A machine learning approach to predict cellular mechanical stresses in response to chemical perturbation.

Mechanical stresses generated at the cell-cell level and cell-substrate level have been suggested to be important in a host of physiological and pathological processes. However, the influence various chemical compounds have on the mechanical stresses mentioned above is poorly understood, hindering the discovery of novel therapeutics, and representing a barrier in the field. To overcome this barrier, we implemented two approaches: 1) monolayer boundary predictor and 2) discretized window predictor utilizing either stepwise linear regression or quadratic support vector machine machine learning model to predict the dose-dependent response of tractions and intercellular stresses to chemical perturbation. We used experimental traction and intercellular stress data gathered from samples subject to 0.2 or 2 µg/mL drug concentrations along with cell morphological properties extracted from the bright-field images as predictors to train our model. To demonstrate the predictive capability of our machine learning models, we predicted tractions and intercellular stresses in response to 0 and 1 µg/mL drug concentrations which were not utilized in the training sets. Results revealed the discretized window predictor trained just with four samples (292 images) to best predict both intercellular stresses and tractions using the quadratic support vector machine and stepwise linear regression models, respectively, for the unseen sample images.

Probing Endothelial Cell Mechanics Through Connexin 43 Disruption.

The endothelium has been established to generate intercellular stresses and suggested to transmit these intercellular stresses through cell-cell junctions, such as VE-Cadherin and ZO-1, for example. Although the previously mentioned molecules reflect the appreciable contributions both adherens junctions and tight junctions are believed to have in endothelial cell intercellular stresses, in doing so they also reveal the obscure relationship that exists between gap junctions and intercellular stresses. Therefore, to bring clarity to this relationship we disrupted expression of the endothelial gap junction connexin 43 (Cx43) by exposing confluent human umbilical vein endothelial cells (HUVECs) to a low (0.2 µg/mL) and high (2 µg/mL) concentration of 2,5-dihydroxychalcone (chalcone), a known Cx43 inhibitor. To evaluate the impact Cx43 disruption had on endothelial cell mechanics we utilized traction force microscopy and monolayer stress microscopy to measure cell-substrate tractions and cell-cell intercellular stresses, respectively. HUVEC monolayers exposed to a low concentration of chalcone produced average normal intercellular stresses that were on average 17% higher relative to control, while exposure to a high concentration of chalcone yielded average normal intercellular stresses that were on average 55% lower when compared to control HUVEC monolayers. HUVEC maximum shear intercellular stresses were observed to decrease by 16% (low chalcone concentration) and 66% (high chalcone concentration), while tractions exhibited an almost 2-fold decrease under high chalcone concentration. In addition, monolayer cell velocities were observed to decrease by 19% and 35% at low chalcone and high chalcone concentrations, respectively. Strain energies were also observed to decrease by 32% and 85% at low and high concentration of chalcone treatment, respectively, when compared to control. The findings we present here reveal for the first time the contribution Cx43 has to endothelial biomechanics.

Disturbed fluid flow reinforces endothelial tractions and intercellular stresses.

Disturbed fluid flow is well understood to have significant ramifications on endothelial function, but the impact disturbed flow has on endothelial biomechanics is not well understood. In this study, we measured tractions, intercellular stresses, and cell velocity of endothelial cells exposed to disturbed flow using a custom-fabricated flow chamber. Our flow chamber exposed cells to disturbed fluid flow within the following spatial zones: zone 1 (inlet; length 0.676-2.027 cm): 0.0037 ± 0.0001 Pa; zone 2 (middle; length 2.027-3.716 cm): 0.0059 ± 0.0005 Pa; and zone 3 (outlet; length 3.716-5.405 cm): 0.0051 ± 0.0025 Pa. Tractions and intercellular stresses were observed to be highest in the middle of the chamber (zone 2) and lowest at the chamber outlet (zone 3), while cell velocity was highest near the chamber inlet (zone 1), and lowest near the middle of the chamber (zone 2). Our findings suggest endothelial biomechanical response to disturbed fluid flow to be dependent on not only shear stress magnitude, but the spatial shear stress gradient as well. We believe our results will be useful to a host of fields including endothelial cell biology, the cardiovascular field, and cellular biomechanics in general.

Extracellular Matrix Composition Alters Endothelial Force Transmission.

