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Behavioral or nutritional drive: which motivation affects rates of food grinding in CD1 mice?
No full textWire mouse feeders used in laboratory mouse cages typically hold enough food to feed 5 mice for two weeks. However, some mice gnaw the food provided into powder, which they do not ingest. The ground-up food fills the cage, resulting in miscalculations of food consumption, welfare issues with blocked water valves and frequent cage handling, more frequent cage changes, and economic issues of food wastage. There is a dearth of studies attempting to understand the causes behind food grinding behavior. This study, following on from previous work, sought to determine if the motivations behind food grinding were related to a mouse’s innate behavioral drive to gnaw or rather a nutritional drive to seek out macronutrient components in the feed. We replicated previous results where high fat treatments (either shell-on sunflower seeds or a diet with macronutrient equivalence to a sunflower kernel), but not the method of delivery of the higher-fat treatments, decreased the amount of ground food produced per cage. Further, the composition of the gnawed food changed over the course of the study, generally with protein increasing and fat decreasing in the gnawed feed, especially after treatments were removed. We also replicated previous results which found differences in the composition of the gnawed feed in comparison to the original diet. These results support the hypothesis that food grinding behavior is driven by a nutritional motivation, not a behavioral one
Finding suitable solvents for the green transformation
No full textSolvent usage is one of the most critical factors for the carbon footprint of the chemical
and pharmaceutical industries. Therefore, finding suitable green solvents that can
be sourced from biomass plays a central role in designing more sustainable manufacturing
processes. One of the greenest solvents is water, and chemical transformations
in or on aqueous solution play an increasingly important role. However, some processes
still require the extraction of the reaction product from water with an organic phase,
and analogous purifications might be necessary for the cleaning of the resulting chemical
waste water. To guide the search for suitable green solvents for extractions from
aqueous solution, the extraction efficiency of a series of green solvents was evaluated
for twelve solutes with molecular simulations. In particular, partition coefficients from
water were evaluated for cyclopentanol, ethyl acetate, 1-butanol, 2-pentanol, 2-methyl
tetrahydrofuran, 1-pentanol, cyclopentyl methyl ether, butyl methyl ether, butyl acetate,
1-octanol, and methyl oleate. Because of the high correlation of the partition
coefficient results of the different solvents with the widely available partition coefficient
of 1-octanol (LogPocoh, also known as logKow), the suitabilty of the selected solvents
for the extraction of a particular solute can be predicted. Cyclopentanol or 1-butanol
are best suited for hydrophilic molecules (LogPocoh < 0.5), while cyclopentyl methyl
ether and butyl methyl ether are particularly efficient for hydrophobic solutes (LogPocoh
2
> 2.6). Ethyl acetate or 1-pentanol turned out to be the most efficient extraction solvents
in the mid-range (LogPocoh between 0.5 to 2.6). The findings are verified based on
experimental extraction efficiencies of an aqueous solution from a micelle-enabled crosscoupling
transformation. The experimental extraction yields confirm that 1-butanol is
a suitable solvent for extractions of hydrophilic molecules from aqueous micellar media.
The experiments also show that only the six most hydrophilic solvents give rise to
a clear phase separation in the presence of residual organic solvents and surfactants.
This indicates that aqueous micellar media might require different extraction solvents
than other chemical reactions. This highlights that both insight at the molecular level
and practical considerations of the application at hand are needed for the selection of
suitable green solvents
Applications of Cell-Based Protein Array Technology to Preclinical Safety Assessment of Biological Products.
No full textOff-target evaluation is essential in preclinical safety assessments of novel biotherapeutics, supporting lead molecule selection, endpoint selection in toxicology studies, and regulatory requirements for first-in-human trials. Off-target interaction of a therapeutic antibody and antibody derivatives has been historically assessed via the Tissue Cross-Reactivity (TCR) study, in which the candidate molecule is used as a reagent in immunohistochemistry (IHC) to assess binding of the candidate molecule to a panel of human tissue sections. The TCR approach is limited by the performance of the therapeutic as an IHC reagent, which is often suboptimal to outright infeasible. Furthermore, binding of the therapeutic in IHC conditions typically has poor in vitro to in vivo translation and lacks qualitative data of the identity of putative off-targets limiting the decisional value of the data. More recently, cell-based protein arrays (CBPA) that allow for screening against a large portion of the human membrane proteome and secretome have emerged as a complement, and likely a higher value alternative, to IHC-based off-target assessment. These arrays identify specific protein interactions and may be useful for testing nontraditional antibody-based therapeutic formats that are unsuitable for TCR studies. This article presents an overview of CBPA technologies in the context of TCR and off-target assessment studies. Selected case examples and strategic considerations covering a range of different modalities are presented
The histone H3 lysine 36 demethylase KDM2A/FBXL11 controls Polycomb-mediated gene repression and germ cell development in male mice.
