Atomic force microscopy for quantitative understanding of peptide-induced lipid bilayer remodeling.

A number of peptides are known to bind lipid bilayer membranes and cause these natural barriers to leak in an uncontrolled manner. Though membrane permeabilizing peptides play critical roles in cellular activity and may have promising future applications in the therapeutic arena, significant questions remain about their mechanisms of action. The atomic force microscope (AFM) is a single molecule imaging tool capable of addressing lipid bilayers in near-native fluid conditions. The apparatus complements traditional assays by providing local topographic maps of bilayer remodeling induced by membrane permeabilizing peptides. The information garnered from the AFM includes direct visualization and statistical analyses of distinct bilayer remodeling modes such as highly localized pore-like voids in the bilayer and dispersed thinned membrane regions. Colocalization of distinct remodeling modes can be studied. Here we examine recent work in the field and outline methods used to achieve precise AFM image data. Experimental challenges and common pitfalls are discussed as well as techniques for unbiased analysis including the Hessian blob detection algorithm, bootstrapping, and the Bayesian information criterion. When coupled with robust statistical analyses, high precision AFM data is poised to advance understanding of an important family of peptides that cause poration of membrane bilayers.

Effect of hepatitis B virus on steatosis in hepatitis C virus co-infected subjects: A multi-centre study and systematic review.

It remains unclear whether hepatitis B virus (HBV) infection may modify the severity of viral steatosis in patients coinfected with chronic hepatitis C virus (HCV). We examined the influence of coinfection with HBV on prevalence of steatosis in chronic hepatitis C in a multi-centre cohort of HBV-HCV subjects, and by performing a systematic review and meta-analysis of the literature. We centrally and blindly assessed steatosis prevalence and severity in a cohort of HBV-HCV coinfected subjects compared to HCV and HBV monoinfected controls and we performed a systematic review of studies addressing the prevalence of steatosis in HBV-HCV subjects compared to HCV controls. In the clinical cohort, we included 85 HBV-HCV, 69 HBV and 112 HCV subjects from 16 international centres. There was no significant difference in steatosis prevalence between the HBV-HCV and the HCV groups (33% vs 45%, P = .11). In subgroup analysis, lean HBV-HCV subjects with detectable HBV DNA had less steatosis than lean HCV subjects matched for HCV viremia (15% vs 45%, P = .02). Our literature search identified 5 additional studies included in a systematic review. Overall, prevalence of steatosis > 5% was similar in HBV-HCV infection compared to HCV (pooled odds ratio [OR] 0.91, 95% CI 0.53-1.6) although there was significant heterogeneity (I2 69%, P = .007). In conclusion, although the prevalence of steatosis is similar in HBV-HCV compared to HCV subjects, our analysis suggests that there may be an inhibitory effect of HCV-induced steatogenesis by HBV in certain subgroups of patients.

Glycaemic control in patients with type 2 diabetes switching from premixed insulin to long-acting basal insulin analogue plus oral antidiabetic drugs: an observational study.

