Transforming the Discovery of Targeted Protein Degraders: The Translational Power of Predictive PK/PD Modeling.

Targeted protein degraders (TPDs), an emerging therapeutic modality, are attracting considerable interest with the promise to address disease-related proteins that are not druggable with conventional small molecule inhibitors. Despite their novel mechanism of action, the PK/PD relationship of degraders is still approached with a mindset deeply rooted in inhibitor drugs. Here, we establish how predictive mechanistic modeling specifically tailored to TPDs can significantly enhance the value of the available information during lead generation and optimization. By integrating the results from in vitro assays with routinely collected PK data, modeling accurately predicts degradation in vivo. These predictions transform the prioritization of compounds for in vivo studies as well as the selection of optimal dose schedules and most informative measurement time points with the least number of animals. Moreover, the comprehensive modeling framework (1) identifies the PK/PD driver of targeted protein degradation and subsequent downstream pharmacodynamic effects, and (2) uncovers the fundamental difference between degrader and inhibitor PK/PD relationships. The practical utility of our predictive modeling is demonstrated with relevant use cases. This framework will allow researchers to transition from current, mostly serendipity-based approaches to more sound model-informed decision making. Going forward, the presented predictive PK/PD modeling framework lays out a rational path to incorporate inter-species differences in the pharmacology and thus promises to help with getting the dose right in clinical trials.

Neuroplastin splice variants Np55 and Np65: Who is doing the job in macrophages?

Neuroplastin, a paralog of CD147/Basigin, is known as a neuronal cell adhesion molecule and as an auxiliary subunit of plasma membrane calcium ATPases in both neurons and adaptive immune cells. Recently, an interesting study by Ren et al. (2022) provided evidence for an important role of neuroplastin in macrophages during bacterial infection. Here, we critically discuss one aspect of this study, the assignment of this role to Np65 as one of two prominent splice variants of neuroplastin.

An informative short-term study on the impacts of a triclocarban/weathered multi-walled carbon nanotube-adsorbed complex to benthic organisms.

Freshwater organisms are suitable models to study the fate of environmental pollutants. Due to their versatile and everyday use, many environmental pollutants such as triclocarban (TCC) or multi-walled carbon nanotubes (MWCNTs) enter environmental compartments very easily. TCC is known as a disinfectant and is declared as a highly aquatic toxicant. Multi-walled carbon nanotubes are used, e.g., in the automotive industry to improve plastic properties. Both TCCs and MWCNTs can pose major pollution hazards to various organisms. In addition, these substances can bind to each other due to their tendency to interact via strong hydrophobic interactions. Therefore, a short-term test was conducted to investigate the effects of the individual chemicals TCC and weathered MWCNTs (wMWCNTs) on a benthic biofilm and a grazing organism, Lymnaea stagnalis. Furthermore, the two compounds were coupled by an adsorption experiment resulting in a coupled complex formation (TCC + wMWCNTs). L. stagnalis showed no effects in terms of mortality. For benthic biofilm, the coupling test (TCC + wMWCNTs) showed a decrease of 58% in chlorophyll a (Chl-a) concentration. The main effect could be attributed to the wMWCNTs' exposure alone (decrease of 82%), but not to presence of TCC. The concentration range of Chl-a upon TCC exposure alone was comparable to that in the control group (32 and 37 µg/cm2). With respect to the particulate organic carbon (POC) concentration, very similar results were found for the solvent control, the TCC, and also for the TCC + wMWCNTs group (3, 2.9, and 2.9 mg/cm2). In contrast to the control, a significant increase in POC concentration (100%) was observed for wMWCNTs, but no synergistic effect of TCC + wMWCNTs was detected.

Drosophila Atlastin regulates synaptic vesicle mobilization independent of bone morphogenetic protein signaling.

