Structural and Functional Organization of Visual Responses in the Inferior Olive of Larval Zebrafish.

The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. Here, we investigate sensory representations in the inferior olive (IO) of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons, we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using two-photon imaging. We found that most IO neurons showed direction-selective and binocular responses to visual stimuli and that the functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between functional properties, soma location, and axonal projection patterns of IO neurons. Taken together, our results suggest that anatomically defined classes of IO neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones.

Target Identification in Anti-Tuberculosis Drug Discovery.

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB), a disease that, although preventable and curable, remains a global epidemic due to the emergence of resistance and a latent form responsible for a long period of treatment. Drug discovery in TB is a challenging task due to the heterogeneity of the disease, the emergence of resistance, and uncomplete knowledge of the pathophysiology of the disease. The limited permeability of the cell wall and the presence of multiple efflux pumps remain a major barrier to achieve effective intracellular drug accumulation. While the complete genome sequence of Mtb has been determined and several potential protein targets have been validated, the lack of adequate models for in vitro and in vivo studies is a limiting factor in TB drug discovery programs. In current therapeutic regimens, less than 0.5% of bacterial proteins are targeted during the biosynthesis of the cell wall and the energetic metabolism of two of the most important processes exploited for TB chemotherapeutics. This review provides an overview on the current challenges in TB drug discovery and emerging Mtb druggable proteins, and explains how chemical probes for protein profiling enabled the identification of new targets and biomarkers, paving the way to disruptive therapeutic regimens and diagnostic tools.

Polysulfone Membranes Doped with Human Neutrophil Elastase Inhibitors: Assessment of Bioactivity and Biocompatibility.

The use of polysulfone (PSU) hemodialysis (HD) membranes modified with bioactive compounds has gained relevance in chronic kidney disease (CKD) management. Compounds based on the 4-oxo-ß-lactam scaffold have outstanding inhibitory ability and selectivity for human neutrophil elastase (HNE). The present work aimed to evaluate the bioactivity and biocompatibility of PSU-based HD membranes doped with HNE inhibitors (HNEIs). For this, two 4-oxo-ß-lactam derivates (D4L-1 and D4L-2) synthesized in house were used, as well as a commercial HNEI (Sivelestat), for comparison purposes. Their HNE inhibition efficacy was evaluated in in vitro and ex vivo (incubations with human plasma) assay conditions. All biomaterials were bioactive and hemocompatible. The inhibitory capacity of the HNEIs and HNEI-PSU membranes in vitro was D4L-1 > D4L-2 > Sivelestat and D4L-2 > Sivelestat > D4L-1, respectively. In ex vivo conditions, both HNEIs and HNEI-PSU materials presented the same relative inhibitory ability (D4L-1 > D4L-2 > Sivelestat). The difference observed between in vitro and ex vivo conditions is most likely due to the inherent lipophilicity/hydrophobicity of each HNEI influencing their affinity and accessibility to HNE when trapped in the membrane. Compared to Sivelestat, both D4L-1 and D4L-2 (and the respective doped membranes) have more potent inhibition capabilities. In conclusion, this work reports the successful development of PSU membranes functionalized with HNEIs.

Chemoproteomics-Enabled Identification of 4-Oxo-ß-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-ß-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

Survival predictors and outcomes of patients with recurrent and/or metastatic head and neck cancer treated with chemotherapy plus cetuximab as first-line therapy: A real-world retrospective study.

BACKGROUND: In patients with recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) the estimated prognosis is usually poor. Patient-specific factors that affect prognosis should be considered when choosing therapy. We conducted a retrospective, single-center analysis in patients treated with first line platinum and antiEGFR antibody-containing regimen. The objective was to generate real-world data considering treatment outcomes and to identify predictors of survival. PATIENTS/METHODS: Clinical charts of patients treated with cetuximab and platinum-based chemotherapy (CT) for R/M HNSCC in first-line setting, between January-2009 and December-2018 were assessed. In these 103 patients, the prognostic value of site of the primary tumor, age at diagnosis, gender, Cetuximab induced skin toxicity and prior treatments were studied multivariately. To evaluate progression free survival (PFS) and overall survival (OS), Kaplan-Meier curves and the log-rank test were used. The Coxregression model was used to investigate the effect of these variables on OS. RESULTS: Longer OS was associated with oral cavity tumor location (p = 0,003), European Cooperative Oncology Group-Performance Status 0 (ECOG-PS) (p = 0,01), complete/partial response (p<0,0001), cetuximab monotherapy until disease progression or unacceptable toxicity (p = 0,037) and Grade 2-4 cetuximab induced skin toxicity (p = 0,037). The median follow-up period was 11,7 months. The mortality rate was 90,3% during this retrospective cohort assessment. The PFS was 7,1 months (95% confidence interval (CI), 5,6-8.6). The OS was 11,7 months (95%CI, 10,5-12,8). CONCLUSIONS: The present study demonstrates that the combination of cetuximab with platinum-based CT was effective in R/M HNSCC. Among patients with R/M HSCC treated with platinum plus cetuximab as first-line therapy, primary site, ECOG-PS, grade 2-4 cetuximab induced toxicity, and weekly cetuximab monotherapy have a marked impact on OS.

Novel and Modified Neutrophil Elastase Inhibitor Loaded in Topical Formulations for Psoriasis Management.

Human neutrophil elastase (HNE) is a serine protease that degrades matrix proteins. An excess of HNE may trigger several pathological conditions, such as psoriasis. In this work, we aimed to synthesize, characterize and formulate new HNE inhibitors with a 4-oxo-ß-lactam scaffold with less toxicity, as well as therapeutic index in a psoriasis context. HNE inhibitors with 4-oxo-ß-lactam scaffolds were synthesized and characterized by NMR, FTIR, melting point, mass spectrometry and elemental analysis. In vitro cytotoxicity and serine protease assays were performed. The compound with the highest cell viability (AAN-16) was selected to be incorporated in an emulsion (AAN-16 E) and in a microemulsion (AAN-16 ME). Formulations were characterized in terms of organoleptic properties, pH, rheology, droplet size distribution, in vitro drug release and in vivo psoriatic activity. All compounds were successfully synthesized according to analytical methodology, with good yields. Both formulations presented suitable physicochemical properties. AAN-16 E presented the most promising therapeutic effects in a murine model of psoriasis. Overall, new HNE inhibitors were synthesized with high and selective activity and incorporated into topical emulsions with potential to treat psoriasis.

Structure of the Zebrafish Locomotor Repertoire Revealed with Unsupervised Behavioral Clustering.

An important concept in ethology is that complex behaviors can be constructed from a set of basic motor patterns. Identifying the set of patterns available to an animal is key to making quantitative descriptions of behavior that reflect the underlying motor system organization. We addressed these questions in zebrafish larvae, which swim in bouts that are naturally segmented in time. We developed a robust and general purpose clustering method (clusterdv) to ensure accurate identification of movement clusters and applied it to a dataset consisting of millions of swim bouts, captured at high temporal resolution from a comprehensive set of behavioral contexts. We identified a set of thirteen basic swimming patterns that are used flexibly in various combinations across different behavioral contexts and show that this classification can be used to dissect the sensorimotor transformations underlying larval social behavior and hunting. Furthermore, using the same approach at different levels in the behavioral hierarchy, we show that the set of swim bouts are themselves constructed from a basic set of tail movements and that bouts are executed in sequences specific to different behaviors.