Modeling Lysosomal Storage Disorders in an Innovative Way: Establishment and Characterization of Stem Cell Lines from Human Exfoliated Deciduous Teeth of Mucopolysaccharidosis Type II Patients.

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

The SCL30a SR protein regulates ABA-dependent seed traits and germination under stress.

SR proteins are conserved RNA-binding proteins best known as splicing regulators that have also been implicated in other steps of gene expression. Despite mounting evidence for a role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain poorly understood. Here we show that the plant-specific SCL30a SR protein negatively regulates ABA signaling to control seed traits and stress responses during germination in Arabidopsis. Transcriptome-wide analyses revealed that loss of SCL30a function barely affects splicing, but largely induces ABA-responsive gene expression and genes repressed during germination. Accordingly, scl30a mutant seeds display delayed germination and hypersensitivity to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA and salt stress sensitivity. An ABA biosynthesis inhibitor rescues the enhanced mutant seed stress sensitivity, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the gene promotes seed germination under stress by reducing sensitivity to the phytohormone. Our results reveal a new player in ABA-mediated control of early development and stress response.

Towards a scalable bioprocess for rAAV production using a HeLa stable cell line.

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.

Gestational exposure to erenumab-The outcome of three pregnancies.

Erenumab is a monoclonal antibody (mAb) approved for the preventive treatment of migraine. While preclinical studies on calcitonin gene-related peptide mAbs did not identify any reproductive toxicity, pregnant and breastfeeding women were excluded from the pivotal human studies, and therefore the safety of calcitonin gene-related peptide medications in this population must be studied. So far, postmarketing data of accidental exposures have not brought to light any specific toxicities. Three women treated with erenumab in our series conceived while exposed to the drug. All had previous successful pregnancies, were on erenumab for more than 6 months, and had ≥80% reduction in headache frequency. The one who stopped erenumab only 1 month before conceiving had a spontaneous abortion during the first trimester due to a gestational trophoblastic neoplasia and has since conceived with an uneventful gestation. The other two women stopped treatment during the first trimester, and both pregnancies went to term with no complications. All babies have shown normal development. No plausible explanation relates the mechanism of action of erenumab and the serious complication that occurred in one patient. Continuous follow-up and reporting of all exposures are encouraged to gather safety data on pregnant and nursing women and on the development of the newborns. So far, immediately stopping the drug is advised and may contribute to decreasing the potential risks.

Downstream processing for influenza vaccines and candidates: An update.

Seasonal and pandemic influenza outbreaks present severe health and economic burdens. To overcome limitations on influenza vaccines' availability and effectiveness, researchers chase universal vaccines providing broad, long-lasting protection against multiple influenza subtypes, and including pandemic ones. Novel influenza vaccine designs are under development, in clinical trials, or reaching the market, namely inactivated, or live-attenuated virus, virus-like particles, or recombinant antigens, searching for improved effectiveness; all these bring downstream processing (DSP) new challenges. Having to deal with new influenza strains, including pandemics, requires shorter development time, driving the development of faster bioprocesses. To cope with better upstream processes, new regulatory demands for quality and safety, and cost reduction requirements, new unit operations and integrated processes are increasing DSP efficiency for novel vaccine formats. This review covers recent advances in DSP strategies of different influenza vaccine formats. Focus is given to the improvements on relevant state-of-the-art unit operations, from harvest and clarification to purification steps, ending with sterile filtration and formulation. The development of more efficient unit operations to cope with biophysical properties of the new candidates is discussed: emphasis is given to the design of new stationary phases, 3D printing approaches, and continuous processing tools, such as continuous chromatography. The impact of the production platforms and vaccine designs on the downstream operations for the different influenza vaccine formats approved for this season are highlighted.

A granular cell tumor: an unusual colon polyp.

We read with interest the article by Sevilla Ribota et al1 that described an unexpected finding of a granular cell tumour (GCT) of the rectum, which was removed by band ligation-assisted mucosectomy. We present a similar case of a GCT of the cecum, which was resected using a different endoscopic procedure.

