Global and local functions of the Fused kinase ortholog CdaH in intracellular patterning in Tetrahymena.

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning.

Deep learning of 2D-Restructured gene expression representations for improved low-sample therapeutic response prediction.

Clinical outcome prediction is important for stratified therapeutics. Machine learning (ML) and deep learning (DL) methods facilitate therapeutic response prediction from transcriptomic profiles of cells and clinical samples. Clinical transcriptomic DL is challenged by the low-sample sizes (34-286 subjects), high-dimensionality (up to 21,653 genes) and unordered nature of clinical transcriptomic data. The established methods rely on ML algorithms at accuracy levels of 0.6-0.8 AUC/ACC values. Low-sample DL algorithms are needed for enhanced prediction capability. Here, an unsupervised manifold-guided algorithm was employed for restructuring transcriptomic data into ordered image-like 2D-representations, followed by efficient DL of these 2D-representations with deep ConvNets. Our DL models significantly outperformed the state-of-the-art (SOTA) ML models on 82% of 17 low-sample benchmark datasets (53% with >0.05 AUC/ACC improvement). They are more robust than the SOTA models in cross-cohort prediction tasks, and in identifying robust biomarkers and response-dependent variational patterns consistent with experimental indications.

The Tetrahymena bcd1 mutant implicates endosome trafficking in ciliate, cortical pattern formation.

Ciliates, such as Tetrahymena thermophila, evolved complex mechanisms to determine both the location and dimensions of cortical organelles such as the oral apparatus (OA: involved in phagocytosis), cytoproct (Cyp: for eliminating wastes), and contractile vacuole pores (CVPs: involved in water expulsion). Mutations have been recovered in Tetrahymena that affect both the localization of such organelles along anterior-posterior and circumferential body axes and their dimensions. Here we describe BCD1, a ciliate pattern gene that encodes a conserved Beige-BEACH domain-containing protein a with possible protein kinase A (PKA)-anchoring activity. Similar proteins have been implicated in endosome trafficking and are linked to human Chediak-Higashi syndrome and autism. Mutations in the BCD1 gene broaden cortical organelle domains as they assemble during predivision development. The Bcd1 protein localizes to membrane pockets at the base of every cilium that are active in endocytosis. PKA activity has been shown to promote endocytosis in other organisms, so we blocked clathrin-mediated endocytosis (using "dynasore") and inhibited PKA (using H89). In both cases, treatment produced partial phenocopies of the bcd1 pattern mutant. This study supports a model in which the dimensions of diverse cortical organelle assembly-platforms may be determined by regulated balance between constitutive exocytic delivery and PKA-regulated endocytic retrieval of organelle materials and determinants.

Escherichia fergusonii bacteremia after esophagectomy: a case report and literature review.

We report the case of a 68-year-old man who experienced Escherichia fergusonii bacteremia after esophageal cancer surgery. The patient presented with complaints of abdominal pain persisting for 1 week. The patient was diagnosed with esophageal malignancy, which was confirmed by surgical exploration and pathological biopsy. The patient developed septic shock on postoperative day 12, and blood culture suggested the growth of E. fergusonii. After treatment with meropenem, the patient's clinical symptoms improved significantly, and the second culture was negative. In this paper, we discuss the characteristics, diagnosis, and treatment of E. fergusonii. E. fergusonii is rarely reported, and its pathogenesis, drug resistance, and potential effects have not been completely confirmed. Thus, this case report adds valuable knowledge to the literature on E. fergusonii.

Enhanced metagenomic deep learning for disease prediction and consistent signature recognition by restructured microbiome 2D representations.

Metagenomic analysis has been explored for disease diagnosis and biomarker discovery. Low sample sizes, high dimensionality, and sparsity of metagenomic data challenge metagenomic investigations. Here, an unsupervised microbial embedding, grouping, and mapping algorithm (MEGMA) was developed to transform metagenomic data into individualized multichannel microbiome 2D representation by manifold learning and clustering of microbial profiles (e.g., composition, abundance, hierarchy, and taxonomy). These 2D representations enable enhanced disease prediction by established ConvNet-based AggMapNet models, outperforming the commonly used machine learning and deep learning models in metagenomic benchmark datasets. These 2D representations combined with AggMapNet explainable module robustly identified more reliable and replicable disease-prediction microbes (biomarkers). Employing the MEGMA-AggMapNet pipeline for biomarker identification from 5 disease datasets, 84% of the identified biomarkers have been described in over 74 distinct works as important for these diseases. Moreover, the method also discovered highly consistent sets of biomarkers in cross-cohort colorectal cancer (CRC) patients and microbial shifts in different CRC stages.

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila.

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.

AggMapNet: enhanced and explainable low-sample omics deep learning with feature-aggregated multi-channel networks.

