Thin Film Plastic Antibody-Based Microplate Assay for Human Serum Albumin Determination.

Herein we demonstrate molecularly imprinted polymers (MIP) as plastic antibodies for a microplate-based assay. As the most abundant plasma protein, human serum albumin (HSA) was selected as the target analyte model. Thin film MIP was synthesized by the surface molecular imprinting approach using HSA as the template. The optimized polymer consisted of acrylic acid (AA) and N-vinylpyrrolidone (VP) in a 2:3 (w/w) ratio, crosslinked with N,N'-(1,2-dihydroxyethylene) bisacrylamide (DHEBA) and then coated on the microplate well. The binding of MIP toward the bound HSA was achieved via the Bradford reaction. The assay revealed a dynamic detection range toward HSA standards in the clinically relevant 1-10 g/dL range, with a 0.01 g/dL detection limit. HSA-MIP showed minimal interference from other serum protein components: γ-globulin had 11% of the HSA response, α-globulin of high-density lipoprotein had 9%, and ß-globulin of low-density lipoprotein had 7%. The analytical accuracy of the assay was 89-106% at the 95% confidence interval, with precision at 4-9%. The MIP-coated microplate was stored for 2 months at room temperature without losing its binding ability. The results suggest that the thin film plastic antibody system can be successfully applied to analytical/pseudoimmunological HSA determinations in clinical applications.

Direct assessment of very-low-density lipoprotein by mass sensitive sensor with molecularly imprinted polymers.

Very-low-density lipoprotein (VLDL) contributes to the buildup of atherosclerotic plaque in the arteries and can lead to coronary heart disease. In clinical laboratory testing, the cholesterol content of VLDL (VLDL-C) cannot be assessed directly by the enzymatic colorimetric assay as it can for other lipoproteins, due to lack of a specific sample pretreatment technique. VLDL concentration relies on analyzing the endogenous triglycerides (TGs) bound in its particles and then converting to the VLDL-C estimate TGs/5. This estimation is valid for at least 12 h-fasted serum when exogenous TGs attached to chylomicrons (CMs) have been cleared from the circulation. A quartz crystal microbalance (QCM)-based sensor was generated using biomimetic sensing elements as a molecularly imprinted polymer (MIP) to directly measure actual VLDL. A novel VLDL-MIP was synthesized using methacrylic acid (MAA) and N-vinylpyrrolidone (VP) in the ratio 1:1 (v/v) as functional monomers in the presence of N, N'-(1,2-dihydroxyethylene) bis(acrylamide) (DHEBA) as a crosslinking agent. The VLDL-MIP sensor showed high sensitivity with a linear response from 2.5 mg dL-1 to 100 mg dL-1 of VLDL-C with a limit of detection at 1.5 mg dL-1. Recoveries of 96-103% were achieved when the VLDL-MIP sensor was used for VLDL assessment at 38-71 mg dL-1 concentrations. Repeatability and reproducibility of the sensor were very good with coefficients of variation at 1.63-4.74% and 4.25-9.04%, respectively. The sensor demonstrated low cross-reactivity with other lipoproteins; 6-7% of low-density lipoprotein (LDL) signals, 2-4% high-density lipoprotein (HDL), and 1% CMs compared to the signal of VLDL. Sensor results for 12 h-fasted serum and non-fasted serum correlated well with VLDL estimates TGs/5, with coefficients of determination (R2) at 0.9967 and 0.9932, respectively. This new sensor offers a new strategy for direct VLDL assessment from non-fasted serum without other sample pretreatment steps than dilution.

Biomimetic sensors targeting oxidized-low-density lipoprotein with molecularly imprinted polymers.

Oxidized-low-density lipoprotein (oxLDL) is well-recognized as an actual patho-atherogenic lipoprotein: elevated serum concentration of oxLDL increases the risk for developing atherosclerosis, leading to coronary artery disease (CAD). Herein, we report an approach for sensing oxLDL directly in serum with molecularly imprinted polymer (MIP) thin films on quartz crystal microbalance (QCM). The resulting MIP sensors show low cross-reaction toward low-density lipoprotein (LDL) and high-density lipoprotein (HDL): signals are around one magnitude smaller. Very-low-density lipoprotein (VLDL) and human serum albumin (HSA) do not lead to any significant sensor response. The sensor allowed for accurately assessing oxLDL over the detection range of 86-5600 µg dL-1, which covers the clinically relevant concentrations. The sensor determines oxLDL with recovery accuracy of 92-107% and a precision of 1-8% coefficient variation. Compared with commercially available oxLDL ELISA test kit our sensor reveals similar characteristics obtaining a correlation coefficient of 0.98. However, the sensors have rapid response times of 10 min compared to 210 min of ELISA, which demonstrates their efficiency in assessing this sensitive atherogenic biomarker for CAD diagnostics.

Alimentary Canal of the Adult Blow Fly, Chrysomya megacephala (F.) (Diptera: Calliphoridae)-Part I: Ultrastructure of Salivary Glands.

