7196 research outputs found
Sort by
Dynamic metabolic modeling of CHO cell metabolism coupled with N-glycosylation in the industrial pharmaceutical production
No full textCellular metabolism consists of a complex network of biochemical reactions and thus presents a challenging modeling problem. Experimental and modeling work, described in this article is focused on the metabolic pathway of Chinese hamster ovary (CHO) cells, which are the preferred expression system for monoclonal antibody (mAb) protein production. CHO cells are one of the primary hosts for mAbs production, which have extensive applications in multiple fields like biochemistry, biology and medicine. Here, an approach to explain cellular metabolism with in-silico modeling of a microkinetic reaction network is presented and validated with unique experimental results. Experimental data of 25 different fed-batch bioprocesses included the variation of multiple process parameters, such as pH, agitation speed, oxygen and carbon dioxide content, and dissolved oxygen. 151 metabolites were involved in our proposed metabolic network, which consisted of 132 chemical reactions that describe the reaction pathways, and include 25 reactions describing N-glycosylation and additional reactions for the accumulation of the produced glycoforms. Additional 8 reactions are considered for accumulation of the N-glycosylation products in the extracellular environment and 1 reaction to correlate cell degradation. The following pathways were considered: glycolysis, pentose phosphate pathway, nucleotide synthesis, tricarboxylic acid cycle, lipid synthesis, protein synthesis, biomass production, anaplerotic reactions and membrane transport. Our contribution to this field is the comparison of unique experimental data to our model, which is coupled with biomass production and N-glycosylation. The effect of various operational conditions was assessed and their effect on the cell production process. The modeling performed is a complementary tool to experimentation, nevertheless, with the applied modeling procedure, different operational scenarios and fed-batch techniques can be tested without the need for long-term experimental campaigns
Transmission Electron Microscopy of the Retina: A Method for Sample Preparation and Evaluation
No full textUltrastructural pathology is critical in the morphologic evaluation and characterization of subcellular structures in nonclinical toxicity and efficacy studies. In murine models of ophthalmologic disease, clinical examination is typically paired with other techniques like electroretinography (ERG) and/or optical coherence tomography (OCT) to more fully characterize a finding. High-quality transmission electron microscopy (TEM) can provide a critical, image-based link between these approaches, providing greater confidence in interpretation of ERG or OCT results. In addition to characterization of disease models, TEM can provide detailed visualization of retinal changes identified by clinical examination or light microscopy in nonclinical toxicity studies. The spherical shape of the eye presents unique challenges for trimming, orientation, imaging, and evaluation by TEM. The varied components of the eye require specialized approaches for embedding to facilitate successful sectioning. Controlling for the orientation of the retina is critical to consistent evaluation, driving the need for an improved method of embedding this unique and complex organ. The authors describe a method of sample preparation resulting in optimal orientation of the posterior aspect of murine eyes (rat and mouse) for TEM of the neural retina, Bruch’s membrane and/or choroid, with examples from mouse ophthalmic disease models
Spontaneous background and procedure-related microscopic findings and common artefacts in ocular tissues of laboratory animals in ocular studies
No full textIdentification of test article-related microscopic findings in ocular toxicology studies requires a working knowledge of the common background and procedure-related findings and artefacts encountered in such studies. The objective of this manuscript is to provide a mini-atlas of the artefacts and the procedure-related and spontaneous background findings commonly observed in ocular tissues from animals in toxicology studies of ocular drug candidates. Artefacts in the eye are most often related to collection and fixation procedures, and include swelling and vacuolation of lens fibers and separation of the neuroretina from the retinal pigment epithelium (RPE). Common in-life procedure-related findings include intravitreal injection needle tracks in the sclera and ciliary body pars plana and foci of RPE hypertrophy and/or hyperpigmentation at subretinal injection sites. Common background findings include corneal mineralization, uveal mononuclear cell infiltrates, and peripheral displacement of photoreceptor nuclei in the retina. A few uncommon spontaneous background findings that may be confused with test article-related findings, such as bilateral optic atrophy in macaques, are also included
Ianalumab (VAY736) in primary Sjögren’s syndrome: assessing disease activity using multi-modal ultrasound
No full textObjective: Apply serial ultrasound (US) assessments to show effects of ianalumab (anti-BAFF-R monoclonal antibody) on inflamed salivary glands of patients with primary Sjögren’s syndrome (pSS).
