A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells

Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions

Biophysical mechanisms of single-cell interactions with microtopographical cues

Biophysical cues encoded in the extracellular matrix (ECM) are increasingly being explored to control cell behavior in tissue engineering applications. Recently, we showed that cell adhesion to microtopographical structures (“micropegs”) can suppress proliferation in a manner that may be blunted by inhibiting cellular contractility, suggesting that this effect is related to altered cell-scaffold mechanotransduction. We now directly investigate this possibility at the microscale through a combination of live-cell imaging, single-cell mechanics methods, and analysis of gene expression. Using time-lapse imaging, we show that when cells break adhesive contacts with micropegs, they form F-actin-filled tethers that extend and then rupture at a maximum, critical length that is greater than trailing-edge tethers observed on topographically flat substrates. This critical tether length depends on myosin activation, with inhibition of Rho-associated kinase abolishing topography-dependent differences in tether length. Using cellular de-adhesion and atomic force microscopy indentation measurements, we show that the micropegs enhance cell-scaffold adhesive interactions without changing whole-cell elasticity. Moreover, micropeg adhesion increases expression of specific mechanotransductive genes, including RhoA GTPase and myosin heavy chain II, and, in myoblasts, the functional marker connexin 43. Together, our data support a model in which microtopographical cues alter the local mechanical microenvironment of cells by modulating adhesion and adhesion-dependent mechanotransductive signaling

Tension, Free Space, and Cell Damage in a Microfluidic Wound Healing Assay

We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method utilizes multiple laminar flows to selectively cleave cells enzymatically, and allows us to present a 'damage free' denudation. We therefore isolate the influence of free space on the onset of sheet migration. First, we observe denudation directly to measure the retraction in the cell sheet that occurs after cell-cell contact is broken, providing direct and quantitative evidence of strong tension within the sheet. We further probe the mechanical integrity of the sheet without denudation, instead using laminar flows to selectively inactivate actomyosin contractility. In both cases, retraction is observed over many cell diameters. We then extend this method and complement the enzymatic denudation with analogies to wounding, including gradients in signals associated with cell damage, such as reactive oxygen species, suspected to play a role in the induction of movement after wounding. These chemical factors are evaluated in combination with the enzymatic cleavage of cells, and are assessed for their influence on the collective migration of a non-abrasively denuded epithelial sheet. We conclude that free space alone is sufficient to induce movement, but this movement is predominantly limited to the leading edge, leaving cells further from the edge less able to move towards the wound. Surprisingly, when coupled with a gradient in ROS to simulate the chemical effects of abrasion however, motility was not restored, but further inhibited.Massachusetts Institute of Technology. Presidential FellowshipNational Institutes of Health (U.S.). Biotechnology Training FellowshipSingapore-MIT Alliance for Research and TechnologyMassachusetts Institute of Biotechnology Training GrantMassachusetts Institute of Technology (Open-source Funding

Regulation of mTORC1 Signaling by pH

BACKGROUND: Acidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known. METHODOLOGY/FINDINGS: We examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1-TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1-TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles. CONCLUSIONS: This study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions

Clients’ satisfaction with quality of childbirth services: A comparative study between public and private facilities in Limuru Sub-County, Kiambu, Kenya

Background: This study intended to compare the clients’ satisfaction with the quality of childbirth services in a private and public facility amongst mothers who have delivered within the last twenty four to seventy hours. Methods: This was a cross-sectional comparative research design with both quantitative and qualitative data collection and analysis methods. Data were collected through a focused group discussion guide and structured questionnaire collecting information on clients’ satisfaction with quality of childbirth services. The study was conducted amongst women of reproductive age (WRA) between 15-49 years in Tigoni District hospital (public hospital) and Limuru Nursing home (private hospital). For quantitative data we conducted descriptive analysis and Mann-Whitney test using SPSS version 20.0 while qualitative data was manually analyzed manually using thematic analysis. Results: A higher proportion of clients from private facility 98.1% were attended within 0-30 minutes of arrival to the facility as compared to 87% from public facility. The overall mean score showed that the respondents in public facility gave to satisfaction with the services was 4.46 out of a maximum of 5.00 score while private facility gave 4.60. The level of satisfaction amongst respondents in the public facility on pain relief after delivery was statistically significantly higher than the respondents in private facilities (U=8132.50, p<0.001) while the level of satisfaction amongst respondents in the public facility on functional equipment was statistically significantly higher than the respondents in private facilities (U=9206.50, p=0.001). Moreover, level of satisfaction with the way staff responded to questions and concerns during labour and delivery was statistically significantly higher than the respondents in private facilities (U=9964.50, p=0.022). Conclusion: In overall, majority of clients from both public and private facilities expressed satisfaction with quality of services from admission till discharge in both public and private facilities and were willing to recommend other to come and deliver in the respective facilities