BACKGROUND: The endoplasmic reticulum (ER) contacts endosomes in all parts of a motor neuron, including the axon and presynaptic terminal, to move structural proteins, proteins that send signals, and lipids over long distances. Atlastin (Atl), a large GTPase, is required for membrane fusion and the structural dynamics of the ER tubules. Atl mutations are the second most common cause of Hereditary Spastic Paraplegia (HSP), which causes spasticity in both sexes' lower extremities. Through an unknown mechanism, Atl mutations stimulate the BMP (bone morphogenetic protein) pathway in vertebrates and Drosophila. Synaptic defects are caused by atl mutations, which affect the abundance and distribution of synaptic vesicles (SV) in the bouton. We hypothesize that BMP signaling, does not cause Atl-dependent SV abnormalities in Drosophila. RESULTS: We show that atl knockdown in motor neurons (Atl-KD) increases synaptic and satellite boutons in the same way that constitutively activating the BMP-receptor Tkv (thick veins) (Tkv-CA) increases the bouton number. The SV proteins Cysteine string protein (CSP) and glutamate vesicular transporter are reduced in Atl-KD and Tkv-CA larvae. Reducing the activity of the BMP receptor Wishful thinking (wit) can rescue both phenotypes. Unlike Tkv-CA larvae, Atl-KD larvae display altered activity-dependent distributions of CSP staining. Furthermore, Atl-KD larvae display an increased FM 1-43 unload than Control and Tkv-CA larvae. As decreasing wit function does not reduce the phenotype, our hypothesis that BMP signaling is not involved is supported. We also found that Rab11/CSP colocalization increased in Atl-KD larvae, which supports the concept that late recycling endosomes regulate SV movements. CONCLUSIONS: Our findings reveal that Atl modulates neurotransmitter release in motor neurons via SV distribution independently of BMP signaling, which could explain the observed SV accumulation and synaptic dysfunction. Our data suggest that Atl is involved in membrane traffic as well as formation and/or recycling of the late endosome.

Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca2+ channels drives sustained active zone potentiation.

At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca2+ channels (VGCCs). While AZs can potentiate SV release in the minutes range, we lack an understanding of how AZ scaffold components and VGCCs engage into potentiation. We here establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel's intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. Our single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation.

Ca2+ Homeostasis by Plasma Membrane Ca2+ ATPase (PMCA) 1 Is Essential for the Development of DP Thymocytes.

The strength of Ca2+ signaling is a hallmark of T cell activation, yet the role of Ca2+ homeostasis in developing T cells before expressing a mature T cell receptor is poorly understood. We aimed to unveil specific functions of the two plasma membrane Ca2+ ATPases expressed in T cells, PMCA1 and PMCA4. On a transcriptional and protein level we found that PMCA4 was expressed at low levels in CD4-CD8- double negative (DN) thymocytes and was even downregulated in subsequent stages while PMCA1 was present throughout development and upregulated in CD4+CD8+ double positive (DP) thymocytes. Mice with a targeted deletion of Pmca1 in DN3 thymocytes had an almost complete block of DP thymocyte development with an accumulation of DN4 thymocytes but severely reduced numbers of CD8+ immature single positive (ISP) thymocytes. The DN4 thymocytes of these mice showed strongly elevated basal cytosolic Ca2+ levels and a pre-mature CD5 expression, but in contrast to the DP thymocytes they were only mildly prone to apoptosis. Surprisingly, mice with a germline deletion of Pmca4 did not show any signs of altered progression through the developmental thymocyte stages, nor altered Ca2+ homeostasis throughout this process. PMCA1 is, therefore, non-redundant in keeping cellular Ca2+ levels low in the early thymocyte development required for the DN to DP transition.

A Mechanistic Pharmacodynamic Modeling Framework for the Assessment and Optimization of Proteolysis Targeting Chimeras (PROTACs).

