Dexamethasone and Surgical-Site Infection.

BACKGROUND: The glucocorticoid dexamethasone prevents nausea and vomiting after surgery, but there is concern that it may increase the risk of surgical-site infection. METHODS: In this pragmatic, international, noninferiority trial, we randomly assigned 8880 adult patients who were undergoing nonurgent, noncardiac surgery of at least 2 hours' duration, with a skin incision length longer than 5 cm and a postoperative overnight hospital stay, to receive 8 mg of intravenous dexamethasone or matching placebo while under anesthesia. Randomization was stratified according to diabetes status and trial center. The primary outcome was surgical-site infection within 30 days after surgery. The prespecified noninferiority margin was 2.0 percentage points. RESULTS: A total of 8725 participants were included in the modified intention-to-treat population (4372 in the dexamethasone group and 4353 in the placebo group), of whom 13.2% (576 in the dexamethasone group and 572 in the placebo group) had diabetes mellitus. Of the 8678 patients included in the primary analysis, surgical-site infection occurred in 8.1% (354 of 4350 patients) assigned to dexamethasone and in 9.1% (394 of 4328) assigned to placebo (risk difference adjusted for diabetes status, -0.9 percentage points; 95.6% confidence interval [CI], -2.1 to 0.3; P<0.001 for noninferiority). The results for superficial, deep, and organ-space surgical-site infections and in patients with diabetes were similar to those of the primary analysis. Postoperative nausea and vomiting in the first 24 hours after surgery occurred in 42.2% of patients in the dexamethasone group and in 53.9% in the placebo group (risk ratio, 0.78; 95% CI, 0.75 to 0.82). Hyperglycemic events in patients without diabetes occurred in 22 of 3787 (0.6%) in the dexamethasone group and in 6 of 3776 (0.2%) in the placebo group. CONCLUSIONS: Dexamethasone was noninferior to placebo with respect to the incidence of surgical-site infection within 30 days after nonurgent, noncardiac surgery. (Funded by the Australian National Health and Medical Research Council and others; PADDI Australian New Zealand Clinical Trials Registry number, ACTRN12614001226695.).

The diabetes management experiences questionnaire: Psychometric validation among adults with type 1 diabetes.

AIMS: To examine the psychometric properties of the Diabetes Management Experiences Questionnaire (DME-Q). Adapted from the validated Glucose Monitoring Experiences Questionnaire, the DME-Q captures satisfaction with diabetes management irrespective of treatment modalities. METHODS: The DME-Q was completed by adults with type 1 diabetes as part of a randomized controlled trial comparing hybrid closed loop (HCL) to standard therapy. Most psychometric properties were examined with pre-randomization data (n = 149); responsiveness was examined using baseline and 26-week follow-up data (n = 120). RESULTS: Pre-randomization, participants' mean age was 44 ± 12 years, 52% were women. HbA1c was 61 ± 11 mmol/mol (7.8 ± 1.0%), diabetes duration was 24 ± 12 years and 47% used an insulin pump prior to the trial. A forced three-factor analysis revealed three expected domains, that is, 'Convenience', 'Effectiveness' and 'Intrusiveness', and a forced one-factor solution was also satisfactory. Internal consistency reliability was strong for the three subscales ( α range = 0.74-0.84) and 'Total satisfaction' ( α = 0.85). Convergent validity was demonstrated with moderate correlations between DME-Q 'Total satisfaction' and diabetes distress (PAID: rs = -0.57) and treatment satisfaction (DTSQ; rs = 0.58). Divergent validity was demonstrated with a weak correlation with prospective/retrospective memory (PRMQ: rs = -0.16 and - 0.13 respectively). Responsiveness was demonstrated, as participants randomized to HCL had higher 'Effectiveness' and 'Total satisfaction' scores than those randomized to standard therapy. CONCLUSIONS: The 22-item DME-Q is a brief, acceptable, reliable measure with satisfactory structural and construct validity, which is responsive to intervention. The DME-Q is likely to be useful for evaluation of new pharmaceutical agents and technologies in research and clinical settings.

Mixed insulin can improve control of prednisolone-induced hyperglycaemia.

