Single-cell analysis of cell competition using quantitative microscopy and machine learning

Cell competition is a widely conserved, fundamental biological quality control mechanism. The cell competition assay of MDCK wild-type versus mutant MDCK Scribble-knockdown (ScribKD) relies on a mechanical mechanism of competition, which posits that the emergence of compressing stresses within the tissue at high confluency drive the competitive outcome. According to this mechanism, proliferating wild-type cells out-compete mutant ScribKD cells, resulting in their apoptosis and apical extrusion. Previous studies show that there is an increased division rate of wild-type cells in neighbourhoods with high numbers of ScribKD cells, but what still remains a mystery is whether this is a cause or consequence of increased apoptosis in the “loser” cell population. This project also interrogated the competitive assay of wild-type versus RasV12 , which is hypothesized to operate on a biochemical mechanism and results in the apical extrusion (but not apoptosis) of the loser RasV12 population. For both these mechanisms of competition it is still unknown which population of cells are driving the winner/loser outcome. Is the winner cell proliferation prompting the loser cell demise? Or is an autonomous loser elimination prompting a subsequent winner cell proliferation? In my research, I have employed multi-modal, time-lapse microscopy to image competition assays continuously for several days. These data were then segmented into wild-type or mutant instances using a Convolutional Neural Network (CNN) that can differentiate between the cell types, after which they were tracked across cellular generations using a Bayesian multi-object tracker. A conjugate analysis of fluorescent cell-cycle indicator probes was then utilised to automatically identify key time points of cellular fate commitment using deep-learning image classification. A spatio-temporal analysis was then conducted in order to quantify any correlation between wild-type proliferation and mutant cell demise. For the case of wild-type versus ScribKD , there was no clear evidence for the wild-type cells mitoses directly impacting upon the ScribKD cell apoptotic elimination. Instead, a subsequent analysis found that a more subtle mechanism of pre-emptive, local density increases around the apoptosis site appeared to be determining the eventual ScribKD fate. On the other hand, there was clear evidence of a direct impact of wild-type mitoses on the subsequent apical extrusion and competitive elimination of RasV12 cells. Both of these conclusions agree with the prevailing classification of cell competition types: mechanical interactions are more diffuse and occur over a larger spatio-temporal domain, whereas biochemical interactions are constrained to nearest neighbour cells. The hypothesized density-dependency of ScribKD elimination was further quantified on a single-cell scale by these analyses, as well as a potential new understanding of RasV12 extrusion. Most interestingly, it appears that there is a clear biophysical mechanism to the elimination in the biochemical RasV12 cell competition. This suggests that perhaps a new semantic approach is needed in the field of cell competition in order to accurately classify different mechanisms of elimination

