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Protein structure is determined by the amino acid sequence and a variety of post-translational modifications, and provides the basis for physiological properties. Not all proteins in the proteome attain a stable conformation; roughly one third of human proteins are unstructured or contain intrinsically disordered regions exceeding 40% of their length. Proteins comprising or containing extensive unstructured regions are termed intrinsically disordered proteins (IDPs). IDPs are known to be overrepresented in protein aggregates of diverse neurodegenerative diseases. We evaluated the importance of disordered proteins in the nematode Caenorhabditis elegans, by RNAi-mediated knockdown of IDPs in disease-model strains that mimic aggregation associated with neurodegenerative pathologies. Not all disordered proteins are sequestered into aggregates, and most of the tested aggregate-protein IDPs contribute to important physiological functions such as stress resistance or reproduction. Despite decades of research, we still do not understand what properties of a disordered protein determine its entry into aggregates. We have employed machine-learning models to identify factors that predict whether a disordered protein is found in sarkosyl-insoluble aggregates isolated from neurodegenerative-disease brains (both AD and PD). Machine-learning predictions, coupled with principal component analysis (PCA), enabled us to identify the physiochemical properties that determine whether a disordered protein will be enriched in neuropathic aggregates. [ABSTRACT FROM AUTHOR]

Protein structure is determined by the amino acid sequence and a variety of post-translational modifications, and provides the basis for physiological properties. Not all proteins in the proteome attain a stable conformation; roughly one third of human proteins are unstructured or contain intrinsically disordered regions exceeding 40% of their length. Proteins comprising or containing extensive unstructured regions are termed intrinsically disordered proteins (IDPs). IDPs are known to be overrepresented in protein aggregates of diverse neurodegenerative diseases. We evaluated the importance of disordered proteins in the nematode Caenorhabditis elegans, by RNAi-mediated knockdown of IDPs in disease-model strains that mimic aggregation associated with neurodegenerative pathologies. Not all disordered proteins are sequestered into aggregates, and most of the tested aggregate-protein IDPs contribute to important physiological functions such as stress resistance or reproduction. Despite decades of research, we still do not understand what properties of a disordered protein determine its entry into aggregates. We have employed machine-learning models to identify factors that predict whether a disordered protein is found in sarkosyl-insoluble aggregates isolated from neurodegenerative-disease brains (both AD and PD). Machine-learning predictions, coupled with principal component analysis (PCA), enabled us to identify the physiochemical properties that determine whether a disordered protein will be enriched in neuropathic aggregates.

Congenital hyperinsulinism (CHI) is a severe inherited neonatal disorder characterized by inappropriate insulin secretion caused by genetic defects of the pancreatic beta cells. Several open questions remain in CHI research, such as the optimal treatment for the most common type of CHI, caused by mutations in the genes encoding ATP-sensitive potassium channels, and the molecular mechanisms of newly identified CHI genes. Answering these questions requires robust preclinical models, particularly since primary patient material is extremely scarce and accurate animal models are not available. In this short review, we explain why pluripotent stem cell derived islets present an attractive solution to these issues and outline the current progress in stem-cell based modeling of CHI. Stem cell derived islets enable the study of molecular mechanisms of CHI and the discovery of novel antihypoglycemic drugs, while also providing a valuable model to study the biology of variable functional states of beta cells. [ABSTRACT FROM AUTHOR]

Congenital hyperinsulinism (CHI) is a severe inherited neonatal disorder characterized by inappropriate insulin secretion caused by genetic defects of the pancreatic beta cells. Several open questions remain in CHI research, such as the optimal treatment for the most common type of CHI, caused by mutations in the genes encoding ATP-sensitive potassium channels, and the molecular mechanisms of newly identified CHI genes. Answering these questions requires robust preclinical models, particularly since primary patient material is extremely scarce and accurate animal models are not available. In this short review, we explain why pluripotent stem cell derived islets present an attractive solution to these issues and outline the current progress in stem-cell based modeling of CHI. Stem cell derived islets enable the study of molecular mechanisms of CHI and the discovery of novel antihypoglycemic drugs, while also providing a valuable model to study the biology of variable functional states of beta cells.