Methods: In a single-center, 24-week double-blind study (NCT02149420), 27 pSS patients of moderate-to-severe activity were randomly assigned to receive a single i.v. dose of either 3 mg/kg or 10 mg/kg ianalumab, or placebo. Concurrent with clinical and laboratory outcomes, multi-modal US images were acquired of bilateral parotid glands (PG) and submandibular glands (SMG) at weeks 0, 6, 12, and 24. Applied US modalities included 1) B-mode echostructure scored by de Vita classification, 2) macrovascular blood flow by power Doppler, and in PG only 3) microvascularization using contrast-enhanced US (area under the curve, time to peak or TTP) and 4) gland stiffness by sonoelastography.
Results: Clinical study results were previously published. US data for PG differed from SMG but were comparable between respective left and right sides of these glands. Numerical improvements in salivary gland quality and declining tissue inflammation were observed in treated versus placebo groups, including more patients achieving ≥1-point reduction from baseline in de Vita score, along with trends towards decreased perfusion and stiffness. Correlations between clinical endpoints and US parameters were largely restricted to microvascular perfusion TTP and at the 12-week timepoint when ianalumab effects were predicted at maximal.
Conclusion: Early, in vivo signs of salivary gland improvement in response to an effective intervention can be shown without need of biopsy by using a non-invasive, comprehensive, ultrasound-based approach over multiple time points
Case-Based Thermal Safety Evaluation on a Pharmaceutical Process Using Dimethyl Sulfoxide as a Solvent
No full textA rupture disc burst was recorded when conducting a calorimetric test by sensitive detection of exothermic reaction. The tested sample was a reaction mixture from a hydroamination process containing the solvent dimethyl sulfoxide (DMSO). Subsequent investigation indicated that the event was caused by the strong autocatalytic decomposition of DMSO. The hazardous decomposition of DMSO that could be initiated by an inadvertent contact with substances involved in the process was studied.
Furthermore, risk evaluation on the process thermal runaway was carried out based on the cooling failure scenario. In this particular case, although Stoessel criticality class 2 was assigned, the boiling point of DMSO could not serve as the safety barrier. On the basis of the thermal safety evaluation, the hydroamination process was safely scaled up in the Novartis pilot plant and successfully launched in a commercial production unit. This investigation highlights that every scale-up process involving DMSO as a solvent should be carefully evaluated owing to the potential safety concern caused by its autocatalytic behavior
Behavioral, respiratory and metabolic consequences of impaired cerebrovascular reactivity
No full textCarbon dioxide (CO2) and protons (H+) have a strong influence on cerebral perfusion, but the function of this is not clear yet. Here, we found that GPR4, a receptor for H+ in the vasculature, sensed CO2/H+ and that an endothelial Gαq/11-dependent signaling pathway mediated the CO2/H+ effect on cerebrovascular reactivity. While CO2/H+-induced Gαq/11 signaling constricted vessels in the retrotrapezoid nucleus, it had a dilative effect in other brain areas explaining why loss of cerebrovascular reactivity in mice differentially modulated CO2 effects: it reduced respiration but aggravated behavioral and metabolic responses to CO2. Even with normal CO2 concentrations mice with impaired cerebrovascular reactivity were more anxious and showed metabolic changes indicating that cerebrovascular reactivity is essential for normal physiology
Fluopack screening platform for unbiased cellular phenotype profiling.