BACKGROUND: Hyperglycaemia is a common side effect of prednisolone, although there are no widely accepted guidelines for the management of glucocorticoid-induced hyperglycaemia (GIH). Our institution uses mixed insulin in a pre-breakfast or pre-breakfast and pre-lunch regimen, with the rationale that this profile of insulin action matches the physiological effect of prednisolone on blood glucose levels (BGLs). AIM: Evaluate the use of the mixed insulin (NovoMix30) in a pre-breakfast or pre-breakfast and pre-lunch regimen as management for GIH in a tertiary hospital setting. METHOD: We retrospectively evaluated all inpatients coprescribed prednisolone ≥7.5 mg and NovoMix30 for at least 48 hours over a 19-month period. BGLs were evaluated with repeated-measures analysis within four time periods across the day, beginning from the day prior to NovoMix30 administration. RESULTS: A total of 53 patients were identified. NovoMix30 significantly reduced BGLs in the morning (mean 12.7 ± 4.5 vs. 9.2 ± 3.9 mmol/L, P < 0.001), afternoon (mean 13.6 ± 3.8 vs. 11.9 ± 3.8 mmol/L, P = 0.001) and evening (12.1 ± 3.8 vs. 10.8 ± 3.8 mmol/L, P = 0.01). With uptitration of insulin over 3 days, 43% of all BGLs were within the target range, compared with 23% on day 0 (P < 0.001). The final median dose of NovoMix30 was 0.15 (0.10-0.22) units/kg bodyweight, or 0.40 (0.23-0.69) units/mg of prednisolone, which is lower than our hospital guideline recommends. One overnight hypoglycaemic event was observed. CONCLUSION: Mixed insulin as a pre-breakfast or pre-breakfast and pre-lunch regimen can target the hyperglycaemic pattern induced by prednisolone and minimise overnight hypoglycaemia. However, higher doses of insulin than those used in our study are likely required for optimal BGL control.

Body mass index is inversely associated with capillary ketones at the time of colonoscopy: Implications for SGLT2i use.

OBJECTIVE: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have been associated with diabetic ketoacidosis at the time of colonoscopy. This study aimed to identify factors associated with ketone concentrations in SGLT2i-treated type 2 diabetes compared with non-SGLT2i-treated diabetes, and those with impaired fasting glycaemia (IFG) and normoglycaemia. DESIGN: Cross-sectional, multicentre, observational study June-December 2020 in four Australian tertiary hospitals. PARTICIPANTS: Capillary glucose and ketones were measured in people undergoing colonoscopy: 37 SGLT2i-treated and 105 non-SGLT2i-treated type 2 diabetes, 65 IFG and 151 normoglycaemia. MEASUREMENTS: Body mass index (BMI), age, glucose, fasting duration and where relevant, HbA1c and time since last SGLT2i dose. RESULTS: In SGLT2i-treated diabetes, BMI (ρ = -0.43 [95% confidence interval: -0.67, -0.11]) and duration since last SGLT2i dose (ρ = -0.33 [-0.60, 0.00]) correlated negatively with increasing ketones, but there was no correlation with fasting duration. In non-SGLT2i-treated diabetes, BMI correlated negatively (ρ = -0.24 [-0.42, -0.05]) and fasting duration positively (ρ = 0.26 [0.07, 0.43]) with ketones. In IFG participants, only fasting duration correlated with ketones (ρ = 0.28 [0.03, 0.49]). In normoglycaemic participants, there were negative correlations with BMI (ρ = -0.20 [-0.35, -0.04]) and fasting glucose (ρ = -0.31 [-0.45, -0.15]) and positive correlations with fasting duration (ρ = 0.20 [0.04, 0.35]) and age (ρ = 0.19 [0.03, 0.34]). Multiple regression analysis of the entire cohort showed BMI, age and fasting glucose remained independently associated with ketones, but in SGLT2i-treated participants only BMI remained independently associated. CONCLUSIONS: In SGLT2i-treated diabetes, lower BMI was a novel risk factor for higher ketones precolonoscopy. Pending larger confirmatory studies, extra vigilance for ketoacidosis is warranted in these people.

Clinical, psychological and demographic factors in a contemporary adult cohort with diabetic ketoacidosis and type 1 diabetes.

