Endoplasmic reticulum stress in diabetes: New insights of clinical relevance

The endoplasmic reticulum (ER) is a cellular compartment responsible for multiple important cellular functions including the biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. A myriad of pathological and physiological factors perturb ER function and cause dysregulation of ER homeostasis, leading to ER stress. Accumulating evidence suggests that ER stress plays a role in the pathogenesis of diabetes, contributing to pancreatic β-cell loss and insulin resistance. ER stress may also link obesity, inflammation and insulin resistance in type 2 diabetes. In this review, we address the transition from physiology to pathology, namely how and why the physiological UPR evolves to a proapoptotic ER stress response in diabetes and its complications. Special attention was given to elucidate how ER stress could explain some of the ‘clinical paradoxes’ such as secondary sulfonylurea failure, initial worsening of retinopathy during tight glycemic control, insulin resistance induced by protease inhibitors and other clinically relevant observations.     

Study of flow behaviors on single-cell manipulation and shear stress reduction in microfluidic chips using computational fluid dynamics simulations

Various single-cell retention structures (SCRSs) were reported for analysis of single cells within microfluidic devices. Undesirable flow behaviors within micro-environments not only influence single-cell manipulation and retention significantly but also lead to cell damage, biochemical heterogeneity among different individual cells (e.g., different cell signaling pathways induced by shear stress). However, the fundamentals in flow behaviors for single-cell manipulation and shear stress reduction, especially comparison of these behaviors in different microstructures, were not fully investigated in previous reports. Herein, flow distribution and induced shear stress in two different single-cell retention structures (SCRS I and SCRS II) were investigated in detail to study their effects on single-cell trapping using computational fluid dynamics (CFD) methods. The results were successfully verified by experimental results. Comparison between these two SCRS shows that the wasp-waisted configuration of SCRS II has a better performance in trapping and manipulating long cylinder-shaped cardiac myocytes and provides a safer “harbor” for fragile cells to prevent cell damage due to the shear stress induced from strong flows. The simulation results have not only explained flow phenomena observed in experiments but also predict new flow phenomena, providing guidelines for new chip design and optimization, and a better understanding of the cell micro-environment and fundamentals of microfluidic flows in single-cell manipulation and analysis.     

Association of Elevated Serum Uric Acid with the Components of Metabolic Syndrome and Oxidative Stress in Abdominal Obesity Subjects

Abdominal obesity (AO) and metabolic syndrome (MetS) are associated with the cardiovascular disease and type 2 diabetes. Serum uric acid (SUA) is often elevated in subjects with the AO. We aimed to investigate the association of elevated SUA with the components of MetS, oxidative stress and TG/HDL-C ratio in AO subjects. This cross-sectional study used data from a Health Survey for Prevention of Hypertension and Type 2 Diabetes Mellitus in residents of two districts in Phitsanulok province, including 443 subjects. Anthropometric, blood pressure (BP) and biochemical variables were measured. We categorized the participants to two-group as 248 AO subjects (median age = 58, interquartile range 50.0–65.0 years) and 195 non-AO subjects (median age = 53, interquartile range 47.0–62.0 years). Waist circumference was significantly correlated with SystBP, DiastBP, Glu and SUA (P < 0.05) and SUA was significantly correlated with Glu, TG, HDL-C and TG/HDL-C ratio (P < 0.05). By using multiple logistic regression, we found the association of elevated SUA with abdominal obesity, hyperglycemia, hypertriglyceridemia, reduced HDL-C, elevated TG/HDL-C ratio, MetS and increased oxidative stress after adjusting for their covariates. Our study demonstrated that circulating UA is a major antioxidant and might help protect against free-radical oxidative damage. However, elevated SUA concentrations associated with oxidative stress, MetS, insulin resistance, and components of MetS. Then, SUA may be a marker of increased oxidative stress, insulin resistance and MetS, implying an increased risk of vascular disease and T2DM.     