The field of targeted protein degradation is growing exponentially. Yet, there is an unmet need for pharmacokinetic/pharmacodynamic models that provide mechanistic insights, while also being practically useful in a drug discovery setting. Therefore, we have developed a comprehensive modeling framework which can be applied to experimental data from routine projects to: (1) assess PROTACs based on accurate degradation metrics, (2) guide compound optimization of the most critical parameters, and (3) link degradation to downstream pharmacodynamic effects. The presented framework contains a number of first-time features: (1) a mechanistic model to fit the hook effect in the PROTAC concentration-degradation profile, (2) quantification of the role of target occupancy in the PROTAC mechanism of action and (3) deconvolution of the effects of target degradation and target inhibition by PROTACs on the overall pharmacodynamic response. To illustrate applicability and to build confidence, we have employed these three models to analyze exemplary data on various compounds from different projects and targets. The presented framework allows researchers to tailor their experimental work and to arrive at a better understanding of their results, ultimately leading to more successful PROTAC discovery. While the focus here lies on in vitro pharmacology experiments, key implications for in vivo studies are also discussed.

Impact of weathered multi-walled carbon nanotubes on the epithelial cells of the intestinal tract in the freshwater grazers Lymnaea stagnalis and Rhithrogena semicolorata.

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p < 0.01).

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform. The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling. The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

Municipal Wastewaters Carry Important Carbapenemase Genes Independent of Hospital Input and Can Mirror Clinical Resistance Patterns.

The spatiotemporal variation of several carbapenemase-encoding genes (CRGs) was investigated in the influent and effluent of municipal WWTPs, with or without hospital sewage input. Correlations among gene abundances, bacterial community composition, and wastewater quality parameters were tested to identify possible predictors of CRGs presence. Also, the possible role of wastewaters in mirroring clinical resistance is discussed. The taxonomic groups and gene abundances showed an even distribution among wastewater types, meaning that hospital sewage does not influence the microbial diversity and the CRG pool. The bacterial community was composed mainly of Proteobacteria, Firmicutes, Actinobacteria, Patescibacteria, and Bacteroidetes. Acinetobacter spp. was the most abundant group and had the majority of operational taxonomic units (OTUs) positively correlated with CRGs. This agrees with recent reports on clinical data. The influent samples were dominated by blaKPC, as opposed to effluent, where blaIMP was dominant. Also, blaIMP was the most frequent CRG family observed to correlate with bacterial taxa, especially with the Mycobacterium genus in effluent samples. Bacterial load, blaNDM, blaKPC, and blaOXA-48 abundances were positively correlated with BOD5, TSS, HEM, Cr, Cu, and Fe concentrations in wastewaters. When influent gene abundance values were converted into population equivalent (PE) data, the highest copies/1 PE were identified for blaKPC and blaOXA-48, agreeing with previous studies regarding clinical isolates. Both hospital and non-hospital-type samples followed a similar temporal trend of CRG incidence, but with differences among gene groups. Colder seasons favored the presence of blaNDM, blaKPC and blaOXA-48, whereas warmer temperatures show increased PE values for blaVIM and blaIMP. IMPORTANCE Wastewater-based epidemiology has recently been recognized as a valuable, cost-effective tool for antimicrobial resistance surveillance. It can help gain insights into the characteristics and distribution of antibiotic resistance elements at a local, national, and even global scale. In this study, we investigated the possible use of municipal wastewaters in the surveillance of clinically relevant carbapenemase-encoding genes (CRGs), seen as critical antibiotic resistance determinants. In this matter, our results highlight positive correlations among CRGs, microbial diversity, and wastewater physical and chemical parameters. Identified predictors can provide valuable data regarding the level of raw and treated wastewater contamination with these important antibiotic resistance genes. Also, wastewater-based gene abundances were used for the first time to observe possible spatiotemporal trends of CRGs incidence in the general population. Therefore, possible hot spots of carbapenem resistance could be easily identified at the community level, surpassing the limitations of health care-associated settings.

Amplification rationale for hearing aids based on characteristics of the Japanese language.