BACKGROUND: Diabetic ketoacidosis (DKA) is a potentially life-threatening but often preventable acute complication of type 1 diabetes (T1D). Understanding clinical and psychosocial characteristics of people with DKA, particularly those with multiple presentations, may aid the development of prevention strategies. AIMS: To describe clinical, psychological and demographic factors in adults with DKA and particularly those factors associated with recurrent admissions. METHODS: A retrospective analysis was performed of all admissions with DKA in people with T1D over a 4-year period from 1 November 2013 to 31 October 2017 at a metropolitan tertiary hospital in Australia. Potential cases were identified by International Classification of Diseases-10th Revision coding data. Data were then manually extracted by clinicians from the electronic medical record. RESULTS: There were 154 clinician-adjudicated admissions for DKA among 128 people with T1D. Of these, 16 (13%) had multiple DKA admissions. Forty-one (32%) had a history of depression. The most common factors contributing to presentation included insulin omission (54%), infection (31%), alcohol excess (26%) and new diabetes diagnosis (16%). Compared to people with single admissions, those with recurrent DKA were more likely to smoke (69% vs 27%, P = 0.003), be unemployed (31% vs 11%, P = 0.04) and use illicit substances (44% vs 17%, P = 0.02). CONCLUSIONS: There is a high prevalence of psychiatric illness, illicit substance use and social disadvantage among people admitted with DKA, particularly those with recurrent presentations. Insulin omission, often due to inappropriate sick day management, was the most common reason for DKA occurrence. Innovative multidisciplinary models of care are required to address these challenges.

Prevalence of hyperglycaemia without previously recognised diabetes mellitus in the emergency department and subsequent management: a retrospective cross-sectional study.

BACKGROUND: Australian hospital data on hyperglycaemia without previously known diabetes are lacking. AIMS: To determine the prevalence of hyperglycaemia without previously recognised diabetes among all patients screened in the emergency department (ED). Secondary aims are to describe the extent of haemoglobin A1c testing for evaluation of new diabetes, adequate glucose monitoring, treatment of significant hyperglycaemia and documented follow-up plans. METHODS: Patients presenting to ED at the Alfred (tertiary hospital in Melbourne) have undergone screening random plasma glucose (RPG) with their first plasma biochemistry since 2015. Of the 16 268 adults screened from July to December 2015, a retrospective, cross-sectional study was undertaken evaluating those with hyperglycaemia (RPG >7.8 mmol/L) but without previously recognised diabetes as determined from coding data. After patient records were reviewed to correct for coding errors, a nested cohort of 200 such patients were further evaluated. Glucose monitoring was deemed adequate if undertaken for ≥48 h. Significant hyperglycaemia (RPG >11 mmol/L) was considered appropriately treated if insulin/hypoglycaemic agents were prescribed. Documented follow-up plans were acceptable if found in the discharge summary. RESULTS: Among all patients screened, 1178 had hyperglycaemia without coded diabetes. After adjusting for coding errors, the prevalence was 5.2%. Within the nested cohort, only 7.5% had a follow-up haemoglobin A1c ordered, 9.5% underwent adequate glucose monitoring, 6.5% had appropriate treatment of significant hyperglycaemia and 2% had documentation of a follow-up plan. CONCLUSIONS: Hyperglycaemia without previously recognised diabetes is commonly seen and justifies ED screening. However, management of newly detected hyperglycaemia in these patients is suboptimal and requires improvement.

Making sense of newer treatment options for type 2 diabetes.

Over the past decade, several new medications have been developed to treat type 2 diabetes mellitus. Large-scale outcome trials have been performed with patients at high cardiovascular risk to assess the cardiovascular safety of these agents. These trials are changing the landscape of diabetes therapy with evidence beyond safety to cardiovascular benefits of sodium-glucose cotransporter 2 (SGLT-2) inhibitors and some glucagon-like peptide-1 receptor agonists. This review provides an overview of incretin-based therapies and SGLT-2 inhibitors with a particular focus on the results of published cardiovascular outcome trials, which have also provided unique opportunities to evaluate uncommon but potentially serious adverse events of these newer agents. The cardiovascular benefits of SGLT-2 inhibitors and some glucagon-like peptide-1 receptor agonists suggest that they may be the preferred choice, usually as an add-on to metformin, for patients with type 2 diabetes mellitus at high cardiovascular risk.

Ezrin contributes to impaired podocyte migration and adhesion caused by advanced glycation end products.

