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Modeling methodology for defining a priori the hydrodynamics of a dynamic suspension bioreactor. Application to human induced pluripotent stem cell culture
No full textno abstrac
Aβ-induced acceleration of Alzheimer-related τ-pathology spreading and its association with prion protein
No full textExtracellular deposition of amyloid β-protein (Aβ) in amyloid plaques and intracellular accumulation of abnormally phosphorylated τ-protein (p-τ) in neurofibrillary tangles (NFTs) represent pathological hallmark lesions of Alzheimer’s disease (AD). Both lesions develop in parallel in the human brain throughout the preclinical and clinical course of AD. Nevertheless, it is not yet clear whether there is a direct link between Aβ and τ pathology or whether other proteins are involved in this process. To address this question, we crossed amyloid precursor protein (APP) transgenic mice overexpressing human APP with the Swedish mutation (670/671 KM → NL) (APP23), human wild-type APP (APP51/16), or a proenkephalin signal peptide linked to human Aβ42 (APP48) with τ-transgenic mice overexpressing human mutant 4-repeat τ-protein with the P301S mutation (TAU58). In 6-month-old APP23xTAU58 and APP51/16xTAU58 mice, soluble Aβ was associated with the aggravation of p-τ pathology propagation into the CA1/subiculum region, whereas 6-month-old TAU58 and APP48xTAU58 mice neither exhibited significant amounts of p-τ pathology in the CA1/subiculum region nor displayed significant levels of soluble Aβ in the forebrain. In APP23xTAU58 and APP51/16xTAU58 mice showing an acceleration of p-τ propagation, Aβ and p-τ were co-immunoprecipitated with cellular prion protein (PrPC). A similar interaction between PrPC, p-τ and Aβ was observed in human AD brains. This association was particularly noticed in 60% of the symptomatic AD cases in our sample, suggesting that PrPC may play a role in the progression of AD pathology. An in vitro pull-down assay confirmed that PrPC is capable of interacting with Aβ and p-τ. Using a proximity ligation assay, we could demonstrate proximity (less than ~ 30–40 nm distance) between PrPC and Aβ and between PrPC and p-τ in APP23xTAU58 mouse brain as well as in human AD brain. Proximity between PrPC and p-τ was also seen in APP51/16xTAU58, APP48xTAU58, and TAU58 mice. Based on these findings, it is tempting to speculate that PrPC is a critical player in the interplay between Aβ and p-τ propagation at least in a large group of AD cases. Preexisting p-τ pathology interacting with PrPC, thereby, appears to be a prerequisite for Aβ to function as a p-τ pathology accelerator via PrPC
Aβ reduction at early disease stages prevents progression of cerebral amyloid angiopathy in a mouse model of hereditary cerebral hemorrhage with amyloidosis - Dutch type
No full textIntroduction: Deposition of -amyloid peptide (A) within cerebral vessels (cerebral amyloid angiopathy, CAA) contributes to intracerebral bleedings and altered brain function. CAA occurs with aging and to various degree in Alzheimer’s disease.
Methods: APPDutch mice model hereditary cerebral hemorrhage with amyloidosis – Dutch-type (HCHWA-D) and develop CAA in the vast absence of parenchymal amyloid. 3D-ultramicroscopy and immunoassays were used to assess CAA onset and progression. An inhibitor to the β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) was used to lower A generation.
Results: CAA was first detected in frontal leptomeningeal and superficial cortical vessels followed by vessels penetrating the cortical layers. Upon CAA onset, A in the CSF was decreased. BACE1-inhibition initiated at the onset of CAA, and continued for 4 months, largely prevented CAA progression and associated microgliosis.