Evaluation of Antioxidants in the Kidney of Streptozotocin Induced Diabetic Rats

Diabetes mellitus is one of the most common endocrine metabolic disorders. Dual endocrine deficits of impaired insulin action (insulin resistance) and inadequate insulin secretion create an environment of chronic hyperglycemia and general metabolic disarray. Oxidative stress plays an important role in diabetic pathogenesis. Oxidative stress induced by streptozotocin (STZ) has been shown to damage pancreatic beta cell and produce hyperglycemia in rats. The present study was made to evaluate the antioxidant activity of ethanolic extract of the Evolvulus alsinoides in STZ induced rats. The antioxidant activities were done by using standard protocols. For histopathological analysis, the pancreatic tissues of all experimental groups were fixed with 10 % formalin for 24 h then the samples were stained with hematoxylin–eosin for the microscopic observation. Our results showed the significant decrease in lipid peroxidation and increases in the antioxidant (both enzymatic and nonenzymatic) levels after treatment with standard as well as the E. alsinoides. There is no significant difference between control and plant alone group rats. The histopathology reports also revealed non-toxic effect and protective effect of E. alsinoides in the kidney of STZ induced diabetic rats. Our result indicated that the E. alsinoides extract effectively increased the antioxidant level thereby it prevents oxidative stress during diabetes mellitus and also it showed the protective effect on kidney of STZ induced rats. Hence it can be used to maintain the antioxidant level during diabetes mellitus.     

Abdominal Obesity Associated with Elevated Serum Butyrylcholinesterase Activity,Insulin Resistance and Reduced High Density Lipoprotein-Cholesterol Levels

Abdominal obesity (AO) has a strong correlation with cardiovascular disease and has been linked to Alzheimer’s disease and type 2 diabetes. We investigated the association between AO and elevated serum butyrylcholinesterase (BChE) activity, insulin resistance and the serum lipid profile, including triglyceride (TG), HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) levels in AO and non-AO women subjects. A total of 500 AO subjects (age 49.1 ± 10.5 years), and 142 non-AO women subjects (age 49.9 ± 11.9 years) were enrolled for the general biochemistry tests, serum BChE, fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR). Body mass index, waist circumference, Blood pressure (BP), plasma glucose (Glu), triglyceride (TG), BChE, insulin, HOMA-IR were significantly higher and HDL-C levels were significantly lower in AO subjects (p < 0.05). Waist circumference was significantly correlated with BP, Glu, TG, BChE, insulin and HOMA-IR in AO subjects. Multiple logistic regression demonstrated that AO was associated with elevated BChE, HOMA-IR, hypertension and reduced HDL-C after adjusting for these variables. AO is associated with elevated BChE, insulin resistance, HT and reduced HDL-C. These may predict the development of type 2 diabetes mellitus and may be associated with cognitive disorder in the future, both are mediated through insulin resistance.     

Biosensors for immune cell analysis��A perspective

Massively parallel analysis of single immune cells or small immune cell colonies for disease detection, drug screening, and antibody production represents a “killer app” for the rapidly maturing microfabrication and microfluidic technologies. In our view, microfabricated solid-phase and flow cytometry platforms of the future will be complete with biosensors and electrical/mechanical/optical actuators and will enable multi-parametric analysis of cell function, real-time detection of secreted signals, and facile retrieval of cells deemed interesting.     