AIM: To evaluate whether administration of long-acting basal insulin analogue plus oral antidiabetic drugs (OADs) improves glycaemic control in type 2 diabetic patients with glycosylated haemoglobin (HbA1c) > 7% (53 mmol/mol) under premixed insulin therapy. METHODS: This is a multicentre, observational, retrospective study performed in type 2 diabetic patients switching from premixed insulin to long-acting basal insulin analogue plus OADs. Data on patients' medical history and assessments were retrieved from patients' medical charts prior to switching the treatment and 6 months thereafter. RESULTS: A total of 131 evaluable patients were enrolled (mean age, 68.2 ± 9.4 years; female, 65.6%; mean diabetes duration, 12.7 ± 6.9 years; mean time on insulin therapy, 53.2 ± 41.9 months). Patients were receiving premixed insulin (once-daily, 4.7%; twice-daily, 85.0%; thrice-daily, 10.2%), 82.4% of whom in combination with OADs (metformin, 79.4%). After the treatment was switched, only 14.5% required intensification of treatment with additional preprandial insulin. HbA1c decreased -1.4% [mean ± SD, 8.4 ± 1.0% (68.7 ± 11.4 mmol/mol) vs. 7.0 ± 1.0% (53.6 ± 10.9 mmol/mol), p < 0.001] and the proportion of patients achieving HbA1c < 7% (53 mmol/mol) increased to 52.7% (p < 0.001). The percentage of patients with hypoglycaemia decreased (19.2% vs. 10.8%, p < 0.05; symptomatic, 17.6% vs. 4.6%, p < 0.01) and body weight diminished by -1.9 kg (mean ± SD, 78.5 ± 14.7 kg vs. 76.6 ± 13.9 kg, p < 0.05). Basal insulin plus OADs was considered more convenient and flexibly adapted to patients' life in 98.4% and 99.2% of patients, respectively. Additionally, 96.9% of patients reported being more satisfied and 96.9% would recommend it. CONCLUSIONS: Switching the treatment from premixed insulin to long-acting basal insulin analogue plus OADs is a feasible and convenient approach to improve glycaemic control of type 2 diabetic patients poorly controlled with premixed insulin under routine clinical practice conditions.

[Management of patient profiles with type2 diabetes mellitus in Primary Care in Spain: CONTROVERTI2 Program]. / Manejo de perfiles de pacientes con diabetes mellitus tipo2 en la práctica clínica de la atención primaria en España: Programa CONTROVERTI2.

AIM: To identify existing controversies in the routine management of patients with T2D and to contrast them with the latest scientific evidence and clinical guidelines, in order to help optimize and homogenize the treatment of patients with T2D in Primary Care (PC) in Spain. MATERIAL AND METHODS: 240 family doctors responded to an online questionnaire about the management of 6 patient profiles with T2D of increasing complexity. RESULTS: The main drivers for the antihyperglycemic treatment choice are an HbA1c>10% and the presence of cardiovascular disease (CVD), although in evolved patients, the estimated glomerular filtration rate and the risk of hypoglycemia become more relevant. In newly diagnosed patients with an HbA1c>9%, treatment is still initiated with monotherapy (24%). In patients not controlled with metformin, dipeptidyl peptidase 4 inhibitors (DPP4-I, 54%) or sodium-glucose cotransporter 2 inhibitors (SGLT2-I, 39%) are usually added. On the other hand, type1 glucagon-like peptide receptor agonists (GLP1-RA) are mainly associated with obese patients with T2D. In patients not controlled with metformin+sulfonylurea (SU), SU replacement is preferred to adding a third antihyperglycemic agent to background therapy (77% vs. 23%). CONCLUSIONS: T2D treatment in PC is still focused on HbA1c reduction and treatment safety. Thus, DPP4-I are widely used. SGLT2-I are usually preferred for patients with T2D and CVD and GLP1-RA for patients with T2D and obesity, although their use in PC is low.

Volumetric assessment of the sella turcica: a re-evaluation.

BACKGROUND: The sella turcica volume is widely measured by the Di Chiro-Nelson method. The purpose is to compare the fidelity of a proposed volumetry method vs. the Di Chiro-Nelson method, using computed tomography (CT) images. MATERIALS AND METHODS: Morphometric examination of 173 CT scans were included, of which 52.6% were female. The mean age was 53.2 ± 17.6 years. Considering the Di Chiro-Nelson method, two measurements were added for each axis in the CT evaluation: length (central, left, and right), width (central, anterior, and posterior), and height (central, left, and right). RESULTS: The mean measurements were length: central 10.11 ± 1.44, left 7.45 ± 1.67, right 7.53 ± 1.59; width: central 12.27 ± 2.11, anterior 10.99 ± 1.92, posterior 10.10 ± 1.74; height: central 7.68 ± 1.38, left 7.16 ± 1.35, right 7.40 ± 1.41. A statistically significant difference between sexes was found only in the anterior width (p = 0.01). Using the proposed method, the volume was 342.2 ± 88.5 and 378. 6 ± 113.9 mm³, respectively for females and males (p = 0.02) vs. 476.1 ± 132.4 and 523.8 ± 186.0 mm3 (p = 0.05) using the Di Chiro-Nelson's method. CONCLUSIONS: Women had significantly smaller sella turcica volume than men. This proposed method considers the sella turcica as a not strictly symmetrical structure and indicates reduced variation between the maximum and minimum values, compared to the Di Chiro-Nelson's. Our findings may be useful for reassessment the volume of the sella turcica as the measurements indicate a higher precision.