Antioxidant proteins can not only balance the oxidative stress in the body, but are also an important component of antioxidant drugs. Accurate identification of antioxidant proteins is essential to help humans fight diseases and develop new drugs. In this paper, we developed a friendly method AOPM to identify antioxidant proteins. 188D and the Composition of k-spaced Amino Acid Pairs were adopted as the feature extraction method. In addition, the Max-Relevance-Max-Distance algorithm (MRMD) and random forest were the feature selection and classifier, respectively. We used 5-folds cross-validation and independent test dataset to evaluate our model. On the test dataset, AOPM presented a higher performance compared with the state-of-the-art methods. The sensitivity, specificity, accuracy, Matthew’s Correlation Coefficient and an Area Under the Curve reached 87.3, 94.2, 92.0%, 0.815 and 0.972, respectively. In addition, AOPM still has excellent performance in predicting the catalytic enzymes of antioxidant drugs. This work proved the feasibility of virtual drug screening based on sequence information and provided new ideas and solutions for drug development.

Antioxidant proteins can not only balance the oxidative stress in the body, but are also an important component of antioxidant drugs. Accurate identification of antioxidant proteins is essential to help humans fight diseases and develop new drugs. In this paper, we developed a friendly method AOPM to identify antioxidant proteins. 188D and the Composition of k-spaced Amino Acid Pairs were adopted as the feature extraction method. In addition, the Max-Relevance-Max-Distance algorithm (MRMD) and random forest were the feature selection and classifier, respectively. We used 5-folds cross-validation and independent test dataset to evaluate our model. On the test dataset, AOPM presented a higher performance compared with the state-of-the-art methods. The sensitivity, specificity, accuracy, Matthew's Correlation Coefficient and an Area Under the Curve reached 87.3, 94.2, 92.0%, 0.815 and 0.972, respectively. In addition, AOPM still has excellent performance in predicting the catalytic enzymes of antioxidant drugs. This work proved the feasibility of virtual drug screening based on sequence information and provided new ideas and solutions for drug development. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBD) with chronic infiltration of immune cells in the gastrointestinal tract are common and largely incurable. The therapeutic targeting of IBD has been hampered by the complex causality of the disease, with environmental insults like cholesterol-enriched Western diets playing a critical role. To address this drug development challenge, we report an easy-to-handle dietary cholesterol-based in vivo assay that allows the screening of immune-modulatory therapeutics in transgenic zebrafish models. An improvement in the feeding strategy with high cholesterol diet (HCD) selectively induces a robust and consistent infiltration of myeloid cells in larvae intestines that is highly suitable for compound discovery efforts. Using transgenics with fluorescent reporter expression in neutrophils, we take advantage of the unique zebrafish larvae clarity to monitor an acute inflammatory response in a whole organism context with a fully functional innate immune system. The use of semi-automated image acquisition and processing combined with quantitative image analysis allows categorizing anti- or pro-inflammatory compounds based on a leukocytic inflammation index. Our HCD gut inflammation (HCD-GI) assay is simple, cost- and time-effective as well as highly physiological which makes it unique when compared to chemical-based zebrafish models of IBD. Besides, diet is a highly controlled, selective and targeted trigger of intestinal inflammation that avoids extra-intestinal outcomes and reduces the chances of chemical-induced toxicity during screenings. We show the validity of this assay for a screening platform by testing two dietary phenolic acids, namely gallic acid (GA; 3,4,5-trihydroxybenzoic acid) and ferulic acid (FA; 4-hydroxy-3-methoxycinnamic acid), with well described anti-inflammatory actions in animal models of IBD. Analysis of common IBD therapeutics (Prednisolone and Mesalamine) proved the fidelity of our IBD-like intestinal inflammation model. In conclusion, the HCD-GI assay can facilitate and accelerate drug discovery efforts on IBD, by identification of novel lead molecules with immune modulatory action on intestinal neutrophilic inflammation. This will serve as a jumping-off point for more profound analyses of drug mechanisms and pathways involved in early IBD immune responses.

