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.

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.

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.

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.

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.

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.

Bicaudal-D is essential for egg chamber formation and cytoskeletal organization in drosophila oogenesis.

Bicaudal-D (Bic-D) is required for the transport of determinant mRNAs and proteins to the presumptive oocyte, an essential step in the differentiation of the oocyte. Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins. We characterized the ovarian phenotypes of females expressing mutant Bic-D proteins (Bic-D(H)) deleted for each of the heptad repeat domains. The altered migration of follicle cells we observe in mutant ovaries suggests that Bic-D functions in the germline and directs the inward migration of somatic follicle cells. In the germarium Bic-D is required for the organization of the egg chamber and the structural integrity of the oocyte and nurse cells. Examination of the polarized microtubule network in Bic-D(H) ovaries shows that Bic-D function is required for both the establishment of the polarized microtubule network and its maintenance throughout oogenesis. To explain the multiple functions suggested by the pleiotropic Bic-D phenotype, we propose that Bic-D protein could form itself a filamentous structure and represent an integral, essential part of the cytoskeleton.

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.

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.

Cactin, a conserved protein that interacts with the Drosophila IkappaB protein cactus and modulates its function.

Rel transcription factors function in flies and vertebrates in immunity and development. Although Rel proteins regulate diverse processes, the control of their function is conserved. In a two-hybrid screen for additional components of the pathway using the Drosophila I-kappaB protein Cactus as a bait, we isolated a novel coiled-coil protein with N-terminal Arg-Asp (RD)- like motifs that we call Cactin. Like the other components of this pathway, Cactin is evolutionarily conserved. Over-expression of cactin in a cactus(A2) heterozygous background results in the enhancement of the cactus phenotype. Both the embryonic lethality and ventralization are strongly increased, suggesting that cactin functions in the Rel pathway controlling the formation of dorsal-ventral embryonic polarity.

Operative management of diverticular emergencies: strategies and outcomes.

HYPOTHESIS: A selective surgical approach using either a 1- or a 2-stage resection is relatively safe and effective in the management of acute complicated colonic diverticulosis. DESIGN: A consecutive cohort study. SETTING: A university hospital. PATIENTS: Eighty-nine consecutive patients who underwent emergency operations for diverticular disease between July 1, 1984, and June 30, 1999. There were 53 male and 36 female patients (mean age, 47 years). The ethnic background was predominantly Mexican American (58 patients [65.2%]). INTERVENTIONS: Resections of the affected colon (n = 83) plus construction of a Hartmann pouch or mucous fistula (n = 72) or primary anastomosis (n = 11). MAIN OUTCOME MEASURES: Morbidity, mortality, and length of hospital stay. RESULTS: Sixty-eight operations were performed for perforation at an annual rate that has increased greater than 75% in the past 15 years. Another 14 patients underwent operations for obstruction, and 7 underwent operations to control unremitting hemorrhage. Surgical therapy included resection of the affected segment of the bowel in 83 (93%) of the 89 patients, and a Hartmann pouch or mucous fistula was added in 72 (81%). A primary anastomosis was performed in 4 (80%) of 5 right-sided lesions but in only 7 (8%) of 84 left-sided lesions. Morbidity occurred in 38 (43%) of the 89 patients, and the mortality was 4%, with 4 deaths occurring secondary to sepsis in high-risk patients with perforations (n = 3) or obstructions (n = 1). The average length of hospital stay was 19.7 days (range, 5-80 days). CONCLUSIONS: Emergency operations for diverticular disease are uncommon but may be associated with substantial morbidity and occasional mortality. Complicated diverticulosis may present at a relatively young age, and perforated forms appear to be increasing rapidly in prevalence. Most diverticular lesions can be satisfactorily managed using a selective approach based on resection with either a primary anastomosis or a temporary colostomy.

The Drosophila shark tyrosine kinase is required for embryonic dorsal closure.