OBJECTIVE: Hearing aid amplification rationales have typically been developed by using global averages of the long-term average speech spectrum (LTASS) from Western European languages. However, there are few reports on hearing-aid amplification based on acoustic-phonetic characteristics of the Japanese language. This study's objective is to investigate the LTASS for Japanese, and to compare a typical amplification rationale originally developed mainly for Western European languages with an amplification rationale specifically adjusted to the LTASS for Japanese. METHODS: LTASS for two speech materials provided by four Japanese talkers were analyzed using 1/3 octave bandwidth filters. The speech was recorded with different levels of vocal effort, yielding three LTASS for "soft", "moderate" and "loud" speech. From these results, a gain offset of the hearing-aid amplification for Japanese was obtained as compared to ANSI S3.5. Speech intelligibility for an amplification rationale for Western European languages and the newly-developed Japanese version was obtained for presentation levels of 50 dB SPL, 65 dB SPL and 80 dB SPL. Nineteen people with mild to moderate hearing loss participated in the speech intelligibility experiment. Scores in% correct were arcsine-transformed and subjected to repeated measures ANOVA with pairwise comparisons of significant main effects using Bonferroni adjustments for multiple comparisons. RESULTS: The LTASS for Japanese was slightly different from the values of previous reports. A comparison of LTASS values to ANSI S3.5 with values for Japanese showed that the Japanese amplification rationale for "moderate" speech levels required more gain in the low-frequency area, and less gain in the high-frequency area. There was no significant difference in the speech intelligibility level between the amplification characteristics of Western European languages and Japanese language at each presentation level. CONCLUSION: It was shown that for hearing-aid amplification for Japanese, adjustments based on LTASS differences for Western European Languages could be made. This preserved speech intelligibility at the same level as the original amplification rationale, suggesting that there was no need to consider differences in phonetics of Japanese to optimize speech understanding.

A workflow to investigate the impacts of weathered multi-walled carbon nanotubes to the mud snail Lymnaea stagnalis.

Although the development and application of nanomaterials is a growing industry, little data is available on the ecotoxicological effects on aquatic organisms. Therefore, we set up a workflow to address the potential uptake of weathered multi-walled carbon nanotubes (wMWCNTs) by a model organism, the pulmonary mud snail Lymnaea stagnalis (L. stagnalis), which plays an important role in the food web. It represents a suitable organism for this approach because as a grazer it potentially ingests large amounts of sedimented wMWCNTs. As food source for L. stagnalis, benthic biofilm was investigated by the use of a transmission electron microscope (TEM) and a scanning electron microscope (SEM) after exposure with wMWCNTs. In addition, isotopic labeling was applied with 14C-wMWCNTs (0.1 mg/L) to quantify fate, behavior, and enrichment of 14C-wMWCNTs in benthic biofilm and in L. stagnalis. Enrichment in benthic biofilm amounted to 529.0 µg wMWCNTs/g dry weight and in L. stagnalis to 79.6 µg wMWCNTs/g dry weight. A bioconcentration factor (BCF) for L. stagnalis was calculated (3500 L/kg). We demonstrate the accumulation of wMWCNTs (10 mg/L) in the digestive tract of L. stagnalis in an effect study. Moreover, the physiological markers glycogen and triglycerides as indicators for the physiological state, as well as the RNA/DNA ratio as growth indicator, were examined. No significant differences between exposed and control animals were analyzed for glycogen and triglycerides after 24 days of exposure, but a decreasing trend is recognizable for triglycerides. In contrast, the significant reduction in the RNA/DNA ratio of L. stagnalis indicated an inhibition of growth with a following recovery after depuration. The described workflow enables a comprehensive determination of the fate and the behavior of wMWCNTs specifically and in general all kinds of CNTs in the aquatic environment and therefore contributes to a holistic risk assessment of wMWCNTs.

Separation of presynaptic Cav2 and Cav1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca2+ pump PMCA.

Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca2+ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)-triggered presynaptic Ca2+ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Cav2 homolog, Dmca1A or cacophony, and the Cav1 homolog, Dmca1D) but with spatial and functional separation. Cav2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Cav1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca2+ influx but has a small impact on release. Instead, L-type calcium currents through Cav1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca2+ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Cav1-mediated Ca2+ influx. We show that the plasma membrane Ca2+ ATPase (PMCA) resides in between active zones and isolates Cav2-triggered release from Cav1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Cav2 may also contribute to. As L-type Cav1 channels also localize next to PQ-type Cav2 channels within axon terminals of some central mammalian synapses, we propose that Cav2, Cav1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.