Discussion: Results provide a preclinical basis for A-reducing treatments in patients at risk of CAA and in presymptomatic HCHWA-D mutation carriers
Partial inhibition of mTORC1 in aged rats counteracts the decline in muscle mass and reverses molecular signaling associated with sarcopenia
No full textThere is a lack of pharmacological interventions available for sarcopenia, a progressive age-associated loss of muscle mass, leading to a decline in mobility and quality of life. We found mTORC1 (mammalian target of rapamycin complex 1), a well-established positive modulator of muscle mass, to be surprisingly hyperactivated in sarcopenic muscle. Furthermore, partial inhibition of the mTORC1 pathway counteracted sarcopenia, as determined by observing an increase in muscle mass and fiber type cross-sectional area in select muscle groups, again surprising because mTORC1 signaling has been shown to be required for skeletal muscle mass gains in some models of hypertrophy. Additionally, several genes related to senescence were downregulated and gene expression indicators of neuromuscular junction denervation were diminished using a low dose of a "rapalog" (a pharmacological agent related to rapamycin). Therefore, partial mTORC1 inhibition may delay the progression of sarcopenia by directly and indirectly modulating multiple age-associated pathways, implicating mTORC1 as a therapeutic target to treat sarcopenia
Development of the synthesis of BEZ235 on ton scale
No full textABSTRACT: The development of a robust synthesis of 2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]propionitrile (NVP-BEZ235 (Dactolisib); compound 10) to a ton-scale process is described. The key step is a Pd-catalyzed Suzuki coupling of 2[4-(8-bromo-3-methyl-2-oxo-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]2-methyl-propionitrile to 3-quinoline boronic acid. A special focus is put on the reduction of the amount of Pd catalyst used in the Suzuki coupling and the removal of traces of Pd from the crude drug substance, as well as the purification of the crude drug substance
A community-based lung cancer rapid tissue donation protocol provides high-quality drug-resistant specimens for proteogenomic analyses
No full textBackground: For the advancement of cancer research, the collection of tissue specimens from drug-resistant tumors after targeted therapy is crucial. Although patients with lung cancer are often provided targeted therapy, post-therapy specimens are not routinely collected due to the risks of collection, limiting the study of targeted therapy resistance mechanisms. Posthumous rapid tissue donation (RTD) is an expedient collection process that provides an opportunity to understand treatment-resistant lung cancers. Methods: Consent to participate in the thoracic RTD protocol was obtained during patient care. When death occurred, tumor and paired non-tumor, cytology, and blood specimens were collected within 48 hours and preserved as formalin-fixed and frozen specimens. Tissue sections were evaluated with hematoxylin and eosin staining and immunohistochemistry (IHC) against multiple biomarkers, including various programmed death ligand 1 (PD-L1) clones. Next-generation sequencing was performed on 13 specimens from 5 patients. Results: Postmortem specimens (N = 180) were well preserved from 9 patients with lung cancer. PD-L1 IHC revealed heterogeneity within and between tumors. An AGK-BRAF fusion was newly identified in tumor from a donor with a known echinoderm microtubule-associated protein-like 4 to anaplastic lymphoma kinase (EML4-ALK) fusion and history of anaplastic lymphoma kinase (ALK) inhibitor therapy. RNA expression analysis revealed a clonal genetic origin of metastatic cancer cells. Conclusions: Post-therapy specimens demonstrated PD-L1 heterogeneity and an acyl glycerol kinase to B-rapidly accelerated fibrosarcoma (AGK-BRAF) fusion in a patient with an EML4-ALK–positive lung adenocarcinoma as a potential resistance mechanism to ALK inhibitor therapy. Rapid tissue donation collection of postmortem tissue from lung cancer patients is a novel approach to cancer research that enables studies of molecular evolution and drug resistance
Shortened abstract for Enhanced CHO clone screening using Next-Generation Sequencing and Targeted Locus Amplification publication
No full textEarly analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next-generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single-nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high-level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays
Benchmarking different QM-levels for usage with COSMO-RS
No full textThe COSMO-RS method is an established method for the prediction of fluid phase properties such as activity coefficients, liquid-liquid equilibria and free energy of solvation. It is also frequently used in quantum-chemistry based chemical reaction modelling to predict the solvation contribution to the reactions. The COSMOtherm software, which features the currently most advanced implementation of COSMO-RS, is based on quantum-chemical COSMO calculations using the BP86 functional with the def2-TZVPD basis set. As the accuracy of COSMO-RS depends on the accuracy of the underlying quantum-chemical (QC) calculation, it is important to validate the currently used level against other common, presumably superior, approaches such as the more recently developed M06-2x hybrid density functional, or wave-function methods such as MP2. As compared to other applications where the electronic energy is the most important result of the QC calculation, the COSMO-RS method has a much higher dependence on the molecular polarity and thus the electron density distribution. We find that MP2, PBE0 and M06-2X perform slightly worse in fully reparametrized COSMO-RS with respect to the prediction of experimentally measured properties like pKa or logP. Although MP2 was reported to yield better polarities than most DFT functionals for spin unpolarized molecules, this theoretical advantage does not manifest in a practical benefit for the prediction of the properties with the refitted COSMO-RS parameters. Other pure DFT functionals such as PBE or TPSS can be used instead of BP86, but again no practical advantage is expected as they yield extremely similar polarities to the original BP86 calculations
Benzimidazolyl-pyrazolo[3,4-b]pyridinones - selective inhibitors of MOLT-4 leukemia cell growth and sea urchin embryo spiculogenesis: Target quest.