Occupancy of nonannular lipid binding sites on KcsA greatly increases the stability of the tetrameric protein.

KcsA, a homotetrameric potassium channel from prokaryotes, contains noncovalently bound lipids appearing in the X-ray crystallographic structure of the protein. The binding sites for such high-affinity lipids are referred to as "nonannular" sites, correspond to intersubunit protein domains, and bind preferentially anionic phospholipids. Here we used a thermal denaturation assay and detergent-phospholipid mixed micelles containing KcsA to study the effects of different phospholipids on protein stability. We found that anionic phospholipids stabilize greatly the tetrameric protein against irreversible, heat-induced unfolding and dissociation into subunits. This occurs in a phospholipid concentration-dependent manner, and phosphatidic acid species with acyl chain lengths ranging 14 to 18 carbon atoms are more efficient than similar phosphatidylglycerols in protecting the protein. A docking model of the KcsA-phospholipid complex suggests that the increased protein stability originates from the intersubunit nature of the binding sites and, thus, interaction of the phospholipid with such sites holds together adjacent subunits within the tetrameric protein. We also found that simpler amphiphiles, such as alkyl sulfates longer than 10 carbon atoms, also increase the protein stability to the same extent as anionic phospholipids, although at higher concentrations than the latter. Modeling the interaction of these simpler amphiphiles with KcsA and comparing it with that of anionic phospholipids serve to delineate the features of a hydrophobic pocket in the nonannular sites. Such pocket is predicted to comprise residues from the M2 transmembrane segment of a subunit and from the pore helix of the adjacent subunit and seems most relevant to protein stabilization.

Sublingual tacrolimus administration provides similar drug exposure to per-oral route employing lower doses in liver transplantation: a pilot study.

BACKGROUND: Per-oral tacrolimus administration is not always practicable. Sublingual administration is a potential alternative, but its feasibility and effectiveness compared with oral route has not been established. AIM: To compare tacrolimus drug exposure after sublingual and oral administration in liver transplant recipients. METHODS: Experimental, open-label, non-randomised, cross-over study. Tacrolimus exposure was evaluated in 32 liver transplant recipients receiving oral administration. 12 h tacrolimus area-under-the-curve (AUC0-12 h ) was calculated using tacrolimus blood concentrations at 0-0.5-1-2-4-6-8-12 hrs post-dose. Recipients were switched to sublingual administration, and dose was adjusted to reach similar trough levels, new AUC0-12 h was calculated. Correlation between AUC0-12 h and trough levels was determined for both oral and sublingual phases. RESULTS: Similar trough levels were accomplished with oral and sublingual administration (6.68 ± 2 ng/mL vs. 6.62 ± 1.9 ng/mL (P = 0.8)). Although concentration 2 h post dose was higher in oral phase (15.36 ± 7.14 vs. 13.18 ± 5.64, P = 0.015), AUC0-12 h was similar in both phases (116.6 ± 34.6 vs. 111.5 ± 36.93 ng/mL* h, P = 0.19). Daily dose of tacrolimus required in sublingual phase was 37% lower than that used in oral phase (P < 0.0001), suggesting significantly increased bioavailability of tacrolimus when employing sublingual route. Good correlation between AUC0-12 h and trough levels was observed in sublingual phase (r2 = 0.74). Twenty-two recipients were maintained on sublingual administration after the end of study (mean follow-up: 18.7 ± 5.8 months). No difference in liver function tests or rejection rates was found during follow-up period. CONCLUSIONS: Sublingual administration of tacrolimus is feasible and provides similar drug exposure compared with oral administration. In our study, at long-term follow-up, sublingual administration was not associated with liver transplant rejection.