Plasma and cellular fibronectin: distinct and independent functions during tissue repair

Fibronectin (FN) is a ubiquitous extracellular matrix (ECM) glycoprotein that plays vital roles during tissue repair. The plasma form of FN circulates in the blood, and upon tissue injury, is incorporated into fibrin clots to exert effects on platelet function and to mediate hemostasis. Cellular FN is then synthesized and assembled by cells as they migrate into the clot to reconstitute damaged tissue. The assembly of FN into a complex three-dimensional matrix during physiological repair plays a key role not only as a structural scaffold, but also as a regulator of cell function during this stage of tissue repair. FN fibrillogenesis is a complex, stepwise process that is strictly regulated by a multitude of factors. During fibrosis, there is excessive deposition of ECM, of which FN is one of the major components. Aberrant FN-matrix assembly is a major contributing factor to the switch from normal tissue repair to misregulated fibrosis. Understanding the mechanisms involved in FN assembly and how these interplay with cellular, fibrotic and immune responses may reveal targets for the future development of therapies to regulate aberrant tissue-repair processes

Putting people first! vs embedded autonomy?

Paper presented at the HSRC Conference on The Potentials for and Challenges of Constructing a Democratic Developmental State in South Africa, Magaliesburg, South Africa, 4-6 Jun

Distribution of parasitic nematodes in Kenyan rice fields and their relation to edaphic factors, rainfall and temperature

Published online: 09 Nov 2017Rice is the third most important crop in Kenya after maize and wheat. Plant parasitic nematodes (PPN) are a major rice production constraint. The objective of this study was to determine the distribution and abundance of rice PPN in Kenya and their association with environmental variables. Soil and plant samples were collected from rainfed upland and irrigated lowland rice fields in Kirogo, Thiba, Nyangati, Ombeyi, Kombura, Kakola and Mwambe Sub-locations. Twenty-two PPN genera were identified across all Sub-locations with the highest (20) number of nematode genera recorded in rainfed upland rice fields. The nematode genera Scutellonema, Hoplolaimus, Ditylenchus, Hemicycliophora, Heterodera, Paralongidorus and Tylenchus were not recorded in the irrigated lowland rice fields while Criconemella and Tylenchorhynchus were not observed in rainfed upland fields. Canonical correspondence analysis of the relationship between soil properties and nematode abundance revealed a significant (P < 0.05) relationship between PPN abundance and potassium, sodium, sand and clay. The relationship between rainfall, minimum temperature and nematode genera was significant (P < 0.05) while maximum temperature was not significant (P = 0.928). Our results provide critical information for designing nematode management strategies in Kenyan rice fields

Termite species and functional groups in maize intercrop systems in Machakos County, Kenya

Termites are important ecosystem engineers in the tropics but certain termite species cause damage to economically important crops. In Africa, termites cause >50% damage in maize. This study evaluated the effect of intercropping maize with soybean, common beans and sorghum on the level of termite damage, abundance of termite species and functional groups in Machakos County, Kenya during two cropping seasons. In both seasons, Macrotermes herus, M. subhyalinus, Coptotermes formosanus, Odontotermes badius, O. longignathus and Cubitermes ugandesis were recorded. There was a low percentage of lodged plants in maize-sorghum intercrop which also had low population densities of M. herus and O. badius in both seasons. There was no difference in the number of C. formosanus in both seasons. The lowest number of fungus-cultivators was in the maize-sorghum intercrop while soil feeders occurred in low populations. Intercropping maize and sorghum can be further explored alongside other integrated termite management techniques. The observed low populations of soil feeders necessitate adoption of farming practices that conserve them in order to improve crop productivity