A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila.

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.

Plasma membrane Ca2+ ATPase 1 (PMCA1) but not PMCA4 is critical for B-cell development and Ca2+ homeostasis in mice.

The amplitude and duration of Ca2+ signaling is crucial for B-cell development and self-tolerance; however, the mechanisms for terminating Ca2+ signals in B cells have not been determined. In lymphocytes, plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 (PMCA1 and PMCA4, aka ATP2B1 and ATP2B4) are the main candidates for expelling Ca2+ from the cell through the plasma membrane. We report here that Pmca4 (Atp2b4) KO mice had normal B-cell development, while mice with a conditional KO of Pmca1 (Atp2b1) had greatly reduced numbers of B cells, particularly splenic follicular B cells, marginal zone B cells, and peritoneal B-1a cells. Mouse and naïve human B cells showed only PMCA1 expression and no PMCA4 by western blot, in contrast to T cells, which did express PMCA4. Calcium handling was normal in Pmca4-/- B cells, but Pmca1 KO B cells had elevated basal levels of Ca2+ , elevated levels in ER stores, and reduced Ca2+ clearance. These findings show that the PMCA1 isoform alone is required to ensure normal B-cell Ca2+ signaling and development, which may have implications for therapeutic targeting of PMCAs and Ca2+ in B cells.

Integration of mechanical conditioning into a high throughput contractility assay for cardiac safety assessment.

INDUCTION: Despite increasing acceptance of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in safety pharmacology, controversy remains about the physiological relevance of existing in vitro models for their mechanical testing. We hypothesize that existing signs of immaturity of the cell models result from an improper mechanical environment. With the presented study, we aimed at validating the newly developed FLEXcyte96 technology with respect to physiological responses of hiPSC-CMs to pharmacological compounds with known inotropic and/or cardiotoxic effects. METHODS: hiPSC-CMs were cultured in a 96-well format on hyperelastic silicone membranes imitating their native mechanical environment. Cardiomyocyte contractility was measured contact-free by application of capacitive displacement sensing of the cell-membrane biohybrids. Acute effects of positive inotropic compounds with distinct mechanisms of action were examined. Additionally, cardiotoxic effects of tyrosine kinase inhibitors and anthracyclines were repetitively examined during repeated exposure to drug concentrations for up to 5 days. RESULTS: hiPSC-CMs grown on biomimetic membranes displayed increased contractility responses to isoproterenol, S-Bay K8644 and omecamtiv mecarbil without the need for additional stimulation. Tyrosine kinase inhibitor erlotinib, vandetanib, nilotinib, gefitinib, A-674563 as well as anthracycline idarubicin showed the expected cardiotoxic effects, including negative inotropy and induction of proarrhythmic events. DISCUSSION: We conclude that the FLEXcyte 96 system is a reliable high throughput tool for invitro cardiac contractility research, providing the user with data obtained under physiological conditions which resemble the native environment of human heart tissue. We showed that the results obtained for both acute and sub-chronic compound administration are consistent with the respective physiological responses in humans.

Cell Survival Failure in Effector T Cells From Patients With Systemic Lupus Erythematosus Following Insufficient Up-Regulation of Cold-Shock Y-Box Binding Protein 1.