Effect of a dual inlet channel on cell loading in microfluidics

Unwanted sedimentation and attachment of a number of cells onto the bottom channel often occur on relatively large-scale inlets of conventional microfluidic channels as a result of gravity and fluid shear. Phenomena such as sedimentation have become recognized problems that can be overcome by performing microfluidic experiments properly, such as by calculating a meaningful output efficiency with respect to real input. Here, we present a dual-inlet design method for reducing cell loss at the inlet of channels by adding a new “ upstream inlet ” to a single main inlet design. The simple addition of an upstream inlet can create a vertically layered sheath flow prior to the main inlet for cell loading. The bottom layer flow plays a critical role in preventing the cells from attaching to the bottom of the channel entrance, resulting in a low possibility of cell sedimentation at the main channel entrance. To provide proof-of-concept validation, we applied our design to a microfabricated flow cytometer system (μFCS) and compared the cell counting efficiency of the proposed μFCS with that of the previous single-inlet μFCS and conventional FCS. We used human white blood cells and fluorescent microspheres to quantitatively evaluate the rate of cell sedimentation in the main inlet and to measure fluorescence sensitivity at the detection zone of the flow cytometer microchip. Generating a sheath flow as the bottom layer was meaningfully used to reduce the depth of field as well as the relative deviation of targets in the z-direction (compared to the x-y flow plane), leading to an increased counting sensitivity of fluorescent detection signals. Counting results using fluorescent microspheres showed both a 40% reduction in the rate of sedimentation and a 2-fold higher sensitivity in comparison with the single-inlet μFCS. The results of CD4⁺ T-cell counting also showed that the proposed design results in a 25% decrease in the rate of cell sedimentation and a 28% increase in sensitivity when compared to the single-inlet μFCS. This method is simple and easy to use in design, yet requires no additional time or cost in fabrication. Furthermore, we expect that this approach could potentially be helpful for calculating exact cell loading and counting efficiency for a small input number of cells, such as primary cells and rare cells, in microfluidic channel applications.     

A Study of Carotid Atherosclerosis in Patients with Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease shares many features of metabolic syndrome and its presence could signify a substantial cardiovascular risk above and beyond that conferred by individual risk factors. This study is an attempt to investigate the association of non-alcoholic fatty liver disease with carotid intima-media thickness and plaque as surrogate measures of increased cardiovascular risk. The study was conducted on 645 non diabetic, non alcoholic subjects in the age range of 20–60 years. Metabolic syndrome was assessed by using ATP III and ADA (2005) criteria. Anthropometric factors—waist circumference and blood pressure were measured. Fasting serum samples were analyzed for glucose, triglyceride, cholesterol and its fractions, insulin, alanine and aspartate transaminases, gamma glutamyl transferase and free fatty acids. Insulin resistance and secretion were calculated by homeostasis model and insulin sensitivity by QUICKI index. Liver ultrasonographic scanning was used for assessing fatty liver. Carotid atherosclerosis was assessed by B-mode ultrasonography of common carotid artery and internal carotid artery. The prevalence of non-alcoholic fatty liver disease was 15.6 % in non alcoholic population and 68.5 % of non-alcoholic fatty liver disease had metabolic syndrome, which was associated with hyperinsulinemia, insulin resistance, insulin insensitivity along with elevated levels of waist circumference, blood pressure, triglyceride, FFA and decreased HDL cholesterol. NAFLD patients had markedly greater carotid intima media thickness than non NAFLD subjects with MCIMT of 591.6 ± 108 and 489.5 ± 132.4 μm (P < 0.001) and plaque prevalence of 19.2 and 2.2 %, respectively, thus the carotid intima media thickness is associated with NAFLD.     

Efficient dielectrophoretic cell enrichment using a dielectrophoresis-well based system

Whilst laboratory-on-chip cell separation systems using dielectrophoresis are increasingly reported in the literature, many systems are afflicted by factors which impede “real world” performance, chief among these being cell loss (in dead spaces, attached to glass and tubing surfaces, or sedimentation from flow), and designs with large channel height-to-width ratios (large channel widths, small channel heights) that make the systems difficult to interface with other microfluidic systems. In this paper, we present a scalable structure based on 3D wells with approximately unity height-to-width ratios (based on tubes with electrodes on the sides), which is capable of enriching yeast cell populations whilst ensuring that up to 94.3% of cells processed through the device can be collected in tubes beyond the output.     