The salivary gland ultrastructure of the adult male blow fly, Chrysomya megacephala (F.) (Diptera: Calliphoridae), was investigated at the ultrastructural level using light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The salivary glands are paired structures composed of a single median deferent duct bifurcated into two long, narrow efferent ducts connected to the coiled tubular glands. The SEM image of the gland surface revealed that the basal lamina is relatively smooth in general, but the whole surface appeared as a trace of rough swollen insertion by intense tracheal ramification. Ultrastructurally, the salivary gland is enclosed within the basal lamina, and interdigitation cytoplasmic extensions were apparent between the adjacent gland cells. The basement membrane appeared infoldings that is similar to the complex of the labyrinth channel. The cytoplasm characteristic of the gland revealed high activity, based on the abundance of noticeable secretory granules, either singly or in an aggregated reservoir. In addition, mitochondria were found to intersperse among rich parallel of arrays rough endoplasmic reticulum. Thick cuticle, which was well-delineated and electron dense, apically lined the gland compartments, with discontinuity of the double-layer cuticle revealing a trace of secretion discharged into the lumen. Gross anatomy of the adult salivary gland was markedly different from that of the third instar of the same species, and structural dissimilarity is discussed briefly.

Alimentary canal of the blow fly Chrysomya megacephala (F.) (Diptera: Calliphoridae): an emphasis on dissection and morphometry.

The alimentary canal is a major organ system that is often involved in the transmission of pathogens to humans from insects that serve as vectors of disease. In this study, we investigated the alimentary canal of the blow fly, Chrysomya megacephala (F.) (Diptera: Calliphoridae), highlighting the description for dissection and morphometric analysis of each organ. Dissection was performed in a phosphate buffer solution (pH=7.4) on 3(rd) instar larvae (three to four days old) and on both male and female adults (seven days old). Larval dissection was accomplished using two fine forceps to open the specimens from the posterior end and proceed anteriorly toward the cephalic segment. Meticulous dissection of the anterior end was vital for observation of the delicate salivary ducts, crop duct, and esophagus. Overall length of the 3(rd) instar alimentary canal measured 89.15 mm (range 81.40-99.70 mm). The midgut comprised the longest portion, measuring 46.35 mm (range 40.00-52.00 mm; n = 30) of the entire canal. Adult dissection was also performed from abdomen to head. Morphometric analyses revealed that the alimentary canal of males and females were relatively similar. No statistical differences were found between the entire length of the alimentary canal from mouth to anus (excluding all branches of the salivary glands, crop, and Malpighian tubules) of males and females. The alimentary canals of males measured 36.23 mm (range 32.60-41.20 mm) in length; whereas, those of females measured 37.23 mm (range 32.70-42.15 mm). Two-thirds of the entire canal length was comprised of midgut in each sex.

Forensic entomology cases in Thailand: a review of cases from 2000 to 2006.

This paper presents and discusses 30 cases of cadavers that had been transferred for forensic entomology investigations to the Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University, northern Thailand, from 2000 to 2006. Variable death scenes were determined, including forested area and suburban and urban outdoor and indoor environments. The fly specimens found in the corpses obtained were the most commonly of the blow fly of family Calliphoridae, and consisted of Chrysomya megacephala (F.), Chrysomya rufifacies (Macquart) Chrysomya villeneuvi Patton, Chrysomya nigripes Aubertin, Chrysomya bezziana Villeneuve, Chrysomya chani Kurahashi, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), and two unknown species. Flies of the family Muscidae [Hydrotaea spinigera Stein, Synthesiomyia nudiseta (Wulp)], Piophilidae [Piophila casei (L.)], Phoridae [Megaselia scalaris (Loew)], Sarcophagidae [Parasarcophaga ruficornis (F.) and three unknown species], and Stratiomyiidae (Sargus sp.) were also collected from these human remains. Larvae and adults of the beetle, Dermestes maculatus DeGeer (Coleoptera: Dermestidae), were also found in some cases. Chrysomya megacephala and C. rufifacies were the most common species found in the ecologically varied death scene habitats associated with both urban and forested areas, while C. nigripes was commonly discovered in forested places. S. nudiseta was collected only from corpses found in an indoor death scene.

Fine structure of the alimentary canal of the larval blow fly Chrysomya megacephala (Diptera: Calliphoridae).

Morphology of the alimentary canal of the mature third instar larva of the blow fly, Chrysomya megacephala (F.), was examined using light, scanning, and transmission electron microscopy. Salivary structures consist of a single median deferent duct that bifurcates into efferent ducts connected to paired, tubular salivary glands comprised of closely packed conical-shaped epithelial cells with large nuclei. The crop occurs as a large, swollen diverticulum of the digestive tube and is lined internally with convoluted cuticle (epicuticle and endocuticle). The esophagus is a simple, straight tube internally lined with cuticle and externally encompassed by muscle fibers. The cardia is a bulb-like structure composed of anterior foregut tissue and posterior midgut tissue from which the peritrophic membrane (PM) is produced. The midgut begins within the cardia which is flanked posteriorly by four tubular gastric caeca that are lined internally with four to five layers of cuboidal epithelial cells bearing microvilli. Midgut tissue is lined with simple cuboidal epithelium whose cells are filled with numerous secretory granules and possessed long microvilli facing the lumen. A peritrophic membrane is contained within the midgut lumen. The larval hindgut consists of the pylorus, Malpighian tubules, ileum, colon, rectum, and anus, posteriorly. The pylorus is characterized by a single layer of epithelial cells encircled by a muscular layer and the presence of PM within the lumen. Malpighian tubules each diverge into two tubular structures totalling four long tubules of long chained cuboidal cells bearing microvilli internally. The wall of the ileum is comprised primarily of a monolayer of cuboidal epithelial cells with large oval nuclei and more intense muscular fibers surrounding the periphery. A cuticular layer surrounds the lumen containing the PM. This inner cuticle consists of a thin epicuticle that is electron-dense; whereas, the endocuticle is much thicker but less electron-dense. Myo-epithelial cells are dense in the anal region, where the PM persists.

