Advancement Towards Microfluidic Approach to Develop Economical Disposable Optical Biosensor for Lead Detection

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Aneconomical single use optical biosensor has been developed for lead detection using a microfluidic approach. The present study represents the initiative step for amalgamation of microfluidic and advanced biosensor technologies which offers rapid analysis with lab-on-a-chip policy. Urease producing Bacillus sphaericus was co-immobilized with phenol red (pH indicator) in the glass capillary which acted as a microchannel. For immobilization, a combination of sol-gel approach and calcium alginate method cited first time in literature was used which reduced the time of solidification to seconds as compared to hours with sol-gel alone. Bioassay principle was based on urease inhibition in the presence of lead. Fiber optic spectrophotometer was used as transducer which measured the intensity of color change. Linear relationship (10-1000 μg/L) was observed between logarithmic concentration of lead and absorbance. The study resulted in the development of cheap, miniaturized, sensitive and reliable lead biosensor with requirement of small sample volume (1 mL).

Increasedenvironmental awareness and stringent environmental regulation led to the dire need of the techniques for the fast, easy and economic detection of various pollutants like heavy metals, pesticides and toxic gases. Heavy metals are among the most hazardous pollutants due to their ubiquitous presence in the biosphere, their bioavailability from both natural and anthropogenic sources as well as their high toxicity even at trace level. Lead is among the toxic heavy metals which harm the body when present above the threshold concentration. The higher concentration of lead (>18 μM) in the blood may cause coma and death. It affects different parts of the body particularly brain and central nervous system. Accumulation of lead in the body produces damaging effects associated with hematology, neurology and nephrology which include paralysis, mental retardation and neural deafness. The substitution of calcium in the body with lead causes impairment in the development of bones and teeth. Human exposure to lead occurs primarily through leadbased paints, industrial waste, water from lead-laden pipes, soil and dust generated from gasoline and food items like milk, dairy products and imported candies. The cattle grazing on the metal contaminated fields transmit lead contamination in the milk and dairy products. A threshold lead limit of 10 μg/L was estimated in food and water by the International Agency for Research on Cancer (IARC) while 5 μg/L in drinking water by Environmental Protection Agency (EPA). It is mandatory, therefore, to detect the lead in food samples and drinking water to prevent the deleterious effects of lead. Conventional methods to detect heavy metals like differential pulse polarography, Atomic Absorption Spectrophotometry (AAS), Differential Pulse Cathodic Stripping Voltametry (DPCSV) are superseded by the use of biosensors.

Biosensorsare analytical tools capable of providing either qualitative or quantitative results, consisting of an immobilized biological recognition element such as an enzyme, antibody or cell receptor immobilized to a physicochemical transducer to create a single unit. Now-a-days biosensor technology has emerged as the most promising tool for detection of heavy metals being costeffective, fast, selective, sensitive, portable, easy to use and reliable. A number of lead biosensors have been developed using various transducers like conductometer, electrochemical and optical  based on whole cell, enzymes or DNAzyme.

Diversity-Oriented Synthesis of a Library of Podocarpic Acid Derivatives

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Podocarpic acid 1 (Figure 1) was first isolated from the resin of Podocarpus cupressins, an important timber tree which is endemic to Java, and later from Podocarpus dacrydioides (“Kahikatea”) and Dacrydium cupressinum (“Rimu”), trees which are found in the timber regions of New Zealand. Since 1968, more than forty oxygenated metabolites of podocarpic acid have been isolated from various species of Podocarpus.Interest in these naturally occurring and synthetic lactones, podolactones, and related podocarpic acid derivatives has been mainly due to the novel structures of these compounds and the various types of biological activity possessed by them.