Analysis of Modification of Liver Proteome in Diabetic Rats by 2D Electrophoresis and MALDI-TOF-MS

The uncontrolled hyperglycemia can lead to disturbances in the cell structure and functions of organs. This study was performed to analyze the “differential proteome” change in rat liver associated with diabetes mellitus in relation to effects of an anti-diabetic herb, Cynodon dactylon leaf extracts. Rats were intraperitoneally injected with alloxan (150 mg/kg/bw) and treated with C. dactylon leaf extracts (450 mg/kg/bw/day/orally). The liver proteins were subjected to proteome analysis using the advanced technologies i.e., 2D electrophoresis (2-DE) and mass spectrometry. Comparison of 2-DE protein distribution profiles among the livers from normal, alloxan-induced diabetic rats and alloxan-induced diabetic rats treated with C. dactylon leaves identified three proteins that were up-regulated in alloxan-induced diabetic rats i.e., nucleophosmin, l-xylulose reductase and carbonic anhydrase III which are known to be mainly involved in ribosome biogenesis, centrosome duplication, cell proliferation, tumor suppression, glucose metabolism, osmo-regulation, water–CO₂ balance and acid–base balance. These results help us to understand the elucidation of molecular mechanism connected to liver function and insulin associated with diabetes mellitus. These identified proteins were primarily involved in cell proliferation and homoeostasis of liver tissues upon the treatment with C. dactylon leaf extracts.     

Type-2 diabetes related intermediate phenotypic traits in north Indian diabetics

Asian Indians are known to be at a higher risk of developing T2DM, but the underlying genetic factor in this population is still not well understood. T2DM is a complex genetic trait and assessment of disease related intermediate phenotypic traits is an important initial step towards any systematic genomic study. Therefore, in the present study we have assessed diabetes related intermediate phenotypic traits of insulin secretion and insulin resistance in the patients belonging to this population. The study included 157 T2DM patients of either sex ranging in age from 45–80 years and 84 non-diabetic subjects with no family history of diabetes, ranging in age from 45 to 75 years served as controls. Intermediate phenotypic traits studied were BMI, W: H ratio, fasting free fatty acid level and Insulin resistance and secretion. Diabetics were found to have significantly higher W: H ratio (p<0.001), FFA (p<0.001) and HOMA-R (p<0.001) as compared to non-diabetics. However, there was no significant difference in their BMI and HOMA-β. There was a positive correlation between FFA level and HOMA-R among diabetics, but not among controls. These findings suggest that in abdominal obesity FFA mediated insulin resistance is an important causative factor underlying T2DM in this population. Moreover, comparable HOMA-β in diabetics reflects compensatory insulin hyper secretion in these subjects. There is a need to examine relative contribution and precise nature of genetic factor in their tendency for central obesity, free fatty acidemia and insulin resistance.     

History of the preanalytical phase: a personal view

In the 70ies of the last century, ther term “preanalytical phase” was introduced in the literature. This term describes all actions and aspects of the “brain to brain circle” of the medical laboratory diagnostic procedure happening before the analytical phase. The author describes his personal experiences in the early seventies and the following history of increasing awareness of this phase as the main cause of “laboratory errors”. This includes the definitions of influence and interference factors as well as the first publications in book, internet, CD-Rom and recent App form over the past 40 years. In addition, a short summary of previous developments as prerequesits of laboratory diagnostic actions is described from the middle age matula for urine collection to the blood collection tubes, anticoagulants and centrifuges. The short review gives a personal view on the possible causes of missing awareness of preanalytical causes of error and future aspects of new techniques in regulation of requests to introduction of quality assurance programs for preanalytical factors.     

A microfluidic device enabling high-efficiency single cell trapping

Single cell trapping increasingly serves as a key manipulation technique in single cell analysis for many cutting-edge cell studies. Due to their inherent advantages, microfluidic devices have been widely used to enable single cell immobilization. To further improve the single cell trapping efficiency, this paper reports on a passive hydrodynamic microfluidic device based on the “least flow resistance path” principle with geometry optimized in line with corresponding cell types. Different from serpentine structure, the core trapping structure of the micro-device consists of a series of concatenated T and inverse T junction pairs which function as bypassing channels and trapping constrictions. This new device enhances the single cell trapping efficiency from three aspects: (1) there is no need to deploy very long or complicated channels to adjust flow resistance, thus saving space for each trapping unit; (2) the trapping works in a “deterministic” manner, thus saving a great deal of cell samples; and (3) the compact configuration allows shorter flowing path of cells in multiple channels, thus increasing the speed and throughput of cell trapping. The mathematical model of the design was proposed and optimization of associated key geometric parameters was conducted based on computational fluid dynamics (CFD) simulation. As a proof demonstration, two types of PDMS microfluidic devices were fabricated to trap HeLa and HEK-293T cells with relatively significant differences in cell sizes. Experimental results showed 100% cell trapping and 90% single cell trapping over 4 × 100 trap sites for these two cell types, respectively. The space saving is estimated to be 2-fold and the cell trapping speed enhancement to be 3-fold compared to previously reported devices. This device can be used for trapping various types of cells and expanded to trap cells in the order of tens of thousands on 1-cm² scale area, as a promising tool to pattern large-scale single cells on specific substrates and facilitate on-chip cellular assay at the single cell level.     