Omics-based biomedical learning frequently relies on data of high-dimensions (up to thousands) and low-sample sizes (dozens to hundreds), which challenges efficient deep learning (DL) algorithms, particularly for low-sample omics investigations. Here, an unsupervised novel feature aggregation tool AggMap was developed to Aggregate and Map omics features into multi-channel 2D spatial-correlated image-like feature maps (Fmaps) based on their intrinsic correlations. AggMap exhibits strong feature reconstruction capabilities on a randomized benchmark dataset, outperforming existing methods. With AggMap multi-channel Fmaps as inputs, newly-developed multi-channel DL AggMapNet models outperformed the state-of-the-art machine learning models on 18 low-sample omics benchmark tasks. AggMapNet exhibited better robustness in learning noisy data and disease classification. The AggMapNet explainable module Simply-explainer identified key metabolites and proteins for COVID-19 detections and severity predictions. The unsupervised AggMap algorithm of good feature restructuring abilities combined with supervised explainable AggMapNet architecture establish a pipeline for enhanced learning and interpretability of low-sample omics data.

Combining kinase inhibitors for optimally co-targeting cancer and drug escape by exploitation of drug target promiscuities.

Cancers resist targeted therapeutics by drug-escape signaling. Multitarget drugs co-targeting cancer and drug-escape mediators (DEMs) are clinically advantageous. DEM coverage may be expanded by drug combinations. This work evaluated to what extent the kinase DEMs (KDEMs) can be optimally co-targeted by drug combinations based on target promiscuities of individual drugs. We focused on 41 approved and 28 clinical trial small molecule kinase inhibitor drugs with available experimental kinome and clinical pharmacokinetic data. From the kinome inhibitory profiles of these drugs, drug combinations were assembled for optimally co-targeting an established cancer target (EGFR, HER2, ABL1, or MEK1) and 9-16 target-associated KDEMs at comparable potency levels as that against the cancer target. Each set of two-, three-, and four-drug combinations co-target 36-71%, 44-89%, 50-88%, and 27-55% KDEMs of EGFR, HER2, ABL1, and MEK1, respectively, compared with the 36, 33, 38, and 18% KDEMs maximally co-targeted by an existing drug or drug combination approved or clinically tested for the respective cancer. Some co-targeted KDEMs are not covered by any existing drug or drug combination. Our work suggested that novel drug combinations may be constructed for optimally co-targeting cancer and drug escape by the exploitation of drug target promiscuities.

Mutual antagonism between Hippo signaling and cyclin E drives intracellular pattern formation.

Not much is known about how organelles organize into patterns. In ciliates, the cortical pattern is propagated during "tandem duplication," a cell division that remodels the parental cell into two daughter cells. A key step is the formation of the division boundary along the cell's equator. In Tetrahymena thermophila, the cdaA alleles prevent the formation of the division boundary. We find that the CDAA gene encodes a cyclin E that accumulates in the posterior cell half, concurrently with accumulation of CdaI, a Hippo/Mst kinase, in the anterior cell half. The division boundary forms between the margins of expression of CdaI and CdaA, which exclude each other from their own cortical domains. The activities of CdaA and CdaI must be balanced to initiate the division boundary and to position it along the cell's equator. CdaA and CdaI cooperate to position organelles near the new cell ends. Our data point to an intracellular positioning mechanism involving antagonistic Hippo signaling and cyclin E.

Clinical trials, progression-speed differentiating features and swiftness rule of the innovative targets of first-in-class drugs.

Drugs produce their therapeutic effects by modulating specific targets, and there are 89 innovative targets of first-in-class drugs approved in 2004-17, each with information about drug clinical trial dated back to 1984. Analysis of the clinical trial timelines of these targets may reveal the trial-speed differentiating features for facilitating target assessment. Here we present a comprehensive analysis of all these 89 targets, following the earlier studies for prospective prediction of clinical success of the targets of clinical trial drugs. Our analysis confirmed the literature-reported common druggability characteristics for clinical success of these innovative targets, exposed trial-speed differentiating features associated to the on-target and off-target collateral effects in humans and further revealed a simple rule for identifying the speedy human targets through clinical trials (from the earliest phase I to the 1st drug approval within 8 years). This simple rule correctly identified 75.0% of the 28 speedy human targets and only unexpectedly misclassified 13.2% of 53 non-speedy human targets. Certain extraordinary circumstances were also discovered to likely contribute to the misclassification of some human targets by this simple rule. Investigation and knowledge of trial-speed differentiating features enable prioritized drug discovery and development.

LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena.

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.

Drug sales confirm clinical advantage of multi-target inhibition of drug escapes by anticancer kinase inhibitors.