AIM: Podocytes are specialized epithelial cells that play a critical role in the glomerular filtration barrier. Podocyte abnormalities are linked to increasing albuminuria and progression to end-stage renal failure as observed in diabetic nephropathy. Advanced glycation end products (AGEs) have been strongly linked to the development of diabetic nephropathy, and we have previously shown that AGEs inhibit ezrin actions in proximal tubule cells. As ezrin plays a key role in controlling podocyte foot process integrity, the present study investigated the contribution of ezrin to AGE-induced podocyte damage. METHODS: Conditionally immortalized human podocytes were cultured with or without AGEs, and changes in morphology, protein expression and cell function were analyzed. RESULTS: Fully differentiated podocytes had long finger-like protrusions forming slit diaphragm structures. Immunofluorescence showed ezrin is mainly localized to the cell membrane with intense fluorescence on cellular protrusions. After AGE-BSA treatment for 6 days, podocytes were rounder with fewer protrusions. Western blotting showed a time-dependent reduction in ezrin levels compared with BSA-treated cells. AGE-BSA treatment reduced podocyte adhesion to fibronectin and inhibited migration. Overexpression of ezrin completely reversed AGE-BSA inhibition of podocyte adhesion to fibronectin and partially reversed AGE-induced inhibition of migration. CONCLUSION: These results show a role for ezrin in AGE-induced podocyte damage and suggest a new avenue for possible therapeutic intervention in diabetic nephropathy.

Insulin-like growth factors and kidney disease.

Insulin-like growth factors (IGF-1 and IGF-2) are necessary for normal growth and development. They are related structurally to proinsulin and promote cell proliferation, differentiation, and survival, as well as insulin-like metabolic effects, in most cell types and tissues. In particular, IGFs are important for normal pre- and postnatal kidney development. IGF-1 mediates many growth hormone actions, and both growth hormone excess and deficiency are associated with perturbed kidney function. IGFs affect renal hemodynamics both directly and indirectly by interacting with the renin-angiotensin system. In addition to the IGF ligands, the IGF system includes receptors for IGF-1, IGF-2/mannose-6-phosphate, and insulin, and a family of 6 high-affinity IGF-binding proteins that modulate IGF action. Disordered regulation of the IGF system has been implicated in a number of kidney diseases. IGF activity is enhanced in early diabetic nephropathy and polycystic kidneys, whereas IGF resistance is found in chronic kidney failure. IGFs have a potential role in enhancing stem cell repair of kidney injury. Most IGF actions are mediated by the tyrosine kinase IGF-1 receptor, and inhibitors recently have been developed. Further studies are needed to determine the optimal role of IGF-based therapies in kidney disease.

Papillary thyroid cancer-derived exosomes contain miRNA-146b and miRNA-222.

BACKGROUND: With the increasing diagnosis of indolent papillary thyroid cancer (PTC), the task of identifying those likely to suffer from recurrence is becoming ever more challenging. MicroRNA (miRNA/miR) in the circulation has been demonstrated as potential biomarkers of recurrence in PTC. This study aimed to investigate in vitro if extracellular miRNAs are contained in exosomes, and their potential effect on other cells. METHODS: TPC-1 (PTC) and NTHY (normal thyroid follicular) cell lines were treated with exosome isolates and conditioned medium (CM), both containing miR-146b and miR-222. The changes in proliferation over a 72-h period of TPC-1 and NTHY were compared. Student t-test and analysis of variance were used for significance testing, and P < 0.05 was considered significant. RESULTS: Exosomes derived from TPC-1 cells were demonstrated to contain miR-146b and miR-222 in relative abundance. These exosomes caused a negative proliferative effect on both TPC-1 and NTHY cells. Exosomes derived from NTHY cells did not exert a significant proliferative effect on either cell line. CM from both cell types caused an initial increase in TPC-1 proliferation at 24 h. No significant change in proliferation was seen with NTHY cells when treated with either of the CM. CONCLUSIONS: The results showed that PTC cells overexpress miR-146b and miR-222 in exosomes; and that factors released by both normal thyroid and PTC cells alter proliferation of other cells in a complex manner. The intercellular interactions were likely conferred in part by exosomal miRNA, which can potentially be developed as biomarkers of PTC recurrence.

Insulin-Like Growth Factor Binding Proteins--an Update.