Dorsal closure (DC) in the Drosophila embryo requires the coordinated interaction of two different functional domains of the epidermal cell layer-the leading edge (LE) and the lateral epidermis. In response to activation of a conserved c-Jun amino-terminal kinase (JNK) signaling module, the dorsal-most layer of cells, which constitute the LE of the stretching epithelial sheet, secrete Dpp, a member of the TGFbeta superfamily. Dpp and other LE cell-derived signaling molecules stimulate the bilateral dorsal elongation of cells of the dorsolateral epidermis over the underlaying amnioserosa and the eventual fusion of their LEs along the dorsal midline. We have found that flies bearing a Shark tyrosine kinase gene mutation, shark(1), exhibit a DC-defective phenotype. Dpp fails to be expressed in shark(1) mutant LE cells. Consistent with these observations, epidermal-specific reconstitution of shark function or overexpression of an activated form of c-Jun in the shark(1) mutant background, rescues the DC defect. Thus, Shark regulates the JNK signaling pathway leading to Dpp expression in LE cells. Furthermore, constitutive activation of the Dpp pathway throughout the epidermis fails to rescue the shark(1) DC defect, suggesting that Shark may function in additional pathways in the LE and/or lateral epithelium.

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.

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.

flex, an X-linked female-lethal mutation in Drosophila melanogaster controls the expression of Sex-lethal.

The Sex-lethal (Sxl) gene is required in Drosophila females for sexual differentiation of the soma, for gem cell differentiation and dosage compensation. We have isolated three new alleles of female-lethal-on-X (flex), an X-linked female-lethal mutation and have characterized its function in sex determination. SXL protein is missing in flex/flex embryos, however transcription from both Sxl(Pe), the early Sxl promoter and Sxl(Pm), the late maintenance promoter, is normal in flex homozygotes. In flex/flex embryos, Sxl mRNA is spliced in the male mode. Analysis of flex germline clones shows that it also functions in oogenesis, but in contrast to Sxl mutants that show an early arrest tumorous phenotype, flex mutant egg chambers develop to stage 10. In flex ovarian clones, Sxl RNA is also spliced in the male form. Hence, flex is a sex-specific regulator of Sxl functioning in both the soma and the germline. Genetic interaction studies show that flex does not enhance female lethality of Sxl loss-of-function alleles but it rescues the male-specific lethality of both of the gain-of-function Sxl mutations, Sxl(M1 )and Sxl(M4.) In contrast to mutations in splicing regulators of Sxl, the female lethality of flex is not rescued by either Sxl(M1 )or Sxl(M4). Based on these observations, we propose that flex regulates Sxl at a post-splicing stage and regulates either its translation or the stability of the SXL protein.

An iterative structure-assisted approach to sequence alignment and comparative modeling.

Correct alignment of the sequence of a target protein with those of homologues of known three-dimensional structure is a key step in comparative modeling. Usually an iterative approach that takes account of the local and overall structural features is required. We describe such an approach that exploits databases of structural alignments of homologous proteins (HOMSTRAD, http:/(/)www-cryst.bioc.cam.ac.uk/ approximately homstrad) and protein superfamilies (CAMPASS, http:/(/)www-cryst.bioc.cam.ac.uk/ approximately campass), in which structure-based alignments are analyzed and formatted with the program JOY (http:/(/)www-cryst.bioc.cam.ac.uk/ approximately joy) to reveal conserved local structural features. The databases facilitate the recognition of a family or superfamily, they assist in the selection of useful parent structures, they are helpful in alignment of the target sequences with the parent set, and are useful for deriving relationships that can be used in validating models. In the iterative approach, a model is constructed on the basis of the proposed sequence alignment and this is then reexpressed in the JOY format and realigned with the parent set. This is repeated until the model and sequence alignment is optimized. We examine the case for comparison and use of multiple structures of family members, rather than a single parent structure. We use the targets attempted by our group in CASP3 to assess the value of such procedures.