Aphid-borne viruses of potato : investigations into virus/host/vector interactions, serological detection using recombinant antibodies and control strategies

Potato is one of the most important food crops in the world, and viruses are largely responsible for the degeneration of this vegetatively propagated crop. At least 35 viruses have been reported to infect potato naturally. The majority and the most economically important ones are vectored by aphids. The objective of this study was to conduct molecular and biological investigations into virus transmission mechanisms, including developing diagnostic methods to help to control the spread of aphid-borne potato viruses, and disrupting the vectoring ability of their aphid vectors by insecticide spray. One way to control the spread of aphid-borne viruses is to control their aphid vector, but this is not always feasible as the majority of aphid-borne potato viruses, including the most important ones, are transmitted non-persistently, being acquired within a very short time before agrochemicals can act. Thus an alternative approach to controlling this class of viruses is through a full understanding of the interaction between the virus, the host plant and the aphid vector, which was the first objective of this project. In this respect, some aphid cuticle proteins were identified to interact with potato virus Y helper component (HC-Pro) through screening of an aphid cDNA expression library, and their potential role in virus transmission was discussed. Moreover, the concept of short retention of non-persistent viruses inside their aphid vectors was challenged; the results show that PVY can be retained inside its aphid vector for a long time but it is not transmissible. This novel finding together with binding to aphid cuticle proteins, generated some new ideas about transmission mechanisms that were proposed and discussed. In addition, the effect on aphid vectoring ability of the plants used to rear aphid colonies, as a virus source, and as a virus recipient was investigated. From laboratory studies of aphid transmission, it was concluded that the transmission efficiency of PVY was significantly affected by the host plant species used to rear M. persicae, or that used as a virus recipient plant. The availability of sensitive and cheap virus detection methods is critical for early detection and control of potato viruses. In this project a sensitive fully recombinant ELISA was developed and validated for routine testing of potato leafroll virus. This technology can be applied to detect other potato viruses and has the potential to replace the commonly used immune reagent antisera.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Time course of normalization of functional β-cell capacity in the Diabetes Remission Clinical Trial after weight loss in type 2 diabetes

Objective: To assess functional β-cell capacity in type 2 diabetes during 2 years of remission induced by dietary weight loss. Research Design and Methods: A Stepped Insulin Secretion Test with Arginine was used to quantify functional β-cell capacity by hyperglycemia and arginine stimulation. Thirty-nine of 57 participants initially achieved remission (HbA1c <6.5% [<48 mmol/mol] and fasting plasma glucose <7 mmol/L on no antidiabetic drug therapy) with a 16.4 ± 7.7 kg weight loss and were followed up with supportive advice on avoidance of weight regain. At 2 years, 20 participants remained in remission in the study. A nondiabetic control (NDC) group, matched for age, sex, and weight after weight loss with the intervention group, was studied once. Results: During remission, median (interquartile range) maximal rate of insulin secretion increased from 581 (480–811) pmol/min/m2 at baseline to 736 (542–998) pmol/min/m2 at 5 months, 942 (565–1,240) pmol/min/m2 at 12 months (P = 0.028 from baseline), and 936 (635–1,435) pmol/min/m2 at 24 months (P = 0.023 from baseline; n = 20 of 39 of those initially in remission). This was comparable to the NDC group (1,016 [857–1,507] pmol/min/m2) by 12 (P = 0.064) and 24 (P = 0.244) months. Median first-phase insulin response increased from baseline to 5 months (42 [4–67] to 107 [59–163] pmol/min/m2; P < 0.0001) and then remained stable at 12 and 24 months (110 [59–201] and 125 [65–166] pmol/min/m2, respectively; P < 0.0001 vs. baseline) but lower than that of the NDC group (250 [226–429] pmol/min/m2; P < 0.0001). Conclusions: A gradual increase in assessed functional β-cell capacity occurred after weight loss, becoming similar to NDC participants by 12 months. This was unchanged at 2 years with continuing remission of type 2 diabetes

Remission of human type 2 diabetes requires decrease in liver and pancreas fat content but is dependent upon capacity for beta cell recovery