OBJECTIVE: The importance of cold-shock Y-box binding protein 1 (YB-1) for cell homeostasis is well-documented based on prior observations of its association with certain cancer entities. This study was undertaken to explore the role of YB-1 in T cell homeostasis and survival and the potential contribution of YB-1 to the pathogenesis of systemic lupus erythematosus (SLE). METHODS: In the peripheral blood from 25 SLE patients and 25 healthy donors, the expression of YB-1 and frequency of T cell apoptosis was analyzed by quantitative polymerase chain reaction (qPCR) and fluorescence-activated cell sorting of CD4+ T cells ex vivo and also analyzed in T cells in vitro after 6 days of stimulation with anti-CD3-coupled or anti-CD3/anti-CD28-coupled microspheres. YB-1 was overexpressed using lentiviral transduction with wild-type green fluorescent protein (wtGFP) YB-1, and knockdown of YB-1 was achieved using specific short hairpin RNA (shRNA) (3-fold reduction; P < 0.0001). RESULTS: YB-1 expression was significantly lower in apoptosis-prone T cells and in activated T cells from SLE patients compared to YB-1 expression in nonapoptotic T cells and activated T cells from healthy donors (P = 0.001). Knockdown of YB-1 in T cells consequently led to expression of proapoptotic molecules and caspase 3 activation (1.6-fold), and subsequently, to apoptosis. Furthermore, YB-1 promoted survival pathways involving enhanced protein expression of the kinase Akt (2-fold) and Bcl-2 (3-fold), even when Fas/CD95 was triggered. YB-1-mediated T cell survival was reversed by Akt and phosphatidylinositol 3-kinase (PI3K) inactivation. In SLE patients, rescue of YB-1 expression strongly promoted survival of T cells and even prevented cell death in T cells that were extremely apoptosis-prone. CONCLUSION: Our data show that failure of YB-1 up-regulation in T cells from SLE patients led to enhanced apoptosis. These findings imply that YB-1 plays a crucial role in the disturbed homeostasis of activated T cells leading to hematopoietic alterations in SLE. These insights may help facilitate the development of new treatment strategies for SLE.

Bassoon proteinopathy drives neurodegeneration in multiple sclerosis.

Multiple sclerosis (MS) is characterized by inflammatory insults that drive neuroaxonal injury. However, knowledge about neuron-intrinsic responses to inflammation is limited. By leveraging neuron-specific messenger RNA profiling, we found that neuroinflammation leads to induction and toxic accumulation of the synaptic protein bassoon (Bsn) in the neuronal somata of mice and patients with MS. Neuronal overexpression of Bsn in flies resulted in reduction of lifespan, while genetic disruption of Bsn protected mice from inflammation-induced neuroaxonal injury. Notably, pharmacological proteasome activation boosted the clearance of accumulated Bsn and enhanced neuronal survival. Our study demonstrates that neuroinflammation initiates toxic protein accumulation in neuronal somata and advocates proteasome activation as a potential remedy.

Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila.

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as 'click chemistry' serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster.

A Presynaptic Function of Shank Protein in Drosophila.

Human genetic studies support that loss-of-function mutations in the SH3 domain and ankyrin repeat containing family proteins (SHANK1-3), the large synaptic scaffolding proteins enriched at the postsynaptic density of excitatory synapses, are causative for autism spectrum disorder and other neuropsychiatric disorders in humans. To better understand the in vivo functions of Shank and facilitate dissection of neuropathology associated with SHANK mutations in human, we generated multiple mutations in the Shank gene, the only member of the SHANK family in Drosophila melanogaster Both male and female Shank null mutants were fully viable and fertile with no apparent morphological or developmental defects. Expression analysis revealed apparent enrichment of Shank in the neuropils of the CNS. Specifically, Shank coexpressed with another PSD scaffold protein, Homer, in the calyx of mushroom bodies in the brain. Consistent with high expression in mushroom body calyces, Shank mutants show an abnormal calyx structure and reduced olfactory acuity. These morphological and functional phenotypes were fully rescued by pan-neuronal reexpression of Shank, and only partially rescued by presynaptic but no rescue by postsynaptic reexpression of Shank. Our findings thus establish a previously unappreciated presynaptic function of Shank.SIGNIFICANCE STATEMENT Mutations in SHANK family genes are causative for idiopathic autism spectrum disorder. To understand the neural function of Shank, a large scaffolding protein enriched at the postsynaptic densities, we examined the role of Drosophila Shank in synapse development at the peripheral neuromuscular junctions and the central mushroom body calyx. Our results demonstrate that, in addition to its conventional postsynaptic function, Shank also acts presynaptically in synapse development in the brain. This study offers novel insights into the synaptic role of Shank.