Metabolic Control in Type 1 Diabetes: Is Adjunctive Therapy the Way Forward?

Despite advances in insulin therapies, patients with type 1 diabetes (T1DM) have a shorter life span due to hyperglycaemia-induced vascular disease and hypoglycaemic complications secondary to insulin therapy. Restricting therapy for T1DM to insulin replacement is perhaps an over-simplistic approach, and we focus in this work on reviewing the role of adjuvant therapy in this population. Current data suggest that adding metformin to insulin therapy in T1DM temporarily lowers HbA1c and reduces weight and insulin requirements, but this treatment fails to show a longer-term glycaemic benefit. Agents in the sodium glucose co-transporter-2 inhibitor (SGLT-2) class demonstrate the greatest promise in correcting hyperglycaemia, but there are safety concerns in relation to the risk of diabetic ketoacidosis. Glucagon-like peptide-1 agonists (GLP-1) show a modest effect on glycaemia, if any, but significantly reduce weight, which may make them suitable for use in overweight T1DM patients. Treatment with pramlintide is not widely available worldwide, although there is evidence to indicate that this agent reduces both HbA1c and weight in T1DM. A criticism of adjuvant studies is the heavy reliance on HbA1c as the primary endpoint while generally ignoring other glycaemic parameters. Moreover, vascular risk markers and measures of insulin resistance—important considerations in individuals with a longer T1DM duration—are yet to be fully investigated following adjuvant therapies. Finally, studies to date have made the assumption that T1DM patients are a homogeneous group of individuals who respond similarly to adjuvant therapies, which is unlikely to be the case. Future longer-term adjuvant studies investigating different glycaemic parameters, surrogate vascular markers and harder clinical outcomes will refine our understanding of the roles of such therapies in various subgroups of T1DM patients

Versatile cell-free protein synthesis systems based on chinese hamster ovary cells

We present an alternative production platform for the synthesis of complex proteins. Apart from conventionally applied protein production using engineered mammalian cell lines, this protocol describes the preparation and principle of cell-free protein synthesis systems based on CHO cell lysates. The CHO cell-free system contains endogenous microsomes derived from the endoplasmic reticulum, which enables a direct integration of membrane proteins into a nature like milieu and the introduction of posttranslational modifications. Different steps of system development are described including the cultivation of CHO cells, cell harvesting and cell disruption to prepare translationally active CHO cell lysates. The requirements for DNA templates and the generation of linear DNA templates suitable for the CHO cell-free reaction is further depicted to underline the opportunity to produce different protein variants in a short period. This experimental setup provides a basis for hig h-throughput applications. The productivity of the CHO cell-free systems is further increased by using a non-canonical translation initiation due to the attachment of an internal ribosomal entry site of the Cricket paralysis virus (CRPV IRES) to the 5´ UTR of the desired gene. In this way, a direct interaction of the IRES structure with the ribosome facilitates a translation factor independent initiation of translation. Cell-free reactions were performed in fast and efficient batch reactions leading to protein yields up to 40 μg/mL. The reaction format was further adjusted to a continuous exchange CHO cell-free reaction (CHO CECF) to prolong reaction time and thereby increase the productivity of the cell-free systems. Finally, protein yields up to 1 g/L were obtained. The CHO CECF system represents a sophisticated resource to address structural and functional aspects of difficult-to-express proteins in fundamental and applied research

Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins. For many viruses, the clustering of fusion proteins—and their distribution on virus particles—is crucial for fusion activity. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection