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Biology subjects

Joglekar, A. P.

Publications and source records attributed to Joglekar, A. P..

5 recordsLinked to original sources

Cell-APP: A generalizable method for microscopic cell annotation, segmentation, and classification

Deep learning-based segmentation models can accelerate the analysis of high-throughput microscopy data by automatically identifying and classifying cells in images. However, the datasets needed to train these models are typically assembled via laborious hand-annotation. This limits their scale and diversity, which in turn limits model performance. We present Cell-APP (Cellular Annotation and Perception Pipeline), a tool that automates the annotation of high-quality training data for transmitted-light (TL) cell segmentation. Cell-APP uses two inputs--paired TL and nuclear fluorescence images--and operates in two main steps. First, it extracts each cells location from the nuclear fluorescence channel and provides these locations to promptable deep learning models to generate cell masks. Then, it classifies each cell as mitotic or non-mitotic based on nuclear features. Together, these masks and classifications form the basis for cell segmentation training data. By training vision-transformer-based models on Cell-APP-generated datasets, we demonstrate how Cell-APP enables the creation of both cell line-specific and multi-cell line segmentation models. Cell-APP thus empowers laboratories to tailor cell segmentation models to their needs, and outlines a scalable path to creating general models for the research community. Significance StatementO_LIDeep learning-based cell segmentation models are typically trained on manually annotated datasets. Manual annotation limits dataset scalability and, consequently, the ability of trained models to generalize across cell types. C_LIO_LICell-APP automates mask generation and cell classification to rapidly create large, custom training datasets. It uses Meta AIs SAM for mask generation and extracts information from fluorescence signals for classification. C_LIO_LIBy reducing the need for hand-annotation, Cell-APP lowers the cost associated with building cell line-specific and generalist segmentation models. It may accelerate high-throughput image analysis and democratize model development across research labs. C_LI

cell biology↗

Aqueous Spice Extracts as Alternative Antimycotics to Control Highly Drug Resistant Extensive Biofilm Forming Clinical Isolates of Candida albicans

Candida albicans forms biofilm by associating with biotic and abiotic surfaces. Biofilm formation by C. albicans is relevant and significant as the organisms residing within, gain resistance to conventional antimycotics and are therefore difficult to treat. This study targeted the potential of spice-based antimycotics to control C. albicans biofilms. Ten clinical isolates of C. albicans along with a standard culture MTCC-3017 (ATCC-90028) were screened for their biofilm-forming ability. C. albicans M-207 and C. albicans S-470 were identified as high biofilm formers by point inoculation on TSA medium as they formed a lawn within 16 h and exhibited resistance to fluconazole and caspofungin at 25 mcg and 8 mcg respectively. Aqueous and organic spice extracts were screened for their antimycotic activity against C. albicans M-207 and S-470 by agar and disc diffusion and Zone of Inhibition was observed. Minimal Inhibitory Concentration was determined based on growth absorbance and cell viability measurements. The whole aqueous extract of garlic inhibited biofilms of C. albicans M-207, whereas whole aqueous extracts of garlic, clove, and Indian gooseberry were effective in controlling C. albicans S-470 biofilm within 12 h of incubation. The presence of allicin, ellagic acid, and gallic acid as dominant compounds in the aqueous extracts of garlic, clove, and Indian gooseberry respectively was determined by High-Performance Thin Layer Chromatography and Liquid Chromatography-Mass Spectrometry. The morphology of C. albicans biofilm at different growth periods was also determined through bright field microscopy, phase contrast microscopy, and fluorescence microscopy. The results of this study indicated that the alternate approach in controlling high biofilm-forming, multi-drug resistant clinical isolates of C. albicans M-207 and S-470 using whole aqueous extracts of garlic, clove, and Indian gooseberry is a safe, potential, and cost-effective one that can benefit the health care needs with additional effective therapeutics to treat biofilm infections.

microbiology↗

Kre28-Spc105 interaction is essential for their recruitment at the kinetochore

Kinetochores are macromolecular protein assemblies that attach sister chromatids to spindle microtubules and mediate accurate chromosome segregation during mitosis. The outer kinetochore consists of the KMN network, a protein super-complex comprising Knl1 (yeast Spc105), Mis12 (yeast Mtw1), and Ndc80 (yeast Ndc80), which harbors sites for microtubule binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signaling. It is known that Spc105 forms a complex with kinetochore component Kre28. However, where Kre28 physically localizes in the budding yeast kinetochore is not clear. The exact function of Kre28 at the kinetochore is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast kinetochores using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the kinetochore with a 1:1 stoichiometry. We also show that the Kre28-Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the kinetochores. We created several truncation mutants of kre28 that affect Spc105 loading at the kinetochores. When over-expressed, these mutants sustain the cell viability, but SAC signaling and kinetochore biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the kinetochores.

cell biology↗

Minimization of cross-talk between Spindle Assembly Checkpoint silencing and error correction

Accurate chromosome segregation during cell division requires that the pair of sister kinetochores on each chromosome attach to microtubules originating from opposite spindle poles. This is ensured by the combined action of the Spindle Assembly Checkpoint (SAC), which detects unattached kinetochores, and an error correction mechanism that destabilizes incorrect attachment of both sister kinetochores to the same spindle pole. These processes are downregulated by Protein Phosphatase 1 (PP1), which both silences the SAC and stabilizes kinetochore-microtubule attachments. We find that this dual PP1 role can be problematic: if PP1 is recruited to the kinetochore for SAC silencing prior to chromosome biorientation, it interferes with error correction. We show that to mitigate this cross-talk, the yeast kinetochore uses independent PP1 sources to stabilize correct attachments and to silence the SAC, and also delays the recruitment of PP1 for SAC silencing. Consequently, chromosome biorientation precedes SAC silencing ensuring accurate chromosome segregation.

cell biology↗

Ectopic activation of the Spindle Assembly Checkpoint reveals its biochemical design and physiological operation

Switch-like activation of the Spindle Assembly Checkpoint (SAC) is critical for accurate chromosome segregation during cell division. To determine the mechanisms that implement it, we engineered an ectopic, kinetochore-independent SAC activator, the \"eSAC\". The eSAC stimulates the SAC signaling cascade by artificially dimerizing the Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the signaling scaffold in the kinetochore. Quantitative analyses and mathematical modeling of the eSAC reveal that the recruitment of multiple SAC proteins by the KNL1 phosphodomain stimulates synergistic signaling, which enables a small number of KNL1 molecules produce a disproportionately strong anaphase-inhibitory signal. However, when multiple KNL1 molecules signal concurrently, they compete for a limited cellular pool of SAC proteins. This frustrates synergistic signaling and modulates signal output. Together, these mechanisms institute automatic gain control - inverse, non-linear scaling between the signal output per kinetochore and the unattached kinetochore number, and thus enact the SAC switch.

cell biology↗