No full textGene and compound functions are often interrogated by perturbation. However, we have limited methods to capture associated phenotypes in an unbiased and holistic manner. Here, we describe Fluopack screening as a novel platform enabling the profiling of subcellular phenotypes associated with perturbation. Our approach leverages imaging of a panel of fluorescent chemical probes to survey cellular processes in an unbiased and high throughput fashion. Segmentation-free, whole image analysis applied to Fluopack images identifies probes revealing distinct phenotypes upon perturbation, thereby informing on the function and mechanism of action of perturbagens. This chemical biology approach allows to interrogate phenotypes that tend to be overlooked by other methods, such as lipid trafficking and ion concentration inside the cell. Fluopack screening is a powerful approach to study orphan protein function, as exemplified by the characterization of TMEM41B as novel regulator of lipid mobilization
Nickel-Catalyzed Cyanation of Aryl Halides and Hydrocyanation of Alkynes via C-CN Bond Cleavage and Cyano Transfer
No full textWe report the nickel-catalyzed cyanation and hydrocynation methods to prepare aryl nitriles and vinyl nitriles from aryl halides and alkynes, respectively. Using cheap and nontoxic 4-cyanopyridine N-oxide as the cyano shuttle, the methods provide an efficient approach to prepare aryl cyanides and vinyl nitriles under mild and operationally simple reaction conditions with a broad range of functional group tolerance. In hydrocyanation of alkynes, the method demonstrated good regioselectivity, producing predominantly E or Z-alkenyl nitriles in a controlled manner and exclusively Markovnikov vinyl nitriles when internal diaryl alkynes and terminal alkynes were applied as the substrates, respectively. The preliminary mechanistic investigation indicated that C-CN bond cleavage process is promoted by oxidative addition to nickel(I) complex in the cyanation of aryl halides, and the further studies via a series of deuterium exchange experiments indicated that water serves as the hydrogen source for the hydrocyanation of alkynes
Characterizing drug-target interactions in complex tissues
No full textRecently, Perrin et al. reported that the coupling of the cellular shift assay with an unbiased proteomics read-out (Thermal Proteome Profiling, TPP) can be applied to complex tissue samples from model organisms and patients. This study demonstrates that TPP enables organ-specific target engagement and identification studies even in patient-derived material
Behavioral and neural network abnormalities of human APP transgenic mice resemble those of App knockin mice but are not prevented by early inhibition of BACE1
No full textAlzheimer’s disease (AD) is the most frequent and costly neurodegenerative disorder and has been recalcitrant to therapeutic interventions. Diverse lines of evidence suggest that the amyloid precursor protein (APP) is involved in its causation. However, the precise mechanisms remain unknown. A favorite hypothesis has been that APP contributes to AD pathogenesis through the cerebral accumulation of the amyloid-peptide (A), which is derived from APP through sequential proteolytic cleavage by BACE1 and-secretase. However, inhibitors of these enzymes have failed in clinical trials despite clear evidence for target engagement. To shed light on this puzzling situation, we compared transgenic mice overexpressing wildtype human APP (hAPP) or hAPP carrying mutations that cause autosomal dominant familial AD (FAD) with knockin mice that do not overexpress hAPP but have two mouse App alleles with FAD mutations and a humanized A sequence. Although these lines of mice had marked differences in cortical and hippocampal levels of APP, APP C-terminal fragments, soluble A, A oligomers, and age- dependent amyloid deposition, they all developed cognitive deficits as well as non-convulsive epileptic activity, a type of network dysfunction that also occurs in a substantive proportion of humans with AD. Pharmacological inhibition of BACE1 effectively reduced levels of amyloidogenic APP C-terminal fragments, soluble A, A oligomers, and amyloid deposits in transgenic mice expressing FAD-mutant hAPP, but did not improve any of their functional deficits, even when initiated at early stages before amyloid deposits were detectable. We conclude that APP contributes to AD-related functional alterations through one or more complex mechanisms that may be impossible to block with treatments selectively aimed at A or other secretase- generated APP metabolites