The insulin-like growth factor (IGF) system is essential for normal growth and development, and its perturbation is implicated in a number of diseases. IGF activity is finely regulated by a family of six high-affinity IGF binding proteins (IGFBPs). 1GFBPs usually inhibit IGF actions but may enhance them under certain conditions. Additionally, IGFBPs bind non-IGF ligands in the extracellular space, cell membrane, cytoplasm and nucleus, thereby modulating cell proliferation, survival and migration in an IGF-independent manner. IGFBP activity is regulated by transcriptional mechanisms as well as by post-translational modifications and proteolysis. Understanding the balance between the various actions of IGFBPs in vivo may lead to novel insights into disease processes and possible IGFBP-based therapeutics.

Prohibitin-2 binding modulates insulin-like growth factor-binding protein-6 (IGFBP-6)-induced rhabdomyosarcoma cell migration.

Insulin-like growth factor (IGF)-binding protein (IGFBP)-6 decreases cancer cell proliferation and survival by inhibiting the effects of IGF-II. More recently, IGFBP-6 was found to promote the migration of rhabdomyosarcoma (RMS) cells in an IGF-independent manner, and MAPK pathways were involved in this process. However, the precise molecular mechanisms of these IGF-independent migratory actions of IGFBP-6 are largely unknown. Here, we report that prohibitin-2 (PHB2), a single-span membrane protein, is a key regulator of IGFBP-6-induced RMS cell migration. PHB2 and IGFBP-6 co-localize on the RMS cell surface, and they specifically interact, as demonstrated by affinity chromatography, co-immunoprecipitation, biosensor analysis, and confocal microscopy. Binding affinities for PHB2 are 9.0 ± 1.0 nM for IGFBP-6 and 10.2 ± 0.5 nM for mIGFBP-6, a non-IGF-binding mutant of IGFBP-6. The C-domain but not the N-domain of IGFBP-6 is involved in PHB2 binding. In addition, IGFBP-6 indirectly increases PHB2 tyrosine phosphorylation on RMS membranes. Importantly, PHB2 knockdown completely abolished IGFBP-6-mediated RMS cell migration. In contrast, IGFBP-6-induced MAPK pathway activation was not affected, suggesting that PHB2 may act as a downstream effector of these pathways. These results indicate that PHB2 plays a key role in this IGF-independent action of IGFBP-6 and suggest a possible therapeutic target for RMS.

Uptake of advanced glycation end products by proximal tubule epithelial cells via macropinocytosis.

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. We describe AGE uptake in LLC-PK1 and HK2 proximal tubule cell lines by macropinocytosis, a non-specific, endocytic mechanism. AGE-BSA induced dorsal circular actin ruffles and amiloride-sensitive dextran-TRITC uptake, significantly increased AGE-BSA-FITC uptake (167±20% vs BSA control, p<0.01) and was ezrin-dependent. AGE-BSA-FITC uptake was significantly inhibited by amiloride and inhibitors of Arf6, Rac1, racGEF Tiam1, PAK1 and actin polymerisation. AGE-BSA-FITC, Arf6 and PIP2 co-localised within dorsal circular actin ruffles. AGE-BSA increased PAK1 kinase activity (212±41% vs control, p<0.05) and protein levels of Tiam1, a Rac1 activator. AGE-BSA significantly increased TGF-ß1 protein levels (160±6%, p<0.001 vs BSA), which were significantly inhibited by inhibitors of Arf6 (82±19%, p<0.001 vs AGE) and actin polymerisation (107±11%, p<0.001 vs AGE), suggesting AGEs partially exert their profibrotic effects via macropinocytosis. PAK1 and PIP5Kγ siRNA significantly decreased AGE-BSA-FITC uptake (81±6% and 64±7%, respectively, p<0.05 vs control for both), and AGE-stimulated TGF-ß1 protein release (99±15% and 49±8% of control, p<0.05 and p<0.001, respectively). Inhibition of AGE uptake by macropinocytosis inhibitors and a neutralising TGF-ß antibody, reversed the AGE-induced decrease in surface Na(+)K(+)ATPase, suggesting AGE uptake by macropinocytosis may contribute to diabetic kidney fibrosis and/or EMT by modulating this pump. Understanding methods of cellular uptake and signalling by AGEs may lead to novel therapies for diabetic nephropathy.

IGF-II and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren's disease.