No full textKDM2A/FBXL11 is a Jumonji-domain containing lysine demethylase catalyzing the removal of mono- and di-methyl modifications of histone H3 lysine 36 (H3K36me1/2). While Kdm2a is required for mouse embryogenesis, its role in adult physiology has been largely unexplored. Using conditional deletion approaches, we demonstrate that Kdm2a deficiency leads to testicular atrophy and male infertility. Although spermatogonial stem cells remain unaffected, proliferating and differentiating spermatogonia exhibit delayed cell cycle progression and apoptosis. RNA-sequencing of purified spermatogonia and spermatocytes reveals Kdm2a-dependent repression of over 750 genes during spermatogonial differentiation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) demonstrates increased H3K36me2 levels at CpG-rich gene promoters in Kdm2a-deficient spermatogonia. KDM2A is required for Polycomb-mediated repression as shown by increased H3K36me2 and reduced H3K27me3 promoter occupancies and failed gene repression in Kdm2a deficient differentiating spermatogonia. Loss of Kdm2a in spermatocytes disrupts progression through meiotic prophase, as evidenced by impaired completion of chromosome synapsis and processing of meiotic double-strand breaks (DSBs), by altered chromatin states and by an impairment of X-linked gene repression. Our study thus identifies critical roles for KDM2A in coordinating gene expression programs during spermatogonial differentiation and meiosis, which are essential for male germ cell development
Practically Significant Method Comparison Protocols for Machine Learning in Small Molecule Drug Discovery.
No full textMachine Learning (ML) methods that relate molecular structure to properties are frequently proposed as in silico surrogates for expensive or time-consuming experiments. In small molecule drug discovery, such methods inform high-stakes decisions like compound synthesis and in vivo studies. This application lies at the intersection of multiple scientific disciplines. When comparing new ML methods to baseline or state-of-the-art approaches, statistically rigorous method comparison protocols and domain-appropriate performance metrics are essential to ensure replicability and ultimately the adoption of ML in small molecule drug discovery. This paper proposes a set of guidelines to incentivize rigorous and domain-appropriate techniques for method comparison tailored to small molecule property modeling. These guidelines, accompanied by annotated examples using open-source software tools, lay a foundation for robust ML benchmarking and thus the development of more impactful methods
Neurotoxicology and Safety Pharmacology Investigations on the Nervous System: 2024 Industry Survey
No full textThe American College of Toxicology (ACT), the Safety Pharmacology Society (SPS) and the Society for Toxicological Pathology (STP) conducted an industry survey in 2024 to assess industry practices as they relate to neurotoxicity and safety testing of neurotherapeutics. One hundred thirty (130) respondents from Asia (5%), Europe (32%) and North America (64%) responded to the survey. Most respondents (54%) were from pharmaceutical companies (>1000 employees). Small molecules (89%), large molecules (73%), gene therapy (52%), cell therapy (41%) and vaccines (38%) were the types of products developed by respondents. Oncology (72%) and neurology/psychiatry (64%) were the most frequent target indications pursued by companies followed by inflammation (56%), cardiovascular (48%), rare/orphan (44%), metabolic (42%), infectious (35%) and respiratory (28%) diseases. Tremors (81%), emesis (75%), gait/coordination abnormalities (67%), convulsion (65%), salivation (61%) and peripheral neuropathy (24%) were the most encountered CNS issues in pre-clinical drug development while headache (59%), emesis/nausea (49%), dizziness (45%), fatigue (30%) and tremors (19%) were the most frequent issues encountered in Phase I clinical trials. Most respondents reported using a modified Irwin’s test (90%) added to toxicology studies (80%) or using a standalone study (71%). Implanted EEG was the most frequently used electrophysiology methodology (66%) while stem cell derived neurons were the most frequent in vitro methodology (50%). The rat was reported as the most common species used for functional observation battery (FOB) (91%), EEG (70%), seizure liability (84%) and juvenile CNS studies (84%)
Designing a Patient Preference Study on Subcutaneous Medical Devices: Incorporating Health Authority Scientific Advice and Patient Perspectives
No full textThis paper describes the planning of a patient preference study for evaluating device features and administrative solutions for the subcutaneous (SC) delivery of high doses / large volumes (e.g., >2mL) of drugs.