Prolactin inhibits the apoptosis of chondrocytes induced by serum starvation.

The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.

[Satisfaction of patients with type 2 diabetes mellitus after starting treatment with insulin]. / Satisfacción de las personas con diabetes mellitus tipo 2 tras iniciar tratamiento con insulina.

OBJECTIVES: The objective of this study is to evaluate if overcoming the barrier of starting treatment with insulin can lead to better clinical control and a higher level of patient satisfaction with their treatment. MATERIAL AND METHODS: This is an observational, multicentre study of patients diagnosed with DM2 who attended primary care centres with poor glycaemic control (A1c≥8%) under treatment with oral antidiabetic drugs (OADs), and who were given motivational treatment to overcome their fear of injections, and started treatment with insulin. The level of satisfaction with the treatment was evaluated using the Diabetes Treatment Satisfaction Questionnaire (DTSQ). The questionnaire was used before initiating the treatment with insulin and in the follow-up visit (3-4 months from the beginning of treatment with basal insulin). RESULTS: A total of 573 patients with a mean age of 64±10 years were recruited. The overall mean score from the DTSQs satisfaction questionnaire was 18.3±6.3, and the change of treatment led to an improvement in patient satisfaction compared to the previous treatment (DTSQc mean score 8.8±5.9). A1c dropped from an initial value of 8.7% (SD 0.8) to 7.5% (SD 0.7) (P<.001). The frequency of hyperglycaemic episodes perceived by the patients was significantly lower after they overcame their fear of injections (35.6% compared to 11.5%; P<.001), but no statistically significant differences were found in the frequency of hypoglycaemic episodes (32% compared to 35%; P=.059). CONCLUSION: In patients with DM2 poorly controlled with OADs, overcoming a fear of injections and starting treatment with insulin was associated with an overall improvement in satisfaction with the new treatment, and decreased the perception of hyperglycaemic episodes. Glycaemic control and the metabolic profile of the patients also improved to a statistically significant degree with the change of treatment.

The influence of a membrane environment on the structure and stability of a prokaryotic potassium channel, KcsA.

The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X-ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. (2004) Curr. Opin. Struct. Biol. 14, 440-446]. Here, we provide infrared spectroscopic evidence that the structure and stability of the potassium channel KcsA and its chymotryptic derivative 1-125 KcsA reconstituted into native-like membranes differ from those exhibited by these proteins in detergent solution, the latter taken as an approximation of the mixed detergent-protein crystal conditions.

Inhibition of rat corneal angiogenesis by 16-kDa prolactin and by endogenous prolactin-like molecules.

PURPOSE: The cornea is an avascular organ, where induction of new blood vessels involves the turn-on of proangiogenic factors and/or the turn-off of antiangiogenic regulators. Prolactin (PRL) fragments of 14 kDa and 16 kDa bind to endothelial cell receptors and inhibit angiogenesis. This study was designed to determine whether antiangiogenic PRL-like molecules are involved in cornea avascularity. METHODS: Sixteen-kDa PRL and basic fibroblast growth factor (bFGF) or anti-PRL antibodies were placed into rat cornea micropockets and neovascularization evaluated by the optical density associated with capillaries stained by the peroxidase reaction and by the number of vessels growing into the implants. Prolactin receptors in corneal epithelium were investigated by immunocytochemistry. RESULTS: bFGF induced a dose-dependent stimulation of corneal neovascularization. This effect was inhibited by coadministration of 16-kDa PRL, as indicated by a 65% reduction in vessel density and a 50% decrement in the incidence of angiogenic responses. Corneal angiogenic reactions of different intensities were induced by implantation of polyclonal and monoclonal anti-PRL antibodies. Corneal epithelial cells were labeled by several anti-PRL receptor monoclonal antibodies. CONCLUSIONS: These findings show that exogenous 16-kDa PRL inhibits bFGF-induced corneal neovascularization and suggest that PRL-like molecules with antiangiogenic actions function in the cornea. PRL receptors in the corneal epithelium may imply that PRL in the cornea derives from lacrimal PRL internalized through an intracellular pathway. These observations are consistent with the notion that members of the PRL family are potential regulators of corneal angiogenesis.