No full text1,3-Substituted pyrazolo[3,4-b]pyridinones 11–18 were synthesized by a three-component condensation of Meldrum’s acid with aryl aldehydes and 1,3-substituted 5-aminopyrazoles. Their biological activity was evaluated using the in vivo phenotypic sea urchin embryo assay and the in vitro cytotoxicity screen against human cancer cell lines. In the sea urchin embryo model, 1-benzimidazolyl-pyrazolo[3,4-b]pyridinones 11 caused inhibition of hatching and spiculogenesis at submicromolar concentrations. These compounds also selectively and potently inhibited growth of the MOLT-4 leukemia cell line. Subsequent structure-activity relationship studies determined the benzimidazolyl fragment as an essential pharmacophore for both effects. We applied numerous techniques for the target identification. A preliminary QSAR target identification search did not result in tangible leads. Attempts to prepare a relevant photoaffinity probe that retained potency in both assays were not successful. Compounds 11 were further characterized for their activity in a wild type vs Notch-mutant leukemia cell lines, and in in vitro panels of kinases and matrix metalloproteinases. Using a series of diverse modulators of spiculogenesis as standards, we excluded multiple signaling networks including Notch, Wnt/-catenin, receptor tyrosine kinases (VEGF/VEGFR, FGF/FGFR), PI3K, and Raf-MEK-ERK as possible targets of 11. On the other hand, matrix metalloproteinase-9/hatching enzyme was identified as one potential target
Inhibiting mGluR5 activity by AFQ056/Mavoglurant rescues circuit-specific functional connectivity in Fmr1 knockout mice
No full textPrevious work has demonstrated that neuroimaging biomarkers which capture functional connectivity of the brain can be used to define a specific and robust endophenotype in Fmr1-/y mice, a well-established animal model of human Fragile-X Syndrome (FXS). However, it is currently unknown whether this macroscopic measure of brain connectivity is sufficiently sensitive to reliably detect changes caused by pharmacological interventions. Here we inhibited the activity of the metabotropic glutamate receptor-5 (mGluR5) using AFQ056/Mavoglurant, a drug that is assumed to normalize excitatory/inhibitory neural signaling imbalances in FXS. We employed resting-state-fMRI (rs-fMRI) and diffusion-weighted imaging (DWI) to test whether Mavoglurant re-established brain connectivity - at least partly - within some of the affected circuits in Fmr1-/y mice that are related to social behavior deficits. In line with previous findings, we observed that Fmr1-/y mice exhibited impaired social interaction, reduced connectivity in three main functional networks and altered network topology. At the group level, Mavoglurant did neither rescue abnormal social behavioral nor white matter abnormalities; however, for some, but not all of these circuits Mavoglurant had a genotype-specific effect of restoring functional connectivity. These results show that rs-fMRI connectivity is sufficiently sensitive to pick up system-level changes after the pharmacological inhibition of mGluR5 activity. However, our results also show that the effects of Mavoglurant are confined to specific networks suggesting that behavioral benefits might be restricted to narrow functional domains