A mixed-methods approach was used including qualitative research studies, the involvement of patients as research partners, and solicitation of advice from the FDA. This resulted in the development of a preference study protocol to investigate what people living with MS (PLwMS) consider important regarding medical device features for high dose SC administration, including refinement of the attributes and levels which will be central to the planned quantitative online patient preference study
HaloPROTAC3 does not trigger the degradation of the halotagged parasitophorous vacuole membrane protein UIS4 during Plasmodium liver stage development.
No full textTargeted protein degradation (TPD) is a novel strategy for developing therapeutics against pathogens. Prior to causing malaria, Plasmodium parasites replicate within hepatocytes as liver stages, surrounded by a parasitophorous vacuole membrane (PVM). We hypothesized that TPD can be employed to trigger host-driven degradation of essential liver stage PVM proteins and lead to parasite death. To explore this, we took advantage of the proteolysis-targeting-chimera HaloPROTAC3, a molecule that recruits the host von Hippel-Lindau (VHL) E3 ligase to the HaloTag (HT). Parasites expressing HT fused to the host cytosol-exposed domain of the PVM protein UIS4 (UIS4-HT) were generated in Plasmodium berghei and Plasmodium cynomolgi, but only P. berghei UIS4-HT enabled productive liver stage infection experiments in vitro. Although HaloPROTAC3 triggered the degradation of HT proteins in host cells, it had no impact on the survival of P. berghei UIS4-HT liver stages. Furthermore, HaloPROTAC3 bound to P. berghei UIS4-HT but did not recruit VHL or trigger ubiquitination of the PVM. Overall, although this study did not establish whether host-driven TPD can degrade Plasmodium PVM proteins, it highlights the challenges of developing TPD approaches against novel targets and offers insights for advancing this therapeutic strategy against pathogens
Mechanistic insights into dengue virus inhibition by a clinical trial compound NITD-688
No full textDengue, caused by the dengue virus (DENV), presents a significant public health challenge with limited effective treatments. NITD-688 is a potent panserotype DENV inhibitor currently in Phase II clinical trials. However, its mechanism of action is not fully understood. Here, we present the molecular details of how NITD-688 inhibits DENV. NITD-688 binds directly to the nonstructural protein 4B (NS4B) with nanomolar affinities across all four DENV serotypes and specifically disrupts the interaction between NS4B and nonstructural protein 3 (NS3) without significantly changing the interactions between NS4B and other viral or host proteins. NS4B mutations that confer resistance to NITD-688 reduce both NITD-688 binding to NS4B and disruption of the NS4B/NS3 interaction. Specifically, NITD-688 blocks the interaction of NS3 with a cytosolic loop within NS4B. This inhibits the formation of new NS4B/NS3 complexes and disrupts preexisting complexes in vitro and DENV-infected cells, ultimately inhibiting viral replication. Consistent with this mechanism, NITD-688 retains greater potency in cellular assays with delayed treatment compared to JNJ-1802, another NS4B inhibitor that has been studied in Phase II clinical trials. Together, these findings provide critical insights into the mechanism of action of NITD-688, facilitating the development of novel flavivirus NS4B inhibitors and informing future clinical interventions against DENV
Host cell protein-mediated adjuvanticity and immunogenicity risks of biotherapeutics
No full textHost cell proteins (HCPs) are process-related impurities of biotherapeutic production that might affect product quality and/or patient safety. In a few cases, adverse events were attributed to HCPs present in the administered biotherapeutic. HCP-associated immune risks include adjuvanticity and immunogenicity with potential cross-reactivity. Based on the published data, some HCPs can act as adjuvants increasing the immunogenicity of the biotherapeutic as a bystander effect. HCPs may also induce immunogenicity against themselves, resulting in anti-HCP T cell responses and anti-HCP antibody formation. Depending on sequence similarities, these anti-HCP immune responses might theoretically be cross-reactive to the biotherapeutic or human endogenous proteins. In this review, we examine HCP-associated immune-related risks reported from non-clinical and clinical studies. We also discuss the potential and limitations of in vitro and in silico methods to evaluate the adjuvanticity and immunogenicity potential of HCPs. A risk-based assessment of the safety impact of HCPs may include the identity of the HCP and similarity to the biotherapeutic and human proteins, as well as product, treatment-, and patient-related factors