Octahydrophenanthrene lactones 2 (Figure 1) and related podocarpic acid derivatives 3 have been reported to possess hormonal and anti-inflammatory properties. Other similar podolactones have been shown to inhibit the expansion and division of plant cells, to have antileukemic activity, to have antibacterial activity, to have insect toxicity properties, and to exhibit antitumor activity.Other reports have indicated that these types of compounds, as a class, possess significant antifungal properties. The lactone 6 (Figure1), first isolated as a mold metabolite, was found to have significant activity against a number of fungi. The momilactones A 7 and B 8 have been shown to be fungitoxic towards Cladosporium cucumerinum. In a recent report several oxidized resin acid derivatives of dehydroabietic acid 9 (a-c) and 13-hydroxypodocarpic acid 9 (d) were found to be highly fungistatic against Dothistroma pini, a conifer pathogenic fungi. It was observed that mature trees were more resistant to fungal infection and contained a greater quantity of oxidized resin acid derivatives in their resin suggesting greater resistance.

More recently, some podocarpic acid analogs have been reported as cytokine release inhibitors and this has consequently led to the discovery of novel anti-inflammatory drugs. Other compounds, including a podocarpic acid anhydride, have been shown to be liver.X receptor α and β agonists. These two nuclear oxysterol receptors in the liver are involved in cholesterol and lipid metabolism. Thus, any of their agonists could be useful in the development of drugs for the treatment of atherosclerosis.In view of their documented biological properties, it appeared worthwhile to prepare a series of synthetic intermediates derived from podocarpic acid for potential biological activity. This report describes the preparation and characterization of these derivatives.Commercial podocarpic acid is derived from natural sources. Several recent studies have been directed towards the total synthesis of this resin acid to assure adequate future supplies of this material for use in agriculture and medicine.The goal of the present study was to prepare a series of derivatives related to podocarpic acid for use in structure/activity studies designed to reveal functional groups responsible for the molecules fungistatic properties. Four specific modifications were planned.

Enhancing Cancer Radiation Therapy with Cell Penetrating Peptide Modified Gold Nanoparticles

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Radiotherapyis one of the most prevalent methods for cancer treatment. However, a challenge for cancer radiotherapy is that therapeutic doses used can damage neighboring normal cells. This paper describes a new method to enhance radiation therapy by delivering gold nanoparticles into cancer cells, where gold nanoparticles were modified with virus-derived cell penetrating peptides (CPPs) and Poly (Ethylene Glycol) (PEG). PEG was used to improve nanoparticles blood circulation time, and CPPs were used to enhance internalization of the nanoparticles into cells. The internalization of CPP-PEG modified gold nanoparticles in cancer cells (HeLa cells) was confirmed with differential interference contrast imaging. A variety of assays (such as bright field imaging, MTT, DNA damage, reactive oxygen species and immunofluorescence) were used to detect cellular and genetic damage in cancer cells. We found that CPP-PEG modified gold nanoparticles caused more cellular and DNA damage than gold nanoparticles at the same radiation doses due to enhanced generation of free radicals. In contrast, damage was not severe for normal fibroblasts cells under the same conditions. This method can potentially be used to severely damage DNA and other cellular structures of cancer cells, while minimizing damage to normal cells during radiation therapy.

Currently, chemotherapy, surgery and radiotherapy are the most effective methods to treat cancer. Radiotherapy targets and destroys tumor with ionizing radiation. The laser generates free radicals that damage various cellular components including DNA. One of the advantages of using radiotherapy is that it can kill tumor even though they are intermix with normal healthy tissue. Hence, more than 50% of cancer patients received radiotherapy treatment. However, the therapeutic doses used during radiotherapy can damage nearby normal cells. Various chemicals and nanoparticles were tested to act as radiosensitlzers to enhance radiotherapy.