Controlled study of early postoperative parenteral nutrition in children.

BACKGROUND/PURPOSE: Nutritional support of surgical patients has improved in recent years because of the possibility of modulating catabolism and anabolism, thus enhancing the immune response and repair processes. The objective of this study was to evaluate metabolic effects of early parenteral nutrition (PN) after major surgery. METHODS: The authors studied 63 children aged 4 to 14 years with diffuse peritonitis caused by perforated-suppurative appendicitis. They were assigned randomly to a study group (SG, n = 31), which received PN for 5 days, starting 24 to 48 hours after surgery or to a control group (CG, n = 32), that received standard treatment (fluids). Weight, C-reactive protein (CRP), albumin, prealbumin, glycemia, nitrogen balance (NB), and insulinlike growth factor (IGF-I), were evaluated on postoperative days 1, 4, and 6. RESULTS: Early nutritional support was associated with a significant improvement in NB and IGF-I (Repeat measures analysis of variance IGF-I, P<.001 and NB P<.01). CONCLUSIONS: The authors conclude that early parenteral nutrition has a positive effect on the anabolic response as shown by improved NB and higher IGF-I levels in pediatric patients after major surgery.

Centrifugal selection of signal-directed pecking.

Pigeons were exposed to a schedule of stimulus-correlated food presentations. When key pecks terminated trial signals and cancelled the delivery of food, pecking was either gradually or rapidly redirected away from the keys, depending on whether the food-omission contingency was introduced from the outset or after exposure to a response-independent baseline. In all cases, the food-omission contingency substantially reduced or eliminated pecking at the keys.

[Cost-effectiveness analysis of 2 strategies of Helicobacter pylori eradication: results of a prospective and randomized study in primary care]. / Análisis coste-efectividad de dos estrategias de erradicación de Helicobacter pylori: resultados de un estudio prospectivo y aleatorizado en atención primaria.

BACKGROUND: To analyze cost-effectiveness of two different strategies to treat H. pylori infection in peptic ulcer in the primary care setting. PATIENTS AND METHODS: Consecutive patients with endoscopic diagnosis of peptic ulcer were randomized to one of two strategies: a) treatment during 7 days with omeprazole, tetracycline, metronidazole and bismuth subcitrate ("quadruple" therapy) and if failure second-line treatment with omeprazole, amoxycillin and clarithromycin during 7 days (OCA7), and b) initial treatment with OCA7 and if failure treatment with "quadruple therapy". End point was eradication 8 weeks after last treatment dose. Direct and indirect costs were estimated (euros, 1997) and a cost-effectiveness analysis using a decision-tree model was undertaken after real clinical data. 95% confidence intervals are given. RESULTS: After screening 255 patients, 97 were finally included. 48 patients were given strategy a and 49 strategy b. Eradication was obtained (intention-to-treat) in 72.9% (CI 95%: 58.2-84.7) in group a versus 91.8% (CI 95%: 80.4-97.7) (p < 0.05) in group b. Mean cost per case treated was lower in group a (237 versus 268 euros) but cost per case eradicated was lower in group b (320 versus 296 euros). The cost was primarily determined by efficacy. CONCLUSIONS: Treatment with OCA7 followed by rescue with "quadruple" therapy if failure is more efficient in our area that the inverse strategy. Efficiency is mostly determined by efficacy.

Is 1-week treatment for peptic ulcer healing sufficient and safe?