Optimization of Chitosan-α-casein Nanoparticles for Improved Gene Delivery: Characterization, Stability, and Transfection Efficiency.

Among non-viral vectors, the cationic polymer chitosan has gained attention as a gene delivery system. We hypothesized that the addition of casein into the nanoparticle's structure would facilitate a proper gene transfer. The work herein presented aimed to optimize the production method of chitosan-casein nanoparticles (ChiCas NPs) and to test their ability as a gene delivery system. ChiCas NPs formulation optimization was carried out by analyzing several characteristics such as NP size, zeta potential, and chitosan and casein incorporation efficacy. The best formulation developed presented small and homogenous particle size (around 335 nm) and positive zeta potential (≈ + 38 mV), and showed to be stable for 34 weeks both, at 4°C and 20°C. The particles were further used to entrap or to adsorb DNA and form NPs-DNA complexes. In vitro transfection studies, carried out in COS-7 cells, suggested a low transfection efficiency of the different NPs:DNA ratios tested, comparatively to the positive control. Nonetheless, we could observe that the complexes with larger sizes presented better transfection results than those with smaller diameters. To conclude, ChiCas NPs have great technological potential since the preparation process is very simple, and the DNA incorporation efficacy is very high and shows to be physically very stable. The NPs:DNA ratio still needs to be optimized with the aim of achieving better transfection results and being able to anticipate a high gene expression on DNA-based vaccination studies.

14-3-3σ Protein Expression in Canine Renal Cell Carcinomas.

14-3-3σ is a protein expressed in many epithelial tissues associated with essential cell functions, including cell-cycle control, apoptosis, and cytoskeletal integrity. There is a paucity of knowledge of the tumorigenesis of canine renal cell carcinomas (RCCs), and the histological origin of this tumor has not been established. This study analyzed the expression of 14-3-3σ, Ki-67, cytokeratins, and vimentin in 40 canine RCCs. Aberrant expression of 14-3-3σ was demonstrated in 15 (38%) cases and was associated with a significantly shorter survival time ( P < .002). In contrast to canine RCC, normal kidney did not express 14-3-3σ. The Ki-67 proliferation index did not show utility as a prognostic factor. The distal convoluted tubular epithelium in normal kidneys coexpressed cytokeratins and vimentin, and thus maintenance of this coexpression pattern in canine RCC suggests that most tumors arise from the distal segment of the nephron. These results suggest that 14-3-3σ is a potential negative prognostic factor and a possible therapeutic target.

Purification of influenza virus-like particles using sulfated cellulose membrane adsorbers.

BACKGROUND: Vaccines based on virus-like particles (VLPs) are an alternative to inactivated viral vaccines that combine good safety profiles with strong immunogenicity. In order to be economically competitive, efficient manufacturing is required, in particular downstream processing, which often accounts for major production costs. This study describes the optimization and establishment of a chromatography capturing technique using sulfated cellulose membrane adsorbers (SCMA) for purification of influenza VLPs. RESULTS: Using a design of experiments approach, the critical factors for SCMA performance were described and optimized. For optimal conditions (membrane ligand density: 15.4 µmol cm-2, salt concentration of the loading buffer: 24 mmol L-1 NaCl, and elution buffer: 920 mmol L-1 NaCl, as well as the corresponding flow rates: 0.24 and 1.4 mL min-1), a yield of 80% in the product fraction was obtained. No loss of VLPs was detected in the flowthrough fraction. Removal of total protein and DNA impurities were higher than 89% and 80%, respectively. CONCLUSION: Use of SCMA represents a significant improvement compared with conventional ion exchanger membrane adsorbers. As the method proposed is easily scalable and reduces the number of steps required compared with conventional purification methods, SCMA could qualify as a generic platform for purification of VLP-based influenza vaccines. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Tender red subcutaneous nodules in an adult female: a challenging diagnosis.

Pancreatic panniculitis is an uncommon and rare skin complication of systemic fat necrosis associated with pancreatitis post-ampullectomy. Besides the rarity of the condition, the clinical history and physical examination for diagnosis is also important.