Ultrastructure of spiracles of Musca domestica and Hydrotaea chalcogaster (Diptera: Muscidae).

Spiracles are major respiratory openings in the exoskeleton of insects. Oxygen, a necessary gas for cell activity, must pass through the spiracle to enter the respiratory system. In this study, we investigated the fine structure of spiracles of adult females of Musca domestica L. and Hydrotaea chalcogaster (Wiedemann), both medically important fly species in many parts of the world, by utilizing scanning electron microscopy. The mesothoracic spiracle of M. domestica is large and elongate-oval in shape, with its anterior end being gradually tapered. The outer surface is densely covered with slender setae of variable distribution and orientation. The metathoracic spiracle is semicircular or D-shaped, with its rim possessing long, fine, inwardly curved setae. A net-like valve or sieve plate, which has a smooth rim with swollen surface, is located within the atrium of this species. The abdominal spiracles are circular with a symmetrically swollen peritreme surrounding the opening. The inner filtering apparatus is composed of many spiral tubes, each possessing many small spines. As for H. chalcogaster, the tapering mesothoracic spiracle is covered with long setae arranged consistently inward from the peritreme, giving it a "combed" appearance. The metathoracic spiracle is similarly arranged but triangularly rounded in shape, with the anterior and posterior rims possessing long fine setae. The net-like valve within the atrium has a smooth, swollen rim, whereas the inner edge of the atrium bears short, slender setae where it meets with the peritreme of the spiracle. The abdominal spiracles of this species look similar to that of M. domestica, with the exception of the filtering apparatus that bears only a few small spines. The function of these spiracles is discussed.

Morphological comparison between Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton (Diptera: Calliphoridae) puparia, forensically important blow flies.

In Thailand, the hairy maggots of the blow flies, Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton, are of forensic importance. Both flies are closely related species, not only in the morphological appearance of their larvae and puparia, but also on the aggressive feeding habit of the former. In our continuing studies of forensically important flies, identification of immature ones needs particular attention. In this study, we reported the morphological comparison between the puparia of these two blow fly species using scanning electron microscopy (SEM). Observation revealed that the cuticular sculpture of tubercles along the dorsal and lateral segments had markedly different features: with C. rufifacies having many sharp spines assembling only at the tip, while of C. villeneuvi bore stout spines throughout the tubercle. A larger number of globules at the bubble membrane on the dorsolateral border of the fifth segment was found in C. villeneuvi (average 225) than in C. rufifacies (average 35), and more papillae was observed on the anterior spiracle in C. villeneuvi (13-15) than in C. rufifacies (9-12). However, the morphology of distinct net-like patches of the integument and structure of the posterior spiracle of both species were almost identical. Morphological comparison in this study permitted identification of the puparia of both fly species, particularly in areas where they co-exist.

Morphology of puparia of Megaselia scalaris (Diptera: Phoridae), a fly species of medical and forensic importance.

Megaselia scalaris (Loew), a scuttle fly, is a fly species of medical and forensic importance. For use in forensic investigation, fly specimens found to associate with human corpses have to be identified at species level. Herein, we present the morphology of the puparia of the above fly species using scanning electron microscopy. The characteristic of the intersegmental spines along the dorsal and lateral segments and sculpture of the pupal respiratory horn of this puparia may be useful in future studies, to distinguish it from other closely related species.

Morphological observation of puparia of Chrysomya nigripes (Diptera: Calliphoridae) from human corpse.

This article presents Chrysomya nigripes Aubertin as a blow fly species of forensic importance in Thailand, and morphological observation of fly puparia using scanning electron microscopy (SEM). Morphologically, we focused on the characteristics of puparia used to accurately identify fly species. Numerous puparia of C. nigripes were found aggregated, adhering side by side, on the tibia of a skeletonized corpse, which was recovered from a forested area of Chiang Mai, northern Thailand. In the triangular shape of the anterior end of the puparia, three thoracic segments and broad hairy patches beginning dorsolaterally at the sixth segments were distinguishing characteristics. This study, showing pupariation of the flies along the bone of a corpse as well as morphological features, provides important guidance in identifying C. nigripes puparia. A key to differentiate puparia of C. nigripes from the other flies of forensic importance in Thailand is given.