In view of the importance of cancer stem cells (CSCs) in chemoresistance, metastasis and recurrence, the biology of CSCs were explored in detail. Based on that, several modalities were proposed to target them. In spite of the several clinical trials, a successful CSC-targeting drug is yet to be identified. The number of molecules screened and entered for clinical trial for CSC-targeting is comparatively low, compared to other drugs. The bottle neck is the lack of a high-throughput adaptable screening strategy for CSCs. This review is aimed to identify suitable reporters for CSCs that can be used to identify the heterogeneous CSC populations, including quiescent CSCs, proliferative CSCs, drug resistant CSCs and metastatic CSCs. Analysis of the tumor microenvironment regulating CSCs revealed that the factors in CSC-niche activates effector molecules that function as CSC markers, including pluripotency markers, CD133, ABCG2 and ALDH1A1. Among these factors OCT4, SOX2, NANOG, ABCG2 and ALDH1A1 are ideal for making reporters for CSCs. The pluripotency molecules, like OCT4, SOX2 and NANOG, regulate self-renewal, chemoresistance and metastasis. ABCG2 is a known regulator of drug resistance while ALDH1A1 modulates self-renewal, chemoresistance and metastasis. Considering the heterogeneity of CSCs, including a quiescent population and a proliferative population with metastatic ability, we propose the use of a combination of reporters. A dual reporter consisting of a pluripotency marker and a marker like ALDH1A1 will be useful in screening drugs that target CSCs.

In view of the importance of cancer stem cells (CSCs) in chemoresistance, metastasis and recurrence, the biology of CSCs were explored in detail. Based on that, several modalities were proposed to target them. In spite of the several clinical trials, a successful CSC-targeting drug is yet to be identified. The number of molecules screened and entered for clinical trial for CSC-targeting is comparatively low, compared to other drugs. The bottle neck is the lack of a high-throughput adaptable screening strategy for CSCs. This review is aimed to identify suitable reporters for CSCs that can be used to identify the heterogeneous CSC populations, including quiescent CSCs, proliferative CSCs, drug resistant CSCs and metastatic CSCs. Analysis of the tumor microenvironment regulating CSCs revealed that the factors in CSC-niche activates effector molecules that function as CSC markers, including pluripotency markers, CD133, ABCG2 and ALDH1A1. Among these factors OCT4, SOX2, NANOG, ABCG2 and ALDH1A1 are ideal for making reporters for CSCs. The pluripotency molecules, like OCT4, SOX2 and NANOG, regulate self-renewal, chemoresistance and metastasis. ABCG2 is a known regulator of drug resistance while ALDH1A1 modulates self-renewal, chemoresistance and metastasis. Considering the heterogeneity of CSCs, including a quiescent population and a proliferative population with metastatic ability, we propose the use of a combination of reporters. A dual reporter consisting of a pluripotency marker and a marker like ALDH1A1 will be useful in screening drugs that target CSCs. [ABSTRACT FROM AUTHOR]