Synchronous intraductal papillary mucinous neoplasm and a pancreatic neuroendocrine tumor: more than a coincidence?

BACKGROUND: Although the association between intraductal papillary mucinous neoplasm of the pancreas (IPMN) and pancreatic neuroendocrine tumor (PNET) has been increasingly reported, whether this association is real or coincidence remains unclear. We report a case of synchronous IPMN and a PNET which were diagnosed preoperatively and discuss the tumorigenesis, clinicopathological features and management of these rare tumors based on the published literature. CASE REPORT: A 56-year-old male was incidentally diagnosed with a 14 mm branch duct IPMN and a 3.6 mm non-functional PNET during an evaluation due to persistent upper abdominal pain via endoscopic ultrasound. Close follow-up of the patient was decided as the IPMN had no worrisome features. A review of twenty-two previously reported cases of synchronous IPMN and PNET indicated that: a) only seven cases were diagnosed preoperatively; b) abdominal pain was the main presenting symptom; c) IPMN was the dominant tumor and presented with low grade dysplasia; d) the PNET was small and non-functional and had an indolent behavior; and e) only one case underwent radiologic follow-up. DISCUSSION: IPMN are associated with other pancreatic and extrapancreatic malignancies. Thus, the entire pancreatic parenchyma should be examined closely during the evaluation of an IPMN in order to exclude other pancreatic lesions, for example, a PNET.

Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles.

Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.

Delayed Emergence From Anesthesia Due to Posterior Reversible Encephalopathy Syndrome (PRES): A Case Report.

Posterior reversible encephalopathy syndrome (PRES) is a rare clinical and radiological syndrome that presents as rapid onset of neurological symptoms such as headache, visual loss, impaired mental status, and seizure activity associated with characteristic focal white matter vasogenic edema. When promptly recognized and managed, these changes are usually reversible. PRES is most commonly associated with hypertensive crises, renal insufficiency, and the use of immunosuppressive therapies, though it may arise in various clinical contexts. Despite its significance, reports of PRES in the field of anesthesiology remain limited. This case report presents the case of a 46-year-old male admitted for elective ambulatory ophthalmic surgery under general anesthesia who developed delayed emergence from anesthesia and post-operative blindness, both attributed to the intraoperative onset of PRES. Anesthesiologists should be vigilant for PRES as a potential complication during the perioperative period, and consider it in the differential diagnosis for delayed emergence from anesthesia. Clinical suspicion should warrant prompt imagiological confirmation by magnetic resonance imaging (MRI), as delayed recognition and management can result in severe and long-term neurological disability.

SWATH-MS as a strategy for CHO host cell protein identification and quantification supporting the characterization of mAb purification platforms.

Host cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics' manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess. Here we explored a mass spectrometry (MS)-based proteomics tool, the sequential window acquisition of all theoretical fragment-ion spectra (SWATH) strategy, as an orthogonal method to traditional ELISA. The SWATH workflow was applied for high-throughput individual HCP identification and quantification, supporting characterization of a mAb purification platform. The design space of HCP clearance of two polishing resins was evaluated through a design of experiment study. Absolute quantification of high-risk HCPs was achieved (reaching 1.8 and 4.2 ppm limits of quantification, for HCP A and B respectively) using HCP-specific synthetic heavy labeled peptide calibration curves. Profiling of other HCPs was also possible using an average calibration curve (using labeled peptides from different HCPs). The SWATH approach is a powerful tool for HCP assessment during bioprocess development enabling simultaneous monitoring and quantification of different individual HCPs and improving process understanding of their clearance.

Neurological Disease Modeling Using Pluripotent and Multipotent Stem Cells: A Key Step towards Understanding and Treating Mucopolysaccharidoses.