Dupuytren's disease (DD) is a common and heritable fibrosis of the palmar fascia that typically manifests as permanent finger contractures. The molecular interactions that induce the development of hyper-contractile fibroblasts, or myofibroblasts, in DD are poorly understood. We have identified IGF2 and IGFBP6, encoding insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-6 respectively, as reciprocally dysregulated genes and proteins in primary cells derived from contracture tissues (DD cells). Recombinant IGFBP-6 inhibited the proliferation of DD cells, patient-matched control (PF) cells and normal palmar fascia (CT) cells. Co-treatments with IGF-II, a high affinity IGFBP-6 ligand, were unable to rescue these effects. A non-IGF-II binding analog of IGFBP-6 also inhibited cellular proliferation, implicating IGF-II-independent roles for IGFBP-6 in this process. IGF-II enhanced the proliferation of CT cells, but not DD or PF cells, and significantly enhanced DD and PF cell contractility in stressed collagen lattices. While IGFBP-6 treatment did not affect cellular contractility, it abrogated the IGF-II-induced contractility of DD and PF cells in stressed collagen lattices. IGF-II also significantly increased the contraction of DD cells in relaxed lattices, however this effect was not evident in relaxed collagen lattices containing PF cells. The disparate effects of IGF-II on DD and PF cells in relaxed and stressed contraction models suggest that IGF-II can enhance lattice contractility through more than one mechanism. This is the first report to implicate IGFBP-6 as a suppressor of cellular proliferation and IGF-II as an inducer of cellular contractility in this connective tissue disease.

The high burden of inpatient diabetes mellitus: the Melbourne Public Hospitals Diabetes Inpatient Audit.

OBJECTIVE: To determine the prevalence of diabetes in inpatients in Melbourne hospitals. DESIGN: Point prevalence survey of all inpatients in each hospital on a single day between 30 November 2010 and 22 November 2012. SETTING: 11 hospitals in metropolitan Melbourne including community, secondary and tertiary hospitals and one aged care and rehabilitation centre. PARTICIPANTS: 2308 adult inpatients in all wards apart from intensive care, emergency, obstetrics and psychiatry. MAIN OUTCOME MEASURES: Point prevalence of self-reported diabetes, details of current medication, self-reported frequency of complications. RESULTS: Diabetes status was obtained in 2273 of 2308 inpatients (98.5%). Of these, 562 (24.7%) had diabetes (95% CI, 22.9%-26.5%). Diabetes prevalence ranged from 15.7% to 35.1% in different hospitals (P < 0.001). Patients with diabetes were older, heavier and more likely to be taking lipid-lowering, antihypertensive and blood-thinning medications. Of 388 patients with complete medication information, 270 (69.6%) were taking oral hypoglycaemic agents alone or in combination with insulin, 158 (40.7%) were treated with insulin (67 [17.3%] with insulin alone) and 51 (13.1%) were not taking medication for diabetes. The frequency of diabetes complications was very high: 207/290 (71.4%) for any microvascular complication, 275/527 (52.2%) for any macrovascular complication and 227/276 (82.2%) for any complication. CONCLUSION: The high burden of diabetes in Melbourne hospital inpatients has major implications for patient health and health care expenditure. Optimising care of these high-risk patients has the potential to decrease inpatient morbidity and length of stay as well as preventing or delaying future complications. A formal Australian national audit of inpatient diabetes would determine its true prevalence and consequences, allowing rational planning to deal with shortcomings in its management.

Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development.

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under in vitro conditions, a fusion protein comprising a J-GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J-GFP-DD). We demonstrate that J-GFP-DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.

Insulin-like growth factor-binding protein-6 and cancer.

The IGF (insulin-like growth factor) system is essential for physiological growth and it is also implicated in a number of diseases including cancer. IGF activity is modulated by a family of high-affinity IGF-binding proteins, and IGFBP-6 is distinctive because of its marked binding preference for IGF-II over IGF-I. A principal role for IGFBP-6 is inhibition of IGF-II actions, but recent studies have indicated that IGFBP-6 also has IGF-independent effects, including inhibition of angiogenesis and promotion of cancer cell migration. The present review briefly summarizes the IGF system in physiology and disease before focusing on recent studies on the regulation and actions of IGFBP-6, and its potential roles in cancer cells. Given the widespread interest in IGF inhibition in cancer therapeutics, increasing our understanding of the mechanisms underlying the actions of the IGF ligands, receptors and binding proteins, including IGFBP-6, will enhance our ability to develop optimal treatments that can be targeted to the most appropriate patients.