Exportable proteins that have significant defects in nascent polypeptide folding or subunit assembly are frequently retained in the endoplasmic reticulum and subject to endoplasmic reticulum-associated degradation by the ubiquitin-proteasome system. In addition to this, however, there is growing evidence for postendoplasmic reticulum quality control mechanisms in which mutant or non-native exportable proteins may undergo anterograde transport to the Golgi complex and post-Golgi compartments before intracellular disposal. In some instances, these proteins may undergo retrograde transport back to the endoplasmic reticulum with re-targeting to the endoplasmic reticulumassociated degradation pathway; in other typical cases, they are targeted into the endosomal system for degradation by vacuolar/lysosomal proteases. Such quality control targeting is likely to involve recognition of features more commonly expressed in mutant proteins, but may also be expressed by wild-type proteins, especially in cells with perturbation of local environments that are essential for normal protein trafficking and stability in the secretory pathway and at the cell surface.

BackgroundDengue imposes a substantial economic and disease burden in most tropical and subtropical countries. Dengue incidence and severity have dramatically increased in Mexico during the past decades. Having objective and comparable estimates of the economic burden of dengue is essential to inform health policy, increase disease awareness, and assess the impact of dengue prevention and control technologies.Methods and FindingsWe estimated the annual economic and disease burden of dengue in Mexico for the years 2010–2011. We merged multiple data sources, including a prospective cohort study; patient interviews and macro-costing from major hospitals; surveillance, budget, and health data from the Ministry of Health; WHO cost estimates; and available literature. We conducted a probabilistic sensitivity analysis using Monte Carlo simulations to derive 95% certainty levels (CL) for our estimates. Results suggest that Mexico had about 139,000 (95%CL: 128,000–253,000) symptomatic and 119 (95%CL: 75–171) fatal dengue episodes annually on average (2010–2011), compared to an average of 30,941 symptomatic and 59 fatal dengue episodes reported. The annual cost, including surveillance and vector control, was US$170 (95%CL: 151–292) million, or $1.56 (95%CL: 1.38–2.68) per capita, comparable to other countries in the region. Of this, $87 (95%CL: 87–209) million or $0.80 per capita (95%CL: 0.62–1.12) corresponds to illness. Annual disease burden averaged 65 (95%CL: 36–99) disability-adjusted life years (DALYs) per million population. Inclusion of long-term sequelae, co-morbidities, impact on tourism, and health system disruption during outbreaks would further increase estimated economic and disease burden.ConclusionWith this study, Mexico joins Panama, Puerto Rico, Nicaragua, and Thailand as the only countries or areas worldwide with comprehensive (illness and preventive) empirical estimates of dengue burden. Burden varies annually; during an outbreak, dengue burden may be significantly higher than that of the pre-vaccine level of rotavirus diarrhea. In sum, Mexico’s potential economic benefits from dengue control would be substantial.

In single-chain insulins (SCIs), the C terminus of the insulin B-chain is contiguous with the N terminus of the A-chain, connected by a short bioengineered linker sequence. SCIs have been proposed to offer potential benefit for gene therapy of diabetes (Lee, H. C., Kim, S. J., Kim, K. S., Shin, H. C., and Yoon, J. W. (2000) Nature 408, 483-488) yet relatively little is known about their folding, intracellular transport, or secretion from mammalian cells. Because SCIs can be considered as mutant proinsulin (with selective shortening of the 35-amino acid connecting peptide that normally includes two sets of flanking dibasic residues), they offer insights into understanding the role of the connecting peptide in insulin biosynthesis. Herein we have explored the relationship of the linker sequence to SCI biosynthesis, folding, and intracellular transport in transiently transfected HEK293 or Chinese hamster ovary cells or in stably transfected AtT20 cells. Despite previous reports that direct linkage of B-and A-chains produces a structure isomorphous with authentic two-chain insulin, we find that constructs with short linkers tend to be synthesized at lower levels, with a significant fraction of molecules exhibiting improper disulfide bonding. Nevertheless, disulfide-mispaired isoforms from a number of different SCI constructs are secreted. While this suggests that a novel folded state goes unrecognized by secretory pathway quality control, we find that misfolded SCIs are detected at higher levels in Chinese hamster ovary cells with artificially activated unfolded protein response mediated by inducible overexpression of active ATF-6. Such a maneuver allows analysis of more seriously misfolded mutants with further foreshortening of the linker sequence or loss (by mutation) of the insulin interchain disulfide bonds.