The clinical advantage of co-targeting cancer drug escape has been indicated by the percentage of these co-targeting drugs among all multi-target drugs in clinics and clinical trials. This clinical advantage needs to be further interrogated from such perspectives as the clinical impact of multi-target inhibition of drug-escape mediators. This impact may be reflected by drug sales data, that is, multi-target inhibition of higher number of drug-escape mediators favors the expanded coverage of drug-resistant patients leading to higher sales. We investigated whether this expectation is followed by the 25 FDA-approved anticancer kinase inhibitors, which were divided into 11 groups of comparable therapeutic mechanisms and approval years. We found 19 (76%) drugs to follow and 3 (12%) drugs not to follow this expectation. The remaining two (8%) and one (4%) drugs cannot be assessed due to insufficient data and incomparability. Therefore, drug sales strongly indicate the clinical advantage of multi-target inhibition of cancer drug escapes.

CMAUP: a database of collective molecular activities of useful plants.

The beneficial effects of functionally useful plants (e.g. medicinal and food plants) arise from the multi-target activities of multiple ingredients of these plants. The knowledge of the collective molecular activities of these plants facilitates mechanistic studies and expanded applications. A number of databases provide information about the effects and targets of various plants and ingredients. More comprehensive information is needed for broader classes of plants and for the landscapes of individual plant's multiple targets, collective activities and regulated biological pathways, processes and diseases. We therefore developed a new database, Collective Molecular Activities of Useful Plants (CMAUP), to provide the collective landscapes of multiple targets (ChEMBL target classes) and activity levels (in 2D target-ingredient heatmap), and regulated gene ontologies (GO categories), biological pathways (KEGG categories) and diseases (ICD blocks) for 5645 plants (2567 medicinal, 170 food, 1567 edible, 3 agricultural and 119 garden plants) collected from or traditionally used in 153 countries and regions. These landscapes were derived from 47 645 plant ingredients active against 646 targets in 234 KEGG pathways associated with 2473 gene ontologies and 656 diseases. CMAUP (http://bidd2.nus.edu.sg/CMAUP/) is freely accessible and searchable by keywords, plant usage classes, species families, targets, KEGG pathways, gene ontologies, diseases (ICD code) and geographical locations.

Two Antagonistic Hippo Signaling Circuits Set the Division Plane at the Medial Position in the Ciliate Tetrahymena.

In a single cell, ciliates maintain a complex pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes. The underlying molecular mechanisms of intracellular pattern formation in ciliates are largely unknown. Ciliates divide by tandem duplication, a process that remodels the parental cell into two daughters aligned head-to-tail. In the elo1-1 mutant of Tetrahymena thermophila, the segmentation boundary/division plane forms too close to the posterior end of the parental cell, producing a large anterior and a small posterior daughter cell, respectively. We show that ELO1 encodes a Lats/NDR kinase that marks the posterior segment of the cell cortex, where the division plane does not form in the wild-type. Elo1 acts independently of CdaI, a Hippo/Mst kinase that marks the anterior half of the parental cell, and whose loss shifts the division plane anteriorly. We propose that, in Tetrahymena, two antagonistic Hippo circuits focus the segmentation boundary/division plane at the equatorial position, by excluding divisional morphogenesis from the cortical areas that are too close to cell ends.

Proteins that control the geometry of microtubules at the ends of cilia.

Cilia, essential motile and sensory organelles, have several compartments: the basal body, transition zone, and the middle and distal axoneme segments. The distal segment accommodates key functions, including cilium assembly and sensory activities. While the middle segment contains doublet microtubules (incomplete B-tubules fused to complete A-tubules), the distal segment contains only A-tubule extensions, and its existence requires coordination of microtubule length at the nanometer scale. We show that three conserved proteins, two of which are mutated in the ciliopathy Joubert syndrome, determine the geometry of the distal segment, by controlling the positions of specific microtubule ends. FAP256/CEP104 promotes A-tubule elongation. CHE-12/Crescerin and ARMC9 act as positive and negative regulators of B-tubule length, respectively. We show that defects in the distal segment dimensions are associated with motile and sensory deficiencies of cilia. Our observations suggest that abnormalities in distal segment organization cause a subset of Joubert syndrome cases.

NPASS: natural product activity and species source database for natural product research, discovery and tool development.