ECM composition is important in a host of pathophysiological processes such as angiogenesis, atherosclerosis, and diabetes, for example and during each of these processes ECM composition has been reported to change over time. However, the impact ECM composition has on the endothelium’s ability to respond mechanically is currently unknown. Therefore, in this study we seeded human umbilical vein endothelial cells (HUVECs) onto soft hydrogels coated with an ECM concentration of 0.1 mg/mL at the following collagen I (Col-I) and fibronectin (FN) ratios: 100%Col-I, 75%Col-I-25%FN, 50%Col-I-50%FN, 25%Col-I-75%FN, and 100%FN. We subsequently measured tractions, intercellular stresses, strain energy, cell morphology, and cell velocity. Our results revealed huvecs seeded on gels coated with 50% Col-I - 50% FN to have the highest intercellular stresses, tractions, strain energies, but the lowest velocities and cell circularity. Huvecs seeded on 100% Col-I had the lowest tractions, cell area while havingthe highest velocities and cell circularity. In addition, cells cultured on 25% Col-I and 75% FN had the lowest intercellular stresses, but the highest cell area. Huvecs cultured on 100% FN yielded the lowest strain energies. We believe these results will be of great importance to the cardiovascular field, biomedical field, and cell mechanics. Summary: Study the influence of different Col-I - FN ECM compositions on endothelial cell mechanics and morphology.

Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration and axonal transport.

Haplo-insufficiency of human Lis1 causes lissencephaly. Reduced Lis1 activity in both humans and mice results in a neuronal migration defect. Here we show that Drosophila Lis1 is highly expressed in the nervous system. Lis1 is essential for neuroblast proliferation and axonal transport, as shown by a mosaic analysis using a Lis1 null mutation. Moreover, it is cell-autonomously required for dendritic growth, branching and maturation. Analogous mosaic analysis shows that neurons containing a mutated cytoplasmic-dynein heavy chain (Dhc64C) exhibit phenotypes similar to Lis1 mutants. These results implicate Lis1 as a regulator of the microtubule cytoskeleton and show that it is important for diverse physiological functions in the nervous system.

Increased platelet:RBC ratios are associated with improved survival after massive transfusion.

BACKGROUND: Several recent military and civilian trauma studies demonstrate that improved outcomes are associated with early and increased use of plasma-based resuscitation strategies. However, outcomes associated with platelet transfusions are poorly characterized. We hypothesized that increased platelet:red blood cells (RBC) ratios would decrease hemorrhagic death and improve survival after massive transfusion (MT). METHODS: A transfusion database of patients transported from the scene to 22 Level I Trauma Centers over 12 months in 2005 to 2006 was reviewed. MT was defined as receiving ≥ 10 RBC units within 24 hours of admission. To mitigate survival bias, 25 patients who died within 60 minutes of arrival were excluded from analysis. Six random donor platelet units were considered equal to a single apheresis platelet unit. Admission and outcome data associated with the low (>1:20), medium (1:2), and high (1:1) platelet:RBC ratios were examined. These groups were based on the median value of the tertiles for the ratio of platelets:RBC units. RESULTS: Two thousand three hundred twelve patients received at least one unit of blood and 643 received an MT. Admission vital signs, INR, temperature, pH, Glasgow Coma Scale, Injury Severity Score, and age were similar between platelet ratio groups. The average admission platelet counts were lower in the patients who received the high platelet:RBC ratio versus the low ratio (192 vs. 216, p = 0.03). Patients who received MT were severely injured, with a mean (± standard deviation) Injury Severity Score of 33 ± 16 and received 22 ± 15 RBCs and 11 ± 14 platelets within 24 hours of injury. Increased platelet ratios were associated with improved survival at 24 hours and 30 days (p < 0.001 for both). Truncal hemorrhage as a cause of death was decreased (low: 67%, medium: 60%, high: 47%, p = 0.04). Multiple organ failure mortality was increased (low: 7%, medium: 16%, high: 27%, p = 0.003), but overall 30-day survival was improved (low: 52%, medium: 57%, high: 70%) in the high ratio group (medium vs. high: p = 0.008; low vs. high: p = 0.007). CONCLUSION: Similar to recently published military data, transfusion of platelet:RBC ratios of 1:1 was associated with improved early and late survival, decreased hemorrhagic death and a concomitant increase in multiple organ failure-related mortality. Based on this large retrospective study, increased and early use of platelets may be justified, pending the results of prospective randomized transfusion data.

Dissociation of the dorsal-cactus complex and phosphorylation of the dorsal protein correlate with the nuclear localization of dorsal.

The formation of dorsal-ventral polarity in Drosophila requires the asymmetric nuclear localization of the dorsal protein along the D/V axis. This process is regulated by the action of the dorsal group genes and cactus. We show that dorsal and cactus are both phosphoproteins that form a stable cytoplasmic complex, and that the cactus protein is stabilized by its interaction with dorsal. The dorsal-cactus complex dissociates when dorsal is targeted to the nucleus. While the phosphorylation of cactus remains apparently unchanged during early embryogenesis, the phosphorylation state of dorsal correlates with its release from cactus and with its nuclear localization. This differential phosphorylation event is regulated by the dorsal group pathway.

Dorsal, an embryonic polarity gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel.