Mechanosensory hair cells of the inner ear transduce auditory and vestibular sensory input. Hair cells are susceptible to death from a variety of stressors, including treatment with therapeutic drugs that have ototoxic side effects. There is a need for co-therapies to mitigate drug-induced ototoxicity, and we showed previously that induction of heat shock proteins (HSPs) protects against hair cell death and hearing loss caused by aminoglycoside antibiotics in mouse. Here, we utilized the library of integrated cellular signatures (LINCS) to identify perturbagens that induce transcriptional profiles similar to that of heat shock. Massively parallel sequencing of RNA (RNA-Seq) of heat shocked and control mouse utricles provided a heat shock gene expression signature that was used in conjunction with LINCS to identify candidate perturbagens, several of which were known to protect the inner ear. Our data indicate that LINCS is a useful tool to screen for compounds that generate specific gene expression signatures in the inner ear. Forty-two LINCS-identified perturbagens were tested for otoprotection in zebrafish, and three of these were protective. These compounds also induced the heat shock gene expression signature in mouse utricles, and one compound protected against aminoglycoside-induced hair cell death in whole organ cultures of utricles from adult mice.

Mechanosensory hair cells of the inner ear transduce auditory and vestibular sensory input. Hair cells are susceptible to death from a variety of stressors, including treatment with therapeutic drugs that have ototoxic side effects. There is a need for co-therapies to mitigate drug-induced ototoxicity, and we showed previously that induction of heat shock proteins (HSPs) protects against hair cell death and hearing loss caused by aminoglycoside antibiotics in mouse. Here, we utilized the library of integrated cellular signatures (LINCS) to identify perturbagens that induce transcriptional profiles similar to that of heat shock. Massively parallel sequencing of RNA (RNA-Seq) of heat shocked and control mouse utricles provided a heat shock gene expression signature that was used in conjunction with LINCS to identify candidate perturbagens, several of which were known to protect the inner ear. Our data indicate that LINCS is a useful tool to screen for compounds that generate specific gene expression signatures in the inner ear. Forty-two LINCS-identified perturbagens were tested for otoprotection in zebrafish, and three of these were protective. These compounds also induced the heat shock gene expression signature in mouse utricles, and one compound protected against aminoglycoside-induced hair cell death in whole organ cultures of utricles from adult mice. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBD) with chronic infiltration of immune cells in the gastrointestinal tract are common and largely incurable. The therapeutic targeting of IBD has been hampered by the complex causality of the disease, with environmental insults like cholesterol-enriched Western diets playing a critical role. To address this drug development challenge, we report an easy-to-handle dietary cholesterol-based in vivo assay that allows the screening of immune-modulatory therapeutics in transgenic zebrafish models. An improvement in the feeding strategy with high cholesterol diet (HCD) selectively induces a robust and consistent infiltration of myeloid cells in larvae intestines that is highly suitable for compound discovery efforts. Using transgenics with fluorescent reporter expression in neutrophils, we take advantage of the unique zebrafish larvae clarity to monitor an acute inflammatory response in a whole organism context with a fully functional innate immune system. The use of semi-automated image acquisition and processing combined with quantitative image analysis allows categorizing anti- or pro-inflammatory compounds based on a leukocytic inflammation index. Our HCD gut inflammation (HCD-GI) assay is simple, cost- and time-effective as well as highly physiological which makes it unique when compared to chemical-based zebrafish models of IBD. Besides, diet is a highly controlled, selective and targeted trigger of intestinal inflammation that avoids extra-intestinal outcomes and reduces the chances of chemical-induced toxicity during screenings. We show the validity of this assay for a screening platform by testing two dietary phenolic acids, namely gallic acid (GA; 3,4,5-trihydroxybenzoic acid) and ferulic acid (FA; 4-hydroxy-3-methoxycinnamic acid), with well described anti-inflammatory actions in animal models of IBD. Analysis of common IBD therapeutics (Prednisolone and Mesalamine) proved the fidelity of our IBD-like intestinal inflammation model. In conclusion, the HCD-GI assay can facilitate and accelerate drug discovery efforts on IBD, by identification of novel lead molecules with immune modulatory action on intestinal neutrophilic inflammation. This will serve as a jumping-off point for more profound analyses of drug mechanisms and pathways involved in early IBD immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Silva, Carregosa, Gonçalves, Vieira, Nunes dos Santos, Jacinto and Crespo.)