There has been renewed interests in the exploration of natural products (NPs) for drug discovery, and continuous investigations of the therapeutic claims and mechanisms of traditional and herbal medicines. In-silico methods have been employed for facilitating these studies. These studies and the optimization of in-silico algorithms for NP applications can be facilitated by the quantitative activity and species source data of the NPs. A number of databases collectively provide the structural and other information of ∼470 000 NPs, including qualitative activity information for many NPs, but only ∼4000 NPs are with the experimental activity values. There is a need for the activity and species source data of more NPs. We therefore developed a new database, NPASS (Natural Product Activity and Species Source) to complement other databases by providing the experimental activity values and species sources of 35 032 NPs from 25 041 species targeting 5863 targets (2946 proteins, 1352 microbial species and 1227 cell-lines). NPASS contains 446 552 quantitative activity records (e.g. IC50, Ki, EC50, GI50 or MIC mainly in units of nM) of 222 092 NP-target pairs and 288 002 NP-species pairs. NPASS, http://bidd2.nus.edu.sg/NPASS/, is freely accessible with its contents searchable by keywords, physicochemical property range, structural similarity, species and target search facilities.

IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium.

Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in Chlamydomonas reinhardtii. IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body. In contrast to this 'open' system, IFT-B proteins from retrograde trains reenter the pool and a portion is reused directly in anterograde trains indicating a 'semi-open' system. Similar IFT systems were also observed in Tetrahymena thermophila and IMCD3 cells. FRAP analysis indicated that IFT proteins and motors of a given train are sequentially recruited to the basal bodies. IFT dynein and tubulin cargoes are loaded briefly before the trains depart. We conclude that the pool contains IFT trains in multiple stages of assembly queuing for successive release into the cilium upon completion.

HEROD: a human ethnic and regional specific omics database.

MOTIVATION: Genetic and gene expression variations within and between populations and across geographical regions have substantial effects on the biological phenotypes, diseases, and therapeutic response. The development of precision medicines can be facilitated by the OMICS studies of the patients of specific ethnicity and geographic region. However, there is an inadequate facility for broadly and conveniently accessing the ethnic and regional specific OMICS data. RESULTS: Here, we introduced a new free database, HEROD, a human ethnic and regional specific OMICS database. Its first version contains the gene expression data of 53 070 patients of 169 diseases in seven ethnic populations from 193 cities/regions in 49 nations curated from the Gene Expression Omnibus (GEO), the ArrayExpress Archive of Functional Genomics Data (ArrayExpress), the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). Geographic region information of curated patients was mainly manually extracted from referenced publications of each original study. These data can be accessed and downloaded via keyword search, World map search, and menu-bar search of disease name, the international classification of disease code, geographical region, location of sample collection, ethnic population, gender, age, sample source organ, patient type (patient or healthy), sample type (disease or normal tissue) and assay type on the web interface. AVAILABILITY AND IMPLEMENTATION: The HEROD database is freely accessible at http://bidd2.nus.edu.sg/herod/index.php. The database and web interface are implemented in MySQL, PHP and HTML with all major browsers supported. CONTACT: phacyz@nus.edu.sg.

The Hippo Pathway Maintains the Equatorial Division Plane in the Ciliate Tetrahymena.

The mechanisms that govern pattern formation within the cell are poorly understood. Ciliates carry on their surface an elaborate pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes by largely unknown mechanisms. Ciliates divide by tandem duplication: the cortex of the predivision cell is remodeled into two similarly sized and complete daughters. In the conditional cdaI-1 mutant of Tetrahymena thermophila, the division plane migrates from its initially correct equatorial position toward the cell's anterior, resulting in unequal cell division, and defects in nuclear divisions and cytokinesis. We used comparative whole genome sequencing to identify the cause of cdaI-1 as a mutation in a Hippo/Mst kinase. CdaI is a cortical protein with a cell cycle-dependent, highly polarized localization. Early in cell division, CdaI marks the anterior half of the cell, and later concentrates at the posterior end of the emerging anterior daughter. Despite the strong association of CdaI with the new posterior cell end, the cdaI-1 mutation does not affect the patterning of the new posterior cortical organelles. We conclude that, in Tetrahymena, the Hippo pathway maintains an equatorial position of the fission zone, and, by this activity, specifies the relative dimensions of the anterior and posterior daughter cell.

Discovery of novel dual VEGFR2 and Src inhibitors using a multistep virtual screening approach.

AIM: Simultaneous inhibition of VEGFR2 and Src may enhance the efficacy of VEGFR2-targeted cancer therapeutics. Hence, development of dual inhibitors on VEGFR2 and Src can be a useful strategy for such treatments. MATERIALS & METHODS: A multistep virtual screening protocol, comprising ligand-based support vector machines method, drug-likeness rules filter and structure-based molecular docking, was developed and employed to identify dual inhibitors of VEGFR2 and Src from a large commercial chemical library. Kinase inhibitory assays and cell viability assays were then used for experimental validation. RESULTS: A set of compounds belonging to six different molecular scaffolds was identified and sent for biological evaluation. Compound 3c belonging to the 2-amino-3-cyanopyridine scaffold exhibited good antiproliferative effect and dual-target activities against VEGFR2 and Src. CONCLUSION: This study demonstrated the ability of the multistep virtual screening approach to identify novel multitarget agents.