The Drosophila gene, dorsal, is a maternal effect locus that is essential for the establishment of dorsal-ventral polarity in the developing embryo. The dorsal protein was predicted from the complementary DNA sequence; it is almost 50 percent identical, over an extensive region, to the protein encoded by the avian oncogene v-rel, its cellular homolog, c-rel, and a human c-rel fragment. The oncogene v-rel is highly oncogenic in avian lymphoid, spleen, and bone marrow cells.

Advice for starting a mechanobiology lab.

Starting a new lab in mechanobiology and a new lab in general can be an exciting, yet daunting experience. Depending on your previous position, you have now found yourself with significantly more responsibility in more ways than one. For those considering taking such a career path, I present in this commentary advice that will hopefully be useful and provide food for thought.

Axon patterning requires DN-cadherin, a novel neuronal adhesion receptor, in the Drosophila embryonic CNS.

We identified DN-cadherin, a novel Drosophila cadherin that is expressed in axons and in the mesoderm. Although DN-cadherin has diverged from vertebrate classic cadherins in terms of its extracellular structure, it still can form a complex with catenins and induce cell aggregation, as do the vertebrate molecules. Loss-of-function mutations of the gene resulted in either embryonic lethality or uncoordinated locomotion of adults. In the central nervous system of null mutant embryos, subsets of ipsilateral axons displayed a variety of aberrant trajectories including failure of position shifts, defective bundling, and errors in directional migration of growth cones. These results suggest that processes of axon patterning critically depend on DN-cadherin-mediated axon-axon interactions.

Dorsal-ventral polarity in the Drosophila embryo.

Embryonic dorsal-ventral polarity in Drosophila is established through a series of successive steps and requires the functions of both maternal and zygotic genes. The graded distribution of the transcription factor dorsal in blastoderm nuclei represents the transition from the maternal to the zygotic program. This results in the activation of specific zygotic genes that act to create the regional pattern along this axis.

Nuclear factor-kappa B pathways in Drosophila.

The nuclear factor kappa B (NF-kappaB) pathways in Drosophila are multi-component pathways, as in vertebrates, that regulate the expression of many genes responsible for the formation of dorsal-ventral polarity in the early embryo, the innate immune response to infection with Gram- negative and positive bacteria and fungi, the cellular immune response and hematopoiesis. Overactivation of the fly pathway can result in developmental defects, overproliferation of hemocytes and the formation of melanotic tumors or nodules. The extracellular events leading to the maturation of the ligand for initiation of the Drosophila NF-kappaB pathway is not conserved between flies and vertebrates, but the Toll receptor and downstream events are remarkably similar. NF-kappaB proteins have been identified in mollusks, and arthropods such as horseshoe crabs and beetles, indicating that this pathway has been established more than 500 million years ago. The fly NF-kappaB pathways are less complex than those in vertebrates, with the involvement of fewer proteins, but they are, nonetheless, just as important as their vertebrate counterparts for the life of the fly.

Cactus-independent regulation of Dorsal nuclear import by the ventral signal.

Rel-family transcription factors function in a variety of biological processes, including development and immunity. During early Drosophila development, the Toll-Cactus-Dorsal pathway regulates the establishment of the embryonic dorsoventral axis. The last step in this pathway is the graded nuclear import of the Rel protein Dorsal. Dorsal is retained in the cytoplasm by the IkappaB-family protein Cactus. Phosphorylation of both Dorsal and Cactus is regulated by a Toll-receptor-dependent ventral signal relayed by the Tube and Pelle proteins. Phosphorylation of Cactus leads to its degradation and to the release of Dorsal to form a ventral-to-dorsal nuclear Dorsal gradient. To understand how the ventral signal regulates the nuclear import and activity of Dorsal, we deleted its conserved nuclear localization signal (NLS). The truncated protein remained in the cytoplasm and could antagonize the function of wild-type Dorsal, suggesting that Dorsal forms a dimer in the cytoplasm. Further, the nuclear import of a mutant Dorsal protein that failed to interact with Cactus was still regulated by the ventral signal. Our results are consistent with a model in which ventral signal-dependent modification of both Cactus and Dorsal is required for the graded nuclear import of Dorsal.

Dorsoventral pattern formation in Drosophila: signal transduction and nuclear targeting.

The maternal determinants of dorsoventral polarity of the Drosophila embryo are derived from somatic and germ-line components of the egg chamber. During oogenesis, asymmetry seems to be established by a signal transduction process. This process is thought to provide the developing embryo with a ventral signal responsible for determining the embryonic axis. Through a set of interactions that may involve signal transduction and proteolytic cascade events, positional information is generated in the form of a graded distribution of dorsal protein in blastoderm nuclei. Different levels of dorsal protein result in asymmetric expression of zygotic genes that ultimately specify cell fate.

Regulated nuclear import of the Drosophila rel protein dorsal: structure-function analysis.