The Diabetes Remission Clinical Trial reported return and persistence of non-diabetic blood glucose control in 46% of people with type 2 diabetes of up to 6 years duration. Detailed metabolic studies were performed on a subgroup (intervention, n = 64; control, n = 26). In the intervention group, liver fat content decreased (16.0% ± 1.3% to 3.1% ± 0.5%, p < 0.0001) immediately after weight loss. Similarly, plasma triglyceride and pancreas fat content decreased whether or not glucose control normalized. Recovery of first-phase insulin response (0.04[−0.05–0.32] to 0.11[0.0005–0.51] nmol/min/m2, p < 0.0001) defined those who returned to non-diabetic glucose control and this was durable at 12 months (0.11[0.005–0.81] nmol/min/m2, p = 0.0001). Responders were similar to non-responders at baseline but had shorter diabetes duration (2.7 ± 0.3 versus 3.8 ± 0.4 years; p = 0.02). This study demonstrates that β cell ability to recover long-term function persists after diagnosis, changing the previous paradigm of irreversible loss of β cell function in type 2 diabetes

2-year remission of type 2 diabetes and pancreas morphology: a post-hoc analysis of the DiRECT open-label, cluster-randomised trial

Background: The pancreas is small and irregular in shape in people with type 2 diabetes. If these abnormalities are caused by the disease state itself rather than being a predisposing factor, remission of type 2 diabetes should restore normal pancreas morphology. The objective of this study was to determine whether changes in pancreas volume and shape occurred during 2 years of remission. Methods: For this post-hoc analysis, we included a subset of adult participants of the Diabetes Remission Clinical Trial (DiRECT), who had type 2 diabetes and were randomly assigned to a weight management intervention or routine diabetes management. Intervention group participants were categorised as responders (HbA1c <6·5% [48 mmol/mol] and fasting blood glucose <7·0 mmol/L, off all anti-diabetes medication) and non-responders, who were classified as remaining diabetic. Data on pancreas volume and irregularity of pancreas border at baseline, 5 months, 12 months, and 24 months after intervention were compared between responders and non-responders; additional comparisons were made between control group participants with type 2 diabetes and a non-diabetic comparator (NDC) group, who were matched to the intervention group by age, sex, and post-weight-loss weight, to determine the extent of any normalisation. We used a mixed-effects regression model based on repeated measures ANOVA with correction for potential confounding. Magnetic resonance techniques were employed to quantify pancreas volume, the irregularity of the pancreas borders, and intrapancreatic fat content. β-cell function and biomarkers of tissue growth were also measured. Findings: Between July 25, 2015, and Aug 5, 2016, 90 participants with type 2 diabetes in the DiRECT subset were randomly assigned to intervention (n=64) or control (n=26) and were assessed at baseline; a further 25 non-diabetic participants were enrolled into the NDC group. At baseline, mean pancreas volume was 61·7 cm3 (SD 16·0) in all participants with type 2 diabetes and 79·8 cm3 (14·3) in the NDC group (p<0·0001). At 24 months, pancreas volume had increased by 9·4 cm3 (95% CI 6·1 to 12·8) in responders compared with 6·4 cm3 (2·5 to 10·3) in non-responders (p=0·0008). Pancreas borders at baseline were more irregular in participants with type 2 diabetes than in the NDC group (fractal dimension 1·138 [SD 0·027] vs 1·097 [0·025]; p<0·0001) and had normalised by 24 months in responders only (1·099 [0·028]). Intrapancreatic fat declined by 1·02 percentage points (95% CI 0·53 to 1·51) in 32 responders and 0·51% (−0·17 to 1·19) in 13 non-responders (p=0·23). Interpretation: These data show for the first time, to our knowledge, reversibility of the abnormal pancreas morphology of type 2 diabetes by weight loss-induced remission

Sex differences in intraorgan fat levels and hepatic lipid metabolism: implications for cardiovascular health and remission of type 2 diabetes after dietary weight loss