Despite its essential role in maintaining cell function, cell membranes present a major barrier for intra-cellular delivery of therapeutic nanoparticles. Hence, even though ions or nanoparticles of high atomic number elements (such as gold, platinum and bismuth) have been used to enhance radiation therapy by absorbing ionizing radiation and generating free radicals at high yield, the measured enhancement effect due to nanoparticles has been negligible, likely because inefficient nanoparticles were present in cancer cells and X-ray generated free radicals cannot reach the vicinity of DNA to cause damage 

Nanoparticles can be modified to have desirable surface properties to allow for uptake and targeted delivery into cells and subcellular locations. Non-viral vectors such as amino-modified silica nanoparticles, iron oxide nanoparticles, carbon nanotubes and gold nanoparticles have been used to deliver nucleic acids in transfection assays. In particular, gold (Au) nanoparticles are stable, non-toxic and easy for surface modification, making them a suitable candidate to deliver molecules into cells. However, to reach their full potential in cellular applications such as radiotherapy, robust methods must be developed to allow for the controlled uptake of gold nanoparticles into cells. This requires the gold nanoparticles to be functionalized with engineered coatings to promote their cellular uptake and targeted delivery.

Poly ethylene glycol (PEG) was used to coat nanoparticles to improve their blood circulation. However, PEG interactions with cell surface ligands prevent nanoparticles intra-cellular uptake. One solution to use cell penetrating peptides (CPPs). CPPs are relatively short cationic and/or amphipathic peptides and are efficient cellular delivery vectors due to their intrinsic ability to enter cells and mediate uptake of a wide range of macromolecular cargo. The various molecular cargo delivered by CPPs ranges from nanosize particles to small chemical molecules and large fragments of DNA. The “cargo” is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions [26]. The function of the CPPs is to deliver the cargo into cells, a process that commonly occurs through endocytosis with the cargo delivered to the endosomes of living mammalian cells.

Proposing Asian Principles of Bioethics from Asian Perspectives

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Much discussion and debate have taken place after the publication of Principles of Biomedical Ethics, co-authored by Georgetown University’s bioethicists, Beauchamp and Childress who implied that the four principles they promoted reflect not only the common concerns of the world but also are trans-cultural in nature. In other words, these four principles, based on common morality theory, can be universally valid.

At first glance, one cannot but agree with this argument. But on a closer examination, one will notice that these four principles are more western than eastern, let alone universally functional despite the fact that Asian religious and philosophical thought share similar concerns.

Biomedical ethics have been guided by a few broad principles, at least from the perspective of principle-ism which, though questioned by some has gradually been accepted by most bioethicists. Some biomedical ethicists list seven, veracity, autonomy, beneficence, nonmaleficence, fidelity, confidentiality and justice, but Beauchamp and Childress listed only four and see the other three as derivatives. Beauchamp and Childress have argued that these principles are trans-cultural in nature, yet Asians have different understandings of their implications. Confucianism, one of the dominate thinking’s in Asia will be selected as an example to illustrate the similarities and differences in the interpretation of these principles.

no harm” hence became the healthprofessionals’ obligation. If a physician cannot benefit someone, at least he/she should do no harm to them. Beneficence is the positive dimension of nonmaleficence. This principle claims that physicians have the duty to help others further their interests. It refers to a positive duty of promoting the health and welfare of patients above all other considerations.

These principles of “do good and do no harm” reflect Confucian teachings. “Do good” is the positive aspect of Jen, the foundation of Confucian ethics. “Do to others what you wish for yourself” is called by Confucius as Chung, or conscientiousness to others. “Do no harm” is the negative aspect -- “do not do to other what you do not wish yourself” which is called Shu, the way to practice Jen.

Furthermore, nonmaleficence and beneficence are understood not only as duties but also as inborn natures of man. Mencius, the second sage in Confucianism, believes that all men are endowed with a concern toward others. In his word: “All men have the mind which cannot bear to see the suffering of others”. He further explained: “When I say that all men have the mind which cannot bear to see the suffering of others, my meaning may be illustrated thus: now when men suddenly see a child about to fall into a well, they all have a feeling of alarm and distress, not to gain friendship with the child’s parents, nor to seek the praise of their neighbors and friends, nor because they dislike the reputation of lack of humanity if they did not rescue the child”. From such a case, we see that beneficence and nonmaleficience are expressions of the feeling of commiseration which Mencius asserted as the beginning of humanity. From this, we see that Confucian thought regards beneficence and malificence as more than principles to be promoted. They are part of human nature.