Mechanisms of therapy resistance in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a highly aggressive pediatric cancer that can affect both B cells and T cells. The advent of new therapies has increased the cure rates for both B-ALL and T-ALL patients. However, some patients still experience relapse with a variable response to the treatment and display poor survival. Thus,identification of novel predictive biomarkers that can predict therapy resistance may help to stratify this group of patients. This could also aid in developing an effective treatment strategy.Glucocorticoids are widely used along with the chemotherapeutic regimens for treating ALL patients. The response to glucocorticoids can predict long-term remission outcome. To understand the mechanisms of resistance to glucocorticoids, such as dexamethasone, we generated dexamethasone-resistant B-ALL cell lines in paper I. One such resistant cell line was found to possess increased FLT3 expression levels with FLT3-ITD and FLT3-R845G mutations that led to the activation of oncogenic RTK signaling. Further, second-generation FLT3 inhibitors, such as AC220 and crenolanib, suppressed this signaling both in vitro and in vivo.We continued exploring the dexamethasone resistance mechanisms in paper II using a different approach. We observed that dexamethasone exposure caused upregulation of Aurora kinase and its various downstream effector kinases such as JAK, p38, mTOR, and S6K. These kinases lead to β-catenin stabilization through phosphorylation-dependent inactivation of GSK-3β either directly or indirectly. Indeed, we observed partial restoration of dexamethasone sensitivity with a combination of dexamethasone and inhibitors targeting either these kinases or β-catenin.The expression of BCL2 varies in T-ALL depending on its stage of maturation, thereby T-ALL displays a heterogenous response to the BCL2-specific inhibitor venetoclax. We thus studied the mechanisms of venetoclax resistance using a panel of T-ALL cell lines in paper III. We observed that all the venetoclax-resistant T-ALL cell lines displayed non-universal changes in the expression of BCL2 family members and cancer stem cell markers, along with specific enrichment of cytokine signaling pathways. However, further investigations are warranted to identify additional mechanisms of venetoclax resistance in T-ALL.Combination therapy is usually the choice of treatment to overcome monotherapy resistance. With this in mind, in paper IV we identified that inhibiting BCL2 by venetoclax synergizes with PLK1 inhibition by volasertib in T-ALL cell lines and PDX models. We observed that BCL2L13 and PMAIP1 genes get upregulated upon PLK1 inhibition, probably through transcriptional regulation by FOXOs in interaction with β-catenin. Thus, the pro-apoptotic functions exhibited by BCL2L13 and PMAIP1 probably synergize with BCL2 inhibition in T-ALL, with the help of sustained β-catenin levels. Moreover, we also identified upregulation of oxidative phosphorylation (OXPHOS) in T-ALL PDXs that didn’t display synergy, which could be treated with a combination of venetoclax and oligomycin. However, additional experiments will be required to verify the above results

Machine Learning Based Classification of Textual Stimuli to Promote Ideation in Bioinspired Design

Bioinspired design uses biological systems to inspire engineering designs. One of bioinspired design’s challenges is identifying relevant information sources in biology for an engineering design task. Currently information can be retrieved by searching biology texts or journals using biology-focused keywords that map to engineering functions. However, this search technique can overwhelm designers with unusable results. This work explores the use of text classification tools to identify relevant biology passages for design. Further, this research examines the effects of using biology passages as stimuli during idea generation. Four human-subjects studies are examined in this work. Two surveys are performed in which participants evaluate sentences from a biology corpus and indicate whether each sentence prompts an idea for solving a specific design problem. The surveys are used to develop and evaluate text classification tools. Two idea generation studies are performed in which participants generate and record solutions for designing a corn shucker using either different sets of biology passages as design stimuli, or no stimuli. Based 286 sentences from the surveys, a k Nearest Neighbor classifier is developed that is able to identify helpful sentences relating to the function “separate” with a precision of 0.62 and recall of 0.48. This classifier could potentially double the number of helpful results found using a keyword search. The developed classifier is specific to the function “separate” and performs poorly when used for another function. Classifiers developed using all sentences and participant responses from the surveys are not able to reliably identify helpful sentences. From the idea generation studies, we determine that using any biology passages as design stimuli increases the quantity and variety of participant solutions. Solution quantity and variety are also significantly increased when biology passages are presented one at a time instead of all at once. Quality and variety are not significantly affected by the presence of design stimuli. Biological stimuli are also found to lead designers to types of solution that are not typically produced otherwise. This work develops a means for designers to find more useful information when searching biology and demonstrates several ways that biology passages can improve ideation

English for Strufents of Biological Departments

Структура пособия позволяет выбрать оптимальные способы организации работы для эффективного усвоения материала и аналитической обработки информации.Данное пособие предназначено для студентов I курса биологического факультета университета. В пособии представлены оригинальные тексты и упражнения к ним, способствующие закреплению лексического и грамматического материала