Aims/hypothesis: Type 2 diabetes confers a greater relative increase in CVD risk in women compared with men. We examined sex differences in intraorgan fat and hepatic VLDL1-triacylglycerol (VLDL1-TG) export before and after major dietary weight loss. Methods: A group with type 2 diabetes (n = 64, 30 male/34 female) and a group of healthy individuals (n = 25, 13 male/12 female) were studied. Intraorgan and visceral fat were quantified by magnetic resonance and VLDL1-TG export by intralipid infusion techniques. Results: Triacylglycerol content of the liver and pancreas was elevated in people with diabetes with no sex differences (liver 16.4% [9.3–25.0%] in women vs 11.9% [7.0–23.1%] in men, p = 0.57, and pancreas 8.3 ± 0.5% vs 8.5 ± 0.4%, p = 0.83, respectively). In the absence of diabetes, fat levels in both organs were lower in women than men (1.0% [0.9–1.7%] vs 4.5% [1.9–8.0%], p = 0.005, and 4.7 ± 0.4% vs 7.6 ± 0.5%, p< 0.0001, respectively). Women with diabetes had higher hepatic VLDL1-TG production rate and plasma VLDL1-TG than healthy women (559.3 ± 32.9 vs 403.2 ± 45.7 mg kg−1 day−1, p = 0.01, and 0.45 [0.26–0.77] vs 0.25 [0.13–0.33] mmol/l, p = 0.02), whereas there were no differences in men (548.8 ± 39.8 vs 506.7 ± 29.2 mg kg−1 day−1, p = 0.34, and 0.72 [0.53–1.15] vs 0.50 [0.32–0.68] mmol/l, p = 0.26). Weight loss decreased intraorgan fat and VLDL1-TG production rates regardless of sex, and these changes were accompanied by similar rates of diabetes remission (65.4% vs 71.0%) and CVD risk reduction (59.8% vs 41.5%) in women and men, respectively. Conclusions/interpretation: In type 2 diabetes, women have liver and pancreas fat levels as high as those of men, associated with raised hepatic VLDL1-TG production rates. Dynamics of triacylglycerol turnover differ between sexes in type 2 diabetes and following weight loss. These changes may contribute to the disproportionately raised cardiovascular risk of women with diabetes

Hepatic lipoprotein export and remission of human type 2 diabetes after weight loss

The role of hepatic lipoprotein metabolism in diet-induced remission of type 2 diabetes is currently unclear. Here, we determined the contributions of hepatic VLDL1-triglyceride production rate and VLDL1-palmitic acid content to changes in intra-pancreatic fat and return of first phase insulin response in a subgroup of the Diabetes Remission Clinical Trial. Liver fat, VLDL1-triglyceride production, and intra-pancreatic fat decreased after weight loss and remained normalized after 24 months of remission. First-phase insulin response remained increased only in those maintaining diabetes remission. Compared with those in remission at 24 months, individuals who relapsed after initial remission had a greater rise in the content of VLDL1-triglyceride and VLDL1-palmitic acid, re-accumulated intra-pancreatic fat, and lost first-phase response by 24 months. Thus, we observed temporal relationships between VLDL1-triglyceride production, hepatic palmitic acid flux, intra-pancreatic fat, and β-cell function. Weight-related disordered fat metabolism appears to drive development and reversal of type 2 diabetes

Durability of a primary care-led weight-management intervention for remission of type 2 diabetes: 2-year results of the DiRECT open-label, cluster-randomised trial