The formation of a gradient of nuclear Dorsal protein in the early Drosophila embryo is the last step in a maternally encoded dorsal-ventral signal transduction pathway. This gradient is formed in response to a ventral signal, which leads to the dissociation of cytoplasmic Dorsal from the I kappa B homolog Cactus. Free Dorsal is then targeted to the nucleus. Dorsal is a Rel-family transcription factor. Signal-dependent nuclear localization characterizes the regulation of Rel proteins. In order to identify regions of Dorsal that are essential for its homodimerization, nuclear targeting, and interaction with Cactus, we have performed an in vivo structure-function analysis. Our results show that all these functions are carried out by regions within the conserved Rel-homology region of Dorsal. The C-terminal divergent half of Dorsal is dispensable for its selective nuclear import. A basic stretch of 6 amino acids at the C terminus of the Rel-homology region is necessary for nuclear localization. This nuclear localization signal is not required for Cactus binding. Removal of the N-terminal 40 amino acids abolished the nuclear import of Dorsal, uncovering a potentially novel function for this highly conserved region.

The shut-down gene of Drosophila melanogaster encodes a novel FK506-binding protein essential for the formation of germline cysts during oogenesis.

In Drosophila melanogaster, the process of oogenesis is initiated with the asymmetric division of a germline stem cell. This division results in the self-renewal of the stem cell and the generation of a daughter cell that undergoes four successive mitotic divisions to produce a germline cyst of 16 cells. Here, we show that shut-down is essential for the normal function of the germline stem cells. Analysis of weak loss-of-function alleles confirms that shut-down is also required at later stages of oogenesis. Clonal analysis indicates that shut-down functions autonomously in the germline. Using a positional cloning approach, we have isolated the shut-down gene. Consistent with its function, the RNA and protein are strongly expressed in the germline stem cells and in 16-cell cysts. The RNA is also present in the germ cells throughout embryogenesis. shut-down encodes a novel Drosophila protein similar to the heat-shock protein-binding immunophilins. Like immunophilins, Shut-down contains an FK506-binding protein domain and a tetratricopeptide repeat. In plants, high-molecular-weight immunophilins have been shown to regulate cell divisions in the root meristem in response to extracellular signals. Our results suggest that shut-down may regulate germ cell divisions in the germarium.

The genetics of the dorsal-Bicaudal-D region of Drosophila melanogaster.

The chromosomal region 36C on 2L contains two maternal-effect loci, dorsal (dl) and Bicaudal-D (Bic-D), which are involved in establishing polarity of the Drosophila embryo along the dorsal-ventral and anterior-posterior axes, respectively. To analyze the region genetically, we isolated X-ray-induced dorsal alleles, which we recognized by virtue of the haplo-insufficient temperature-sensitive dorsal-dominant phenotype in progeny of single females heterozygous for a mutagenized chromosome. From the 20,000 chromosomes tested, we isolated three deficiencies, two inversions with breakpoint in dl and one apparent dl point mutant. One of the deficiencies, Df(2L)H20 (36A6,7; 36F1,2) was used to screen for EMS-induced lethal- and maternal-effect mutants mapping in the vicinity of dl and Bic-D. We isolated 44 lethal mutations defining 11 complementation groups. We also recovered as maternal-effect mutations four dl alleles, as well as six alleles of quail and one allele of kelch, two previously identified maternal-effect genes. Through complementation tests with various viable mutants and deficiencies in the region, a total of 18 loci were identified in an interval of about 30 cytologically visible bands. The region was subdivided into seven subregions by deficiency breakpoints. One lethal complementation group as well as the two maternal loci, Bic-D and quail, are located in the same deficiency interval as is dl.

Functional domains of the Drosophila bicaudal-D protein.

The localization of oocyte-specific determinants in the form of mRNAs to the pro-oocyte is essential for the establishment of oocyte identity. Localization of the Bicaudal-D (Bic-D) protein to the presumptive oocyte is required for the accumulation of Bic-D and other mRNAs to the pro-oocyte. The Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins, and several of the mutations in Bic-D map to these conserved domains. We have undertaken a structure-function analysis of Bic-D by testing the function of mutant Bic-D transgenes (Bic-D(H)) deleted for each of the heptad repeat domains in a Bic-D null background. Our transgenic studies indicate that only the C-terminal heptad repeat deletion results in a protein that has lost zygotic and ovarian functions. The three other deletions result in proteins with full zygotic function, but with affected ovarian function. The functional importance of each domain is well correlated with its conservation in evolution. The analysis of females heterozygous for Bic-D(H) and the existing alleles Bic-D(PA66) or Bic-D(R26) reveals that Bic-D(R26) as well as some of Bic-D(H) transgenes have antimorphic effects. The yeast two-hybrid interaction assay shows that Bic-D forms homodimers. Furthermore, we found that Bic-D exists as a multimeric protein complex consisting of Egl and at least two Bic-D monomers.