Asthma and Cesarean Section

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Asthma is chronic disease ofmultifactorial etiologic and exposure perinatal factors can determine the development of airway inflammation. High prevalence of asthma in developing countries accompanies the increase in the Cesarean Section (CS) rate in the same period. Cesarean births in some countries of Latin America reach the 50% prevalence, in the Brazilian, public and private health services the rate CS is considerate highest in the world. However, the association between CS and asthma is still a controversial issue with conflicting data in the literature due to potential confounding of several other determinants of asthma.

The mechanism to explain its association between CS and asthma refers to the hygiene hypothesis. Decrease exposure to certain types of microorganisms early in life would lead to insufficient stimulation of T1 lymphocytes and consequences predominance of allergic response of the type T2, responsible for the development of asthma and allergic reactions. In children born CS the initial colonization of intestinal micro flora is composed by bacteria from colonization of the skin and those related to the hospital environment and these bacteria are capable of modulating the type of immune response to the type T2.

The EPIC hypothesis (EpigeneticImpact of Childbirth) has also been proposed for explain the association between CS and asthma; this suggests that the genome of fetus undergoes remodelling in chromantin architecture and DNA methylation during the intrapartum period and cause susceptibility to diseases. Children born CS have higher DNA methylation than children born of vaginal delivery. The third hypothesis to explain the association between CS and asthma is the lowest rate of onset and duration of exclusive breastfeeding in children born cesarean, the maternal milk would have a protective effect for asthma due to the immunomodulators, low proportion of IL10 and more of IL13 and gamma interferon.

Studies that assessed the association between CS and asthma presented different results. Association between asthma and CS was reported by Roduit, et al. [10], in a prospective cohort study of 2.917 children, found a significant association between delivery by CS and asthma OR = 1.77 CI 95% (1.03-1.32) and the risk was increased in the presence of parental history of atopy. Almqvist, et al. evaluated a retrospective cohort of 87,500 cesarean births and risk of asthma in children and adolescents CS was associated with the use of asthma.

drugs OR: 1.13; 95% CI (1.04-1.24) and diagnosis of asthma OR: 1.20 95% CI (1.05-1.37), adjusted by family history of atopy, weight at birth, gestational age, gender, Apgar score and age maternal. Tollanes, et al. found a high risk for asthma in children born of emergency CS with OR: 1.42; CI 95 (1.12-1.62) and elective CS with OR 1.59; 95% CI (1.44-1.55) being the highest risk for asthma in preterm infants. Kero, et al. evaluated records of 59,927 children and found association between CS and asthma with OR: 1.21; CI 95% (1.08-1.36) and atopic asthma OR: 2.2; 95% CI (1.06-4.64), adjusted for maternal age, gender and born. Salam et al. Evaluated a cohort of 3,464 children born ≥37 weeks of gestation and birth weight ≥2,500 kg and a structured asthma questionnaire was applied. CS represented risk factor for asthma OR: 1.33; CI 95% (1.01-1.75). Bager, et al. evaluated 9,722 women from a cohort in the Denmark for type of delivery, gestational age, weight and height at birth, asthma and rhinitis obtained by the local registry system and CS associated with asthma OR: 1.33;CI 95% (1.2-1.74) but not allergic rhinitis OR: 1.16; 95% CI (0.90-1.49). Gessner and Chimonas 23 evaluated 37,349 children aged 5-9 years of a retrospective birth cohort. Asthma was confirmed by the Standard International Classification of Diseases 9^th Revision codes. Asthma was associated with preterm birth but not with small for gestational age status. CS represented risk factor for asthma or hospitalizations for asthma OR: 1.4; 95% CI (1.08-2.2).