Background: The DiRECT trial assessed remission of type 2 diabetes during a primary care-led weight-management programme. At 1 year, 68 (46%) of 149 intervention participants were in remission and 36 (24%) had achieved at least 15 kg weight loss. The aim of this 2-year analysis is to assess the durability of the intervention effect. Methods: DiRECT is an open-label, cluster-randomised, controlled trial done at primary care practices in the UK. Practices were randomly assigned (1:1) via a computer-generated list to provide an integrated structured weight-management programme (intervention) or best-practice care in accordance with guidelines (control), with stratification for study site (Tyneside or Scotland) and practice list size (>5700 or ≤5700 people). Allocation was concealed from the study statisticians; participants, carers, and study research assistants were aware of allocation. We recruited individuals aged 20–65 years, with less than 6 years' duration of type 2 diabetes, BMI 27–45 kg/m2, and not receiving insulin between July 25, 2014, and Aug 5, 2016. The intervention consisted of withdrawal of antidiabetes and antihypertensive drugs, total diet replacement (825–853 kcal per day formula diet for 12–20 weeks), stepped food reintroduction (2–8 weeks), and then structured support for weight-loss maintenance. The coprimary outcomes, analysed hierarchically in the intention-to-treat population at 24 months, were weight loss of at least 15 kg, and remission of diabetes, defined as HbA1c less than 6·5% (48 mmol/mol) after withdrawal of antidiabetes drugs at baseline (remission was determined independently at 12 and 24 months). The trial is registered with the ISRCTN registry, number 03267836, and follow-up is ongoing. Findings: The intention-to-treat population consisted of 149 participants per group. At 24 months, 17 (11%) intervention participants and three (2%) control participants had weight loss of at least 15 kg (adjusted odds ratio [aOR] 7·49, 95% CI 2·05 to 27·32; p=0·0023) and 53 (36%) intervention participants and five (3%) control participants had remission of diabetes (aOR 25·82, 8·25 to 80·84; p<0·0001). The adjusted mean difference between the control and intervention groups in change in bodyweight was −5·4 kg (95% CI −6·9 to −4·0; p<0·0001) and in HbA1c was −4·8 mmol/mol (–8·3 to −1·4 [–0·44% (–0·76 to −0·13)]; p=0·0063), despite only 51 (40%) of 129 patients in the intervention group using anti-diabetes medication compared with 120 (84%) of 143 in the control group. In a post-hoc analysis of the whole study population, of those participants who maintained at least 10 kg weight loss (45 of 272 with data), 29 (64%) achieved remission; 36 (24%) of 149 participants in the intervention group maintained at least 10 kg weight loss. Serious adverse events were similar to those reported at 12 months, but were fewer in the intervention group than in the control group in the second year of the study (nine vs 22). Interpretation: The DiRECT programme sustained remissions at 24 months for more than a third of people with type 2 diabetes. Sustained remission was linked to the extent of sustained weight loss

β-Cell Dysfunction, Hepatic Lipid Metabolism, and Cardiovascular Health in Type 2 Diabetes: New Directions of Research and Novel Therapeutic Strategies

Cardiovascular disease (CVD) remains a major problem for people with type 2 diabetes mellitus (T2DM), and dyslipidemia is one of the main drivers for both metabolic diseases. In this review, the major pathophysiological and molecular mechanisms of β-cell dysfunction and recovery in T2DM are discussed in the context of abnormal hepatic lipid metabolism and cardiovascular health. (i) In normal health, continuous exposure of the pancreas to nutrient stimulus increases the demand on β-cells. In the long term, this will not only stress β-cells and decrease their insulin secretory capacity, but also will blunt the cellular response to insulin. (ii) At the pre-diabetes stage, β-cells compensate for insulin resistance through hypersecretion of insulin. This increases the metabolic burden on the stressed β-cells and changes hepatic lipoprotein metabolism and adipose tissue function. (iii) If this lipotoxic hyperinsulinemic environment is not removed, β-cells start to lose function, and CVD risk rises due to lower lipoprotein clearance. (iv) Once developed, T2DM can be reversed by weight loss, a process described recently as remission. However, the precise mechanism(s) by which calorie restriction causes normalization of lipoprotein metabolism and restores β-cell function are not fully established. Understanding the pathophysiological and molecular basis of β-cell failure and recovery during remission is critical to reduce β-cell burden and loss of function. The aim of this review is to highlight the link between lipoprotein export and lipid-driven β-cell dysfunction in T2DM and how this is related to cardiovascular health. A second aim is to understand the mechanisms of β-cell recovery after weight loss, and to explore new areas of research for developing more targeted future therapies to prevent T2DM and the associated CVD events

A fully recombinant ELISA using in vivo biotinylated antibody fragments for the detection of potato leafroll virus

A recombinant antibody fusion protein, V3HCL, which was shown previously to have specific reactivity for potato leafroll virus (PLRV), was labeled with biotin using standard chemical coupling procedures and by an in vivo method. The in vivo method proved superior giving reproducible VMCL-biotin preparations. A fully recombinant ELISA was devised incorporating V3HCL, V3HCL-biotin and streptavidin alkaline phosphatase conjugate. This assay gave comparable results for PLRV detection in potato to an assay based on immunoglobulins. The V3HCL-biotin preparations were stable and retained specific activity for more than 1 year when stored at 4 degrees C or -20 degrees C. The results demonstrate that scFv reagents derived from synthetic phage display platforms can provide effective alternatives to assays incorporating immune reagents. (C) 2009 Elsevier B.V. All rights reserved.</p