Despite extensive research, the links between the accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of mucopolysaccharidoses (MPSs) have yet to be further elucidated. This is particularly true for the neuropathology of these disorders; the neurological symptoms are currently incurable, even in the cases where a disease-specific therapeutic approach does exist. One of the best ways to get insights on the molecular mechanisms driving that pathogenesis is the analysis of patient-derived cells. Yet, not every patient-derived cell recapitulates relevant disease features. For the neuronopathic forms of MPSs, for example, this is particularly evident because of the obvious inability to access live neurons. This scenario changed significantly with the advent of induced pluripotent stem cell (iPSC) technologies. From then on, a series of differentiation protocols to generate neurons from iPSC was developed and extensively used for disease modeling. Currently, human iPSC and iPSC-derived cell models have been generated for several MPSs and numerous lessons were learnt from their analysis. Here we review most of those studies, not only listing the currently available MPS iPSC lines and their derived models, but also summarizing how they were generated and the major information different groups have gathered from their analyses. Finally, and taking into account that iPSC generation is a laborious/expensive protocol that holds significant limitations, we also hypothesize on a tempting alternative to establish MPS patient-derived neuronal cells in a much more expedite way, by taking advantage of the existence of a population of multipotent stem cells in human dental pulp to establish mixed neuronal and glial cultures.

Digital pathology implementation in a private laboratory: The CEDAP experience.

Introduction: The transition to digital pathology has been carried out by several laboratories across the globe, with some cases described in Portugal. In this article, we describe the transition to digital pathology in a high-volume private laboratory, considering the main challenges and opportunities. Material and methods: Our process started in 2020, with laboratory workflow adaptation and we are currently using a high-capacity scanner (Aperio GT450DX) to digitize slides at 20×. The visualization system, Aperio eSlide Manager WebViewer, is integrated into the Laboratory System. The validation process followed the Royal College of Pathologists Guidelines. Results: Regarding validation, the first phase detected an error rate of 6.8%, mostly due to digitization errors. Phase optimization and collaboration with technical services led to improvements in this process. In the second validation phase, most of the slides had the desired quality for evaluation, with only an error rate of 0.6%, corrected with a new scan. The interpathologist correlation had a total agreement rate of 96.87% and 3.13% partial agreement. Conclusion: The implementation and validation of digital pathology was a success, being ready for prime time. The total integration of all laboratory systems and the acquisition of new equipment will maximize their use, especially with the application of artificial intelligence algorithms.

Help Comes from Unexpected Places: How a Tiny Fairy and a Tropical Fish may help us Model Mucopolysaccharidoses.

INTRODUCTION: When it comes to disease modeling, countless models are available for Lysosomal Storage Diseases (LSD). Historically, two major approaches are well-established: in vitro assessments are performed in patient fibroblasts, while in vivo pre-clinical studies are performed in mouse models. Still, both platforms have a series of drawbacks. Thus, we implemented two alternative and innovative protocols to mimic a particular sub-group of LSDs, the Mucopolysaccharidoses both in vitro and in vivo. METHODS: The first one relies on a non-invasive approach using dental pulp stem cells from deciduous teeth (SHEDs). SHEDs are multipotent neuronal precursors that can easily be collected. The second uses a state-of-the-art gene editing technology (CRISPR/Cas9) to generate zebrafish disease models. RESULTS: Even though this is an ongoing project, we have already established and characterized two MPS II and one MPS VI SHED cell models. These cells self-maintain through several passages and can give rise to a variety of cells including neurons. Furthermore, all MPS-associated sub-cellular phenotypes we have assessed so far are easily observable in these cells. Regarding our zebrafish models, we have successfully knocked down both naglu and hgsnat and the first results we got from the behavioral analysis are promising ones, as we can observe altered activity and sleep patterns in the genetically modified fish. For this particular approach we chose MPS III forms as our target disorders, since their neurological features (hyperactivity, seizures and motor impairment) and lifespan decrease would be easily recognizable in zebrafish. CONCLUSION: Now that these methods are well-established in our lab, their potential is immense. On one hand, the newly developed models will be of ultimate value to understand the mechanisms underlying MPS sub-cellular pathology, which have to be further elucidated. On the other hand, they will constitute an optimal platform for drug testing in house. Also noteworthy, our models will be published as lab resources and made available for the whole LSD community.