In a prospective cohort study of 12,367 children Maitra, et al. Found no association between delivery by CS and asthma (OR= 1.14; 95% CI 0.9–1.4). Brandão, et al. evaluated 672 children with asthma and allergic rhinitis found no association between CS and asthma however there was association between CS and allergic rhinitis, adjusted for the main confounding variables for asthma. Mallen, et al. using population-based registers evaluated conditions of birth and asthma, allergic rhinitis, eczema and hay fever in 567 adults. CS was not associated with asthma OR: 1.71; 95% CI (0.76-6.84), rhinitis, eczema or hay fever. Vonk, et al. evaluated 597 adults using birth data on labour for delivery type and diagnosis for asthma and atopy after 20-year follow-up, CS was not risk factor for asthma OR: 1.77; 95% CI (0.98-3.51). Bernsen, et al. evaluated 1,797 children in a cohort through birth data records on type of birth and asthma, eczema and allergy at six years of age. CS was not a risk for asthma OR: 1.03; 95% CI (0.51-2.08). Nathan, et al. evaluated 156 children in a control case study at the age of 3-15 years. Seventy-eight children with diagnosis of asthma and seventy-eight controls were evaluated for CS. There was no association between CS and asthma OR: 1.21; 95% CI (0.6-2.41). Rusconi, et al. performed a cross-sectional study in 15,609 children diagnosed with asthma less than five years old of age. CS was associated with asthma and atopy. The association was of greater magnitude in children of no atopic parent. The perinatal variables, gestational age and low birth weight had low frequency in children of the CS.

Thimble - Type Prostheses: A Successful Option for Distal Finger Loss

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Finger and partial finger amputations are frequently encountered problems leading to partial hand loss, which occur commonly due to trauma, congenital absence or malformations. All of these may present similar clinical challenges which can vary from loss of fingertip to complete finger amputation. Frequently, the loss of even the tip of a digit can be so emotionally disturbing to the patient that it deserves serious attention. Creating a life like prosthesis with characteristics such as pleasing shape, thin margins, lifelike fingernails, and realistic color, contours and details are essential for patient satisfaction, but to maintain all these qualities it needs good suspension. Recording a suitable impression of the residual supporting structure is primary requirement for obtaining better fit of the prosthesis.

A 24-year-old female patient came to the department with the chief complain of loss of upper phalange of the middle finger of left hand due to childhood injury (Figure 1). The patient was more esthetically and socially concerned with no functional problem reported as she was well adapted with time. Remnant of the nail was seen on the tip of amputated part. The distal amputation level was sub zone II as classified by Ishikawa et al. The patient was advised to cut her nail at timely interval, so that it may not affect the fitting of the prosthesis. A plastic cylindrical small open container (Impression Cap) was used for making silicone impression of the defected finger (Figure 2). Putty and light body addition silicone impression material (Express, 3M ESPE; St. Paul, MN) was used and after that cast was poured. The wax pattern was made using alginate impression (Zelgan, Dentsply India Pvt. Ltd.) of the same fingers of right hand.

was poured in alginate impression and after cooling of wax, minor modification of the pattern was done to simulate it according to the fingers of left hand. Wax pattern was modified and terminated till next joint of the missing finger as margins were least visible at joint area. This pattern closely resembles the shape and size of the missing finger and after hollowing with the hot wax spatula this pattern was properly fitted over the cast (Figure 3). After taking final opinion of the patient pattern was flasked for curing with silicone material (bredent Multisil-Epithetic set, Senden, Germany). Care was taken to avoid undercuts during counter- flasking. The pattern was flasked in such a way that dorsal and the ventral aspects of the finger were separable to enhance the accuracy at the stage of shade matching. Suitable quantity of Multisil-Epithetic transparent was first poured onto a mixing pad. Intensive stains were mixed into the epithetic transparent and direct color comparison of the palmer and dorsal surface of the hand were carried out separately in natural day light. After all stains had been mixed, thickener was used to increase viscosity of silicon material to avoid running of the different stains while adding them into the flask. For the characterization of fingernail, palmer surface shade was used to simulate natural nails. After curing the final prosthesis was retrieved, the flash was trimmed using a sharp blade and the final finishing was completed using fine sand paper. Patient was well satisfied with its retention and esthetic appearance (Figure 4). Proper instructions regarding the follow- ups and handling of the prosthesis were given.

