Critical user-configurations in mHealth design: How mHealth-app design practices come to bias design against chronically ill children and young people as mHealth users.

Mobile health smartphone applications (mHealth-apps) are increasingly emerging to assist children's and young people's management of chronic conditions. However, difficulties persist in applying design approaches in mHealth projects that return apps that are useful to this group. In this article, we explore ethnographically two self-proclaimed 'user-driven' projects designing mHealth apps for Danish patients below the age of 18 living with, respectively, haemophilia and rheumatoid arthritis. These projects initially included the perspectives of children and young people to inform the designs, however, eventually launched the final apps for adult patients only. Through a concept of 'critical user-configuration', we examine the projects' challenges with attuning the designs to children and young people and how these drove their exclusion as users of the emerging mHealth apps. Critical user-configuration draws attention to critical moments in design practices where significant shifts in user-configurations take place, shaping who can become a user. More specifically, we uncover three critical moments: where mHealth projects expand the group of prospective users; where test subjects are selected; and where data governance systems and digital health infrastructures are mobilised in the design process. Throughout these critical moments, there is a drift from user-driven to data-driven design approaches which increasingly exclude groups of users who are less datafiable - in our case children and young people. We argue that besides giving voice to minors in mHealth design processes, we need to be mindful of the design practices that become decisive for - often implicitly - who can be configured as a user.

HIV Nef-mediated major histocompatibility complex class I down-modulation is independent of Arf6 activity.

HIV Nef has a number of important biological effects, including the down-modulation of several immunological important molecules (CD4, major histocompatibility complex [MHC] class I). Down-modulation of CD4 seems to be via clathrin-dependent endocytosis, whereas down-modulation of MHC class I remains unexplained. Several mutant proteins, including mutations in the small GTPase Arf6, have been used to probe membrane traffic pathways. One such mutant has recently been used to propose that Nef acts through Arf6 to activate the endocytosis of MHC class I. Here, we show that MHC class I down-modulation is unaffected by other Arf6 mutants that provide more specific perturbations in the GDP-GTP cycling of Arf6. Inhibition of phosphatidylinositol-3-phosphate kinase, an upstream activator of Arf6, also had no effect on the internalization step, but its activity is required to direct MHC class I to the trans-Golgi network. We conclude that the apparent Arf6 dependency of Nef-mediated MHC class I down-modulation is due to nonspecific perturbations in membrane traffic.

Cholera toxin toxicity does not require functional Arf6- and dynamin-dependent endocytic pathways.

Cholera toxin (CT) and related AB(5) toxins bind to glycolipids at the plasma membrane and are then transported in a retrograde manner, first to the Golgi and then to the endoplasmic reticulum (ER). In the ER, the catalytic subunit of CT is translocated into the cytosol, resulting in toxicity. Using fluorescence microscopy, we found that CT is internalized by multiple endocytic pathways. Inhibition of the clathrin-, caveolin-, or Arf6-dependent pathways by overexpression of appropriate dominant mutants had no effect on retrograde traffic of CT to the Golgi and ER, and it did not affect CT toxicity. Unexpectedly, when we blocked all three endocytic pathways at once, although fluorescent CT in the Golgi and ER became undetectable, CT-induced toxicity was largely unaffected. These results are consistent with the existence of an additional retrograde pathway used by CT to reach the ER.

BAMLET activates a lysosomal cell death program in cancer cells.

A complex of human alpha-lactalbumin and oleic acid (HAMLET) was originally isolated from human milk as a potent anticancer agent. It kills a wide range of transformed cells of various origins while leaving nontransformed healthy cells largely unaffected both in vitro and in vivo. Importantly, purified alpha-lactalbumins from other mammals form complexes with oleic acid that show biological activities similar to that of HAMLET. The mechanism by which these protein-lipid complexes kill tumor cells is, however, largely unknown. Here, we show that complex of bovine alpha-lactalbumin and oleic acid (BAMLET), the bovine counterpart of HAMLET, kills tumor cells via a mechanism involving lysosomal membrane permeabilization. BAMLET shows potent cytotoxic activity against eight cancer cell lines tested, whereas nontransformed NIH-3T3 murine embryonic fibroblasts are relatively resistant. BAMLET accumulates rapidly and specifically in the endolysosomal compartment of tumor cells and induces an early leakage of lysosomal cathepsins into the cytosol followed by the activation of the proapoptotic protein Bax. Ectopic expression of three proteins known to stabilize the lysosomal compartment, i.e. heat shock protein 70 (Hsp70), Hsp70-2, and lens epithelium-derived growth factor, confer significant protection against BAMLET-induced cell death, whereas the antiapoptotic protein Bcl-2, caspase inhibition, and autophagy inhibition fail to do so. These data indicate that BAMLET triggers lysosomal cell death pathway in cancer cells, thereby clarifying the ability of alpha-lactalbumin:oleate complexes to kill highly apoptosis-resistant tumor cells.

Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking.

Tubules and vesicles are membrane carriers involved in traffic along the endocytic and secretory routes. The small GTPase Arf6 regulates a recycling branch of short dynamic tubular intermediates used by major histocompatibility class I (MHC-I) molecules to traffic through vesicles between endosomes and the plasma membrane. We observed that Arf6 also affects a second network of very long and stable tubules containing MHC-I, many of which correspond to deep invaginations of the plasma membrane. Treatment with wortmannin, an inhibitor of phosphatidylinositol-3-phosphate kinase, prevents formation of the short dynamic tubules while increasing the number of the long and very stable ones. Expression of NefAAAA, a mutant form of HIV Nef, increases the number of cells containing the stable tubules, and is used here as a tool to facilitate their study. Photoactivation of NefAAAA-PA-GFP demonstrates that this molecule traffics from endosomes to the tubules. Finally, live-cell imaging also shows internalization of MHC-I molecules into these tubules, suggesting that this is an additional route for MHC-I traffic.

Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells.

Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity of secretion is determined by conformational sensitive sorting signals.

Serpins are apically secreted from MDCK cells independently of their raft association.

It has been suggested that detergent-resistant membranes (DRMs), also known as lipid rafts, are involved in vectorial transport of proteins to the apical surface. In this report we use Madin-Darby canine kidney (MDCK) cells expressing the apically secreted C1-esterase inhibitor, the non-sorted antithrombin or chimeras of serpins to study the possible connection between DRM association and apical targeting of secretory proteins. We found newly synthesised C1-esterase inhibitor associated with DRMs in MDCK cells, whereas antithrombin was not. However, two chimeric proteins, secreted mainly from the apical membrane, do not associate with DRMs. Based on these observations we suggest that apical targeting and association with DRMs are two independent events for secretory serpins.