of the disease (8–10). In particular, they can disrupt membranes, induce oxidative stress, dysregulate calcium homeostasis, cause mitochondria dysfunction, or impair the proteasome system (11). Furthermore, α-syn oligomers have been implicated in the spreading of the disease, as these aggregates can be transmitted between cells (12, 13). Small fibrils have also been related to intercellular spreading and propagation of neurodegeneration (14–18). In contrast, large amyloid aggregates are believed to be relatively inert, as their highly ordered packing and slow diffusion reduces undesired interactions with cellular components. Even so, large aggregates can generate intermediate species that contribute to cytotoxicity through secondary processes such as fragmentation or nucleation on the aggregate surface (19, 20).To counteract the toxic effect of protein aggregates, cells have evolved a sophisticated protein homeostasis network that coordinates protein synthesis, folding, disaggregation and degradation (21). This network is composed of the translational machinery, molecular chaperones and cochaperones, the ubiquitin-proteasome system, and the autophagy machinery. The way this network tackles amyloid aggregates remains poorly understood. It has been previously reported that the constitutive human Hsp70 (Hsc70) in collaboration with its Hsp40 cochaperone (Hdj1 or DnaJB1) slowly disassembles preformed α-syn fibrils (22). This activity was further stimulated by adding the NEF Hsp110 (Apg2). HspB5, a small heat shock protein also known as αB-crystallin, potentiated α-syn fibril disassembly by the ternary chaperone mixture. Although this chaperone combination was able to disaggregate fibrils, they did it in a timescale of weeks through a depolymerization process. Only when Hsp104, a yeast representative of the Hsp100 family able of fragmenting fibrils, was added to the mixture, disassembly occurred within hours (22). The lack of Hsp104 homologs in metazoans questioned whether this activity was physiologically relevant in humans. A later study revealed that a chaperone complex composed solely of members of the Hsp70, Hsp40, and Hsp110 families was able to efficiently reverse α-syn amyloid fibrils through both fragmentation and depolymerization, generating smaller fibrils, oligomers, and, ultimately, monomers (23). Despite the importance of this emerging disaggregase functionality, its mechanism of action remains largely unknown. Recently, the same chaperone mixture has been reported to also disaggregate tau and Htt fibrils (24–26), pointing to this Hsp70-based machinery as a potential human amyloid disaggregase.The two-fold aim of this work is, firstly, to test whether human disaggregase remodels with the same efficiency the different aggregates that populate the complex process of amyloid formation and, secondly, to shed light on the key mechanisms involved in the disassembly of amyloids. We show that the human disaggregase system disassembles toxic oligomers and short fibrils much better than large, less toxic fibrils, and that it does so by an enhanced destabilization of the small aggregated forms. Explicitly, fibril disassembly involves destabilization of the fibril ends and unzipping of the protofilaments, which allow depolymerization. The fast propagation of protofilament depolymerization toward the opposite fibril end is consistent with entropic pulling forces exerted by Hsc70 upon binding the fibril surface.     

Pediatric transplantation in Europe during the COVID-19 pandemic: Early impact on activity and healthcare

The current pandemic SARS-CoV-2 has required an unusual allocation of resources that can negatively impact chronically ill patients and high-complexity procedures. Across the European Reference Network on Pediatric Transplantation (ERN TransplantChild), we conducted a survey to investigate the impact of the COVID-19 outbreak on pediatric transplant activity and healthcare practices in both solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT). The replies of 30 professionals from 18 centers in Europe were collected. Twelve of 18 centers (67%) showed a reduction in their usual transplant activity. Additionally, outpatient visits have been modified and restricted to selected ones, and the use of telemedicine tools has increased. Additionally, a total of 14 COVID-19 pediatric transplanted patients were identified at the time of the survey, including eight transplant recipients and six candidates for transplantation. Only two moderate-severe cases were reported, both in HSCT setting. These survey results demonstrate the limitations in healthcare resources for pediatric transplantation patients during early stages of this pandemic. COVID-19 disease is a major worldwide challenge for the field of pediatric transplantation, where there will be a need for systematic data collection, encouraging regular discussions to address the long-term consequences for pediatric transplantation candidates, recipients, and their families.     

Multifocal lymphangioendotheliomatosis without thrombocytopenia or clinical signs of systemic bleeding

Multifocal lymphangioendotheliomatosis with thrombocytopenia (MLT) is an extremely rare recently described disorder characterized by diffuse congenital skin and gastrointestinal vascular lesions that may be associated with gastrointestinal bleeding and thrombocytopenia. We herein present a case report of multifocal lymphangioendotheliomatosis without thrombocytopenia or extensive extracutaneous involvement (gastrointestinal bleeding). Given the high morbidity and mortality associated with this disease, it is important for clinicians to recognize this disorder in order to select the most appropriate therapeutic approach.