Defining laboratory medicine: a circle cannot be squared

At the down of the third millennium, it is rather misleading to consider the “whole population” as a conceptual entity, whereby the population is actually composed by single individuals, who differ broadly in terms of age, sex, ethnic origin, occupation, health, wellbeing, lifestyle and risk factors. While reaffirming strongly that laboratory medicine shall aim to provide data that could be translated into actionable information on “BOTH” an individual and universal level, we confute and refuse the naive and too simplistic approach that the common beneficence shall always be prioritized over the individual good, since the common good is just the sum of many individual beneficences.     

A liver-on-a-chip for hepatoprotective activity assessment

Hepatoprotectant is critical for the treatment of liver disease. This study first reported the application of a liver chip in the hepatoprotective effect assessment. We first established a biomimetic sinusoid-on-a-chip by laminating four types of hepatic cell lines (HepG2, HUVEC, LX-2, and U937 cells) in a single microchannel with the help of laminar flow in the microchannel and some micro-fences. This chip was straightforward to fabricate and operate and was able to be long-term cultured. It also demonstrated better hepatic activity (cell viability, albumin synthesis, urea secretion, and cytochrome P450 enzyme activities) over the traditional planar cell culture model. Then, we loaded three hepatoprotectants (tiopronin, bifendatatum, and glycyrrhizinate) into the chip followed by the addition of acetaminophen as a toxin. We successfully observed the hepatoprotective effect of these hepatoprotectants in the chip, and we also found that bifendatatum predominantly reduced alanine transaminase secretion, tiopronin predominantly reduced lactate dehydrogenase secretion, and glycyrrhizinate predominantly reduced aspartate transaminase secretion, which revealed the different mechanisms of these hepatoprotectants and provided a clue for following molecular biological study of the protecting mechanism.     

Association of Resistin with Insulin Resistance and Factors of Metabolic Syndrome in North Indians

Metabolic syndrome (MetS) is a cluster of interrelated common clinical disorders. The role of resistin in insulin sensitivity and MetS is controversial till date. So, the aim of the present study was to investigate the relationship of plasma resistin levels with markers of the MetS in Indian subjects. In a case control study, total 528 subjects were selected for the study. 265 (194 male and 71 female) were cases (with MetS) and 263 (164 male and 99 female) were controls (without MetS). Required anthropometric measurements and calculations were carried out accordingly. All the Biochemical estimations were carried out according to standard protocol. Resistin level was measured by the standard protocol (By ELISA i.e. enzyme linked immunosorbent assay) as illustrated in the kit. Insulin level was also measured by the standard protocol as illustrated in the kit and insulin resistance was calculated by the standard procedures. Plasma resistin levels were significantly higher in cases compared with controls (male = 13.05 ± 4.31 vs. 7.04 ± 2.09 ng/ml; p ≤ 0.001 and female = 13.53 ± 4.14 vs. 7.42 ± 2.30 ng/ml; p ≤ 0.001). Plasma resistin levels were well correlated with waist circumference, glucose, triglycerides, waist/hip ratio, systolic and diastolic blood pressure, high density lipoprotein, total cholesterol, serum low density lipoprotein, serum very low density lipoprotein, insulin and insulin resistance. Plasma resistin levels were elevated in presence of the MetS and were associated with increased metabolic risk factors.     