Англійська мова для біологів

Навчальний посібник укладений відповідно до вимог програми рівневого вивчення іноземної мови в університеті і призначений для студентів 1-3 курсів біологічного факультету, які вивчають мову у групах середнього та вищого рівнів. Мета посібника - поглиблення теоретичних і практичних знань студентів з англійської мови, формування та розвиток умінь і навичок сприймати і відтворювати іншомовний науковий фаховий дискурс, розширення словникового запасу загально-наукової та професійної лексики. Посібник містить сучасний автентичний текстовий матеріал, який охоплює базову лексику основних галузей біологічної науки. Практичні завдання укладено з урахуванням новітніх методичних стратегій викладання іноземної мови професійного спрямування. Посібник може бути корисним для магістрантів, аспірантів та науковців-біологів, які самостійно удосконалюють свої знання з англійської мови.CONTENTS : Передмова 5; UNIT 1 7; Lesson 1. Biology – the Science of Life 7; Lesson 2. Life 15; Lesson 3. The Origin of Life 23; Unit 1 Focus Words and Phrases 31; Revision and Additional Practice 1 32; UNIT 2 42; Lesson 1. Macromolecules 42; Lesson 2. Cell as a Basic Unit of Life 53; Lesson 3. Cell Structure 64; Lesson 4. Cell Division 74; Unit 2 Focus Words and Phrases 84; Revision and Additional Practice 2 85; UNIT 3 96; Lesson 1. The Protista 96; Lesson 2. The Bacteria 109; Lesson 3. Viruses 119; Unit 3 Focus Words and Phrases 131; Revision and Additional Practice 3 132; UNIT 4 143 ; Lesson 1. Vascular Plants 143 ; Lesson 2. Development of Gametophytes in Angiosperms 153 ; Lesson 3. Animals 165; Lesson 4. Phylum Chordata 175; Unit 4 Focus Words and Phrases 186; Revision and Additional Practice 4 188; UNIT 5 199; Lesson 1. Anthropogenesis 199; Lesson 2. Evolution 212; Lesson 3. Genetics 225; Lesson 4. Ecology 236; Unit 5 Focus Words and Phrases 248; Revision and Additional Practice 5 250; List of Biology Terms and Biology Related Words 261; References 277; List of Sources 278; Appendix 279; Tapescripts 280

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Using Haptic Virtual Reality to Increase Learning Gains and Construct Knowledge of Unobservable Phenomena

This project is designed to be a compilation of ten haptic virtual reality labs using the software zSpace. The labs will follow the NYS Living Environment Standards as well as the Next Generation Science Standards for living environment as well as physical/general science topics for middle school students. The project will be a list of available laboratories along with their appropriate fit into the curriculum and a description of how they fit New York State curriculum standards for the appropriate discipline. The goal of these laboratory assignments is to increase learning gains in students by allowing them to experience scientific phenomena that can often be unrelatable and unobservable

Machine Learning Based Classification of Textual Stimuli to Promote Ideation in Bioinspired Design

Bioinspired design uses biological systems to inspire engineering designs. One of bioinspired design’s challenges is identifying relevant information sources in biology for an engineering design task. Currently information can be retrieved by searching biology texts or journals using biology-focused keywords that map to engineering functions. However, this search technique can overwhelm designers with unusable results. This work explores the use of text classification tools to identify relevant biology passages for design. Further, this research examines the effects of using biology passages as stimuli during idea generation. Four human-subjects studies are examined in this work. Two surveys are performed in which participants evaluate sentences from a biology corpus and indicate whether each sentence prompts an idea for solving a specific design problem. The surveys are used to develop and evaluate text classification tools. Two idea generation studies are performed in which participants generate and record solutions for designing a corn shucker using either different sets of biology passages as design stimuli, or no stimuli. Based 286 sentences from the surveys, a k Nearest Neighbor classifier is developed that is able to identify helpful sentences relating to the function “separate” with a precision of 0.62 and recall of 0.48. This classifier could potentially double the number of helpful results found using a keyword search. The developed classifier is specific to the function “separate” and performs poorly when used for another function. Classifiers developed using all sentences and participant responses from the surveys are not able to reliably identify helpful sentences. From the idea generation studies, we determine that using any biology passages as design stimuli increases the quantity and variety of participant solutions. Solution quantity and variety are also significantly increased when biology passages are presented one at a time instead of all at once. Quality and variety are not significantly affected by the presence of design stimuli. Biological stimuli are also found to lead designers to types of solution that are not typically produced otherwise. This work develops a means for designers to find more useful information when searching biology and demonstrates several ways that biology passages can improve ideation