OBJECTIVE: To confirm whether 1-week anti-Helicobacter therapy to achieve ulcer healing is sufficient and safe. METHODS: We retrospectively analyzed patients with peptic ulcer who were infected with Helicobacter pylori and treated with 3 different 7-day regimens, according to predefined protocols in 3 different centers in the same geographical area (Aragón, Spain). Three combinations commonly described in the literature were used: a) omeprazole (40 mg/24 h), tetracycline hydrochloride (2 g/24 h), colloidal bismuth subcitrate (480 mg/24 h) and metronidazole (750 mg/24 h) (OBTM, n = 105); b) omeprazole (40 mg/24 h), clarithromycin (1.5 g/24 h) and amoxicillin (3 g/24 h) (O40C1.5A3, n = 13); and c) omeprazole (40 mg/24 h), clarithromycin (1 g/24 h) and amoxicillin (2 g/24 h) (O40C1A2, n = 4). In all patients the diagnosis of peptic ulcer disease was confirmed endoscopically, and H. pylori infection was verified with urease testing and histological analysis. After treatment ended, no other antacids were allowed until after endoscopic examination to check eradication and ulcer healing. RESULTS: 122 patients were included (107 with duodenal ulcer, 12 with gastric ulcer and 3 with both). Compliance was good and side effects infrequent and mild. Eradication rates were 88.5% (93/105) in the OBTM group, 100% (13/13) with O40C1.5A3, and 75% (3/4) with O40C1A2. Healing was achieved in 98.16% (107/109) of the patients in whom the bacterial infection was eradicated, and in 23.07% (3/13) of those in whom it was not (p < 0.0001). No patient had any complications during the period without treatment. CONCLUSIONS: 1-week eradication therapy with previously described combinations commonly used in clinical practice achieves high ulcer healing rates with no complications in the period without antacid treatment. We consider that it is not necessary, at least in most patients, to prolong antacid therapy.

An analysis of timeout and response cost in a programmed environment.

A group of mildly retarded adolescents with high rates of antisocial behavior was exposed to two parameters of timeout and response cost within the context of a programmed environment. For five of the six subjects, the two higher values (30 tokens response cost or 30 min timeout) were significantly more suppressive than the lower values (five tokens or 5 min). For the one remaining subject, there was a strong relationship in the opposite direction. Also, the timeout and response cost of higher value became increasingly more suppressive over time, whereas those of lower value did not. There were few appreciable differences between the timeout and response cost of similar magnitude. A discussion of these results is presented in support of the notion that the functional aversiveness of timeouts (and response costs) appears to be critically dependent upon interactions with the environmental conditions in which they are implemented and the reinforcement histories of the subjects.

Familiarity, proximity and self-injurious behaviors.

This paper introduces familiarity and proximity of direct-care staff as possible contributors to the etiology of self-injurious behaviors. Analysis suggests that research workers consider these two variables when evaluating the etiology of such behaviors with specific reference to positive and negative reinforcement paradigms.

[Role of classical oral glucose-lowering medications in current treatment]. / Papel de los hipoglucemiantes orales clásicos en el tratamiento actual.

Classical oral glucose were discovered in the mid twentieth century. Despite the time elapsed since then and the lack of large studies to support the use of some of these drugs, they continue to be employed, are indicated in all clinical practice guidelines and consensus documents and, overall, remain among the most widely prescribed drugs in the national health system. The main arguments for their continued use are their widespread and prolonged prescription, their effectiveness, and cost. Their main disadvantages have always been and continue to be their adverse gastrointestinal effects, weight gain, the risk of hypoglycemia and other adverse effects, which have encouraged the development of new glucose-lowering drugs with an improved pharmacological profile that would cover the various mechanisms of hyperglycemia. Currently, deep knowledge of glucose-lowering drugs is required in the patient-centered management of diabetes. Furthermore, this knowledge should be adapted to each individual patient to acquire the experience necessary to achieve effective metabolic control, delay the development of chronic complications, and improve the quality of life and life expectancy of patients with diabetes.