Erectile Dysfunction as an Early Marker of Microangiopathic Complications in Type 2 Diabetes Mellitus

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Erectiledysfunction (ED), a very frequent finding among type 2 diabetes patients (T2DM), is associated with cardiovascular disease. To investigate the prevalence of ED among our T2DM population and its association with microangiopathic complications (diabetic retinopathy (DR) and microalbuminuria [mAlb]), we performed a retrospective cross-sectional study involving 121 patients attending the Diabetology Unit of Padua Hospital. All subjects were studied with accurate anamnesis, IIEF-5 questionnaire, microalbuminuria determined in spot urine sample, fundus examination and carotid artery echo-color-doppler. ED prevalence was 64.8% while DR and mAlb prevalence was 25.6% and 23.1% respectively. In ED group vs. non-ED, DR prevalence was 32.9% vs. 11.9% (p=0.012) and mAlb prevalence was 26.6% vs. 16.7% (p=0.218). ED group had a worse glycemic control (HbA1c 7.6 ± 1.6 vs. 7.0 ± 1.0 %, p=0.010) and a longer T2DM duration (10.3 ± 9.2 vs. 6.0 ± 5.7 years, p=0.002). Furthermore, ED was associated with a higher carotid intima-media thickness (IMT 0.9 ± 0.2 vs. 0.8 ± 0.2 mm, p=0.049). ED was the first vascular complication in 57% of patients, occurring some years before DR and mAlb. Association with DR and mAlb is independent of common cardiovascular risk factors. In conclusion, ED onset in diabetic subjects is a very important finding that can be considered an early microangiopathic marker in T2DM subjects, suggesting the evaluation for the presence of other microangiopathic complications and a more intense control of cardiovascular risk factors.

ED: Erectile Dysfunction; T2DM: Type 2 Diabetes Mellitus; DR: Diabetic Retinopathy; Malb: Microalbuminuria; DN: Diabetic Neuropathy; IIEF-5: International Index Of Erectile Function – 5; MI: Myocardial Infarction; CVD: Cardiovascular Disease; CHD: Coronary Heart Disease; Hba1c: Glycated Hemoglobin; LH: Luteinizing Hormone; FSH: Follicle-Stimulating Hormone; E2: Estradiol; PSA: Prostatic-Specific Antigen; Egfr: Estimated Glomerular Filtration Rate; LDL: Low-Density Lipoprotein; HDL: High-Density Lipoprotein; IMT: Intima-Media Thickness; BMI: Body Mass Index; NO: Nitric Oxide.

Type 2 diabetes mellitus (T2DM) is not merely a disorder of carbohydrate metabolism, but a cause of vascular diseases affecting nearly all arterial vessels which are classically divided in microangiopathic and microangiopathic. The link between diabetes and macroangiopathic disease was suggested many years ago, observing a higher risk of myocardial infarction (MI) and cardiovascular death in several diabetic populations. In Italy, diabetic patients have a cardiovascular mortality excess of about 30-40% vs. non diabetic individuals. Microangiopathic disease is characterized by three major manifestations: diabetic retinopathy (DR), diabetic neuropathy (DN) and diabetic nephropathy. Around 30% of diabetic patients suffer from DR, ranging from mild to severe. Male sex, higher glycated haemoglobin levels, longer duration of diabetes mellitus, higher blood pressure values and use of insulin are all associated with the development of retinopathy. Diabetic nephropathy in T2DM occurs in 20-40% of patients and microalbuminuria (mAlb) is a marker of early nephropathy.