Angiogenic Potential,Circulating Angiogenic Factors and Insulin Resistance in Subjects with Obesity

Altered vascular function and pathological angiogenesis are important factors common to the development of obesity and obesity-associated diseases. Most human studies relating obesity and angiogenesis have compared levels of angiogenic factors in obesity without looking at the serum angiogenic capacity which reflects the balance between the effects of angiogenic and angiostatic factors. Therefore, in this cross-sectional study, the serum angiogenic potential and levels of angiogenic factors in serum of obese (BMI > 25 kg/m²) and lean subjects (BMI < 23 kg/m²), with no history of obesity associated co-morbidities, were assessed. Serum angiogenic potential was significantly higher (p < 0.0001) in both male (n = 67) and female (n = 35) obese subjects and showed a positive correlation (r = 0.4, p < 0.0001) with BMI. Serum levels of the angiogenic factors, vascular endothelial growth factor (VEGF) and angiopoietin were significantly higher in obese subjects. Levels of angiostatic factors such as angiostatin, endostatin were not altered in obese male subjects but were elevated in female obese subjects. Angiogenic potential and levels of VEGF did not vary in obese subjects with high HOMA-IR compared to obese subjects with low HOMA-IR. These results suggest that the angiogenic potential of serum was elevated in obesity and that insulin resistance may not contribute to the increased angiogenic potential in obesity.     

Survey on reporting of epithelial cells in urine sediment as part of external quality assessment programs in Brazilian laboratories

IntroductionEpithelial cells (ECs) are structures regularly observed during urine microscopy analysis. The correct identification of EC subtypes can be useful since renal tubular epithelial cells (RTECs) are clinically relevant. We investigate the urinary ECs report and the judgement of its clinical importance by Brazilian laboratories.Materials and methodsA survey with four questions was made available to participants of the Urinalysis External Quality Assessment Program (EQAP) from Controllab. Laboratories composed 3 groups: (1) differentiating ECs subtypes: “squamous”, “transitional” and “RTECs”; (2) differentiating ECs subtypes: “squamous” or “non-squamous” cells; (3) without ECs subtype identification. Participants did not necessarily answer to all questions and the answers were evaluated both within the same laboratory’s category and within different categories of laboratories.ResultsA total of 1336 (94%) laboratories answered the survey; Group 1, 119/140 (85%) reported that ECs differentiation is important to the physician and 62% want to be evaluated by EQAP, while in Group 3, 455/1110 (41%) reported it is useful to them, however only 25% want be evaluated by EQAP. Group 2 laboratories 37/51 (73%) reported that the information is important, but only 13/52 (25%) are interested in an EQAP with differentiation of the 3 ECs subtypes.ConclusionMost of the laboratories do not differentiate ECs in the three subtypes, despite the clinical importance of RTECs. Education of laboratory staff about the clinical significance of urinary particles should be considered a key priority.     

Microfluidics platform for measurement of volume changes in immobilized intestinal enteroids

Intestinal enteroids are ex vivo primary cultured single-layer epithelial cell spheroids of average diameter ∼150 μm with luminal surface facing inward. Measurement of enteroid swelling in response to secretagogues has been applied to genetic testing in cystic fibrosis and evaluation of drug candidates for cystic fibrosis and secretory diarrheas. The current measurement method involves manual addition of drugs and solutions to enteroids embedded in a Matrigel matrix and estimation of volume changes from confocal images of fluorescently stained enteroids. We developed a microfluidics platform for efficient trapping and immobilization of enteroids for quantitative measurement of volume changes. Multiple enteroids are trapped in a “pinball machine-like” array of polydimethylsiloxane posts for measurement of volume changes in unlabeled enteroids by imaging of an extracellular, high-molecular weight fluorescent dye. Measurement accuracy was validated using slowly expanding air bubbles. The method was applied to measure swelling of mouse jejunal enteroids in response to an osmotic challenge and cholera toxin-induced chloride secretion. The microfluidics platform allows for parallel measurement of volume changes on multiple enteroids during continuous superfusion, without an immobilizing matrix, and for quantitative volume determination without chemical labeling or assumptions about enteroid shape changes during swelling.