In-vivo monitoring of infectious diseases in living animals using bioluminescence imaging.

Traditional methods of localizing and quantifying the presence of pathogenic microorganisms in living experimental animal models of infections have mostly relied on sacrificing the animals, dissociating the tissue and counting the number of colony forming units. However, the discovery of several varieties of the light producing enzyme, luciferase, and the genetic engineering of bacteria, fungi, parasites and mice to make them emit light, either after administration of the luciferase substrate, or in the case of the bacterial lux operon without any exogenous substrate, has provided a new alternative. Dedicated bioluminescence imaging (BLI) cameras can record the light emitted from living animals in real time allowing non-invasive, longitudinal monitoring of the anatomical location and growth of infectious microorganisms as measured by strength of the BLI signal. BLI technology has been used to follow bacterial infections in traumatic skin wounds and burns, osteomyelitis, infections in intestines, Mycobacterial infections, otitis media, lung infections, biofilm and endodontic infections and meningitis. Fungi that have been engineered to be bioluminescent have been used to study infections caused by yeasts (Candida) and by filamentous fungi. Parasitic infections caused by malaria, Leishmania, trypanosomes and toxoplasma have all been monitored by BLI. Viruses such as vaccinia, herpes simplex, hepatitis B and C and influenza, have been studied using BLI. This rapidly growing technology is expected to continue to provide much useful information, while drastically reducing the numbers of animals needed in experimental studies.

Rapid, label-free CD4 testing using a smartphone compatible device.

The most recent guidelines have called for a significant shift towards viral load testing for HIV/AIDS management in developing countries; however point-of-care (POC) CD4 testing still remains an important component of disease staging in multiple developing countries. Advancements in micro/nanotechnologies and consumer electronics have paved the way for mobile healthcare technologies and the development of POC smartphone-based diagnostic assays for disease detection and treatment monitoring. Here, we report a simple, rapid (30 minutes) smartphone-based microfluidic chip for automated CD4 testing using a small volume (30 µL) of whole blood. The smartphone-based device includes an inexpensive (<$5) cell phone accessory and a functionalized disposable microfluidic device. We evaluated the performance of the device using spiked PBS samples and HIV-infected and uninfected whole blood, and compared the microfluidic chip results with the manual analysis and flow cytometry results. Through t-tests, Bland-Altman analyses, and regression tests, we have shown a good agreement between the smartphone-based test and the manual and FACS analysis for CD4 count. The presented technology could have a significant impact on HIV management in developing countries through providing a reliable and inexpensive POC CD4 testing.

An automated smartphone-based diagnostic assay for point-of-care semen analysis.

Male infertility affects up to 12% of the world's male population and is linked to various environmental and medical conditions. Manual microscope-based testing and computer-assisted semen analysis (CASA) are the current standard methods to diagnose male infertility; however, these methods are labor-intensive, expensive, and laboratory-based. Cultural and socially dominated stigma against male infertility testing hinders a large number of men from getting tested for infertility, especially in resource-limited African countries. We describe the development and clinical testing of an automated smartphone-based semen analyzer designed for quantitative measurement of sperm concentration and motility for point-of-care male infertility screening. Using a total of 350 clinical semen specimens at a fertility clinic, we have shown that our assay can analyze an unwashed, unprocessed liquefied semen sample with <5-s mean processing time and provide the user a semen quality evaluation based on the World Health Organization (WHO) guidelines with ~98% accuracy. The work suggests that the integration of microfluidics, optical sensing accessories, and advances in consumer electronics, particularly smartphone capabilities, can make remote semen quality testing accessible to people in both developed and developing countries who have access to smartphones.

Printed Flexible Plastic Microchip for Viral Load Measurement through Quantitative Detection of Viruses in Plasma and Saliva.

We report a biosensing platform for viral load measurement through electrical sensing of viruses on a flexible plastic microchip with printed electrodes. Point-of-care (POC) viral load measurement is of paramount importance with significant impact on a broad range of applications, including infectious disease diagnostics and treatment monitoring specifically in resource-constrained settings. Here, we present a broadly applicable and inexpensive biosensing technology for accurate quantification of bioagents, including viruses in biological samples, such as plasma and artificial saliva, at clinically relevant concentrations. Our microchip fabrication is simple and mass-producible as we print microelectrodes on flexible plastic substrates using conductive inks. We evaluated the microchip technology by detecting and quantifying multiple Human Immunodeficiency Virus (HIV) subtypes (A, B, C, D, E, G, and panel), Epstein-Barr Virus (EBV), and Kaposi's Sarcoma-associated Herpes Virus (KSHV) in a fingerprick volume (50 µL) of PBS, plasma, and artificial saliva samples for a broad range of virus concentrations between 10(2) copies/mL and 10(7) copies/mL. We have also evaluated the microchip platform with discarded, de-identified HIV-infected patient samples by comparing our microchip viral load measurement results with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) as the gold standard method using Bland-Altman Analysis.

Microfluidics for sperm research.

One in six couples of reproductive age worldwide are affected at least once by some form of infertility. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are widely-available assisted reproductive technologies (ART). The identification and isolation of the most-motile sperm with DNA integrity are essential to IVF and ICSI, ultimately affecting treatment consequences and the health of offspring. Recently, microfluidic technologies been developed to sort sperm according to sperm morphology, motility, DNA integrity, and functionality for IVF techniques. There have also been emerging applications in wildlife conservation, high-throughput single-sperm genomics, sperm-driven robotics, and in-home fertility testing. We review a broad range of studies applying the principles of microfluidics to sperm research.

Potentiation of photoinactivation of Gram-positive and Gram-negative bacteria mediated by six phenothiazinium dyes by addition of azide ion.

Antimicrobial photodynamic inactivation (APDI) using phenothiazinium dyes is mediated by reactive oxygen species consisting of a combination of singlet oxygen (quenched by azide), hydroxyl radicals and other reactive oxygen species. We recently showed that addition of sodium azide paradoxically potentiated APDI of Gram-positive and Gram-negative bacteria using methylene blue as the photosensitizer, and this was due to electron transfer to the dye triplet state from azide anion, producing azidyl radical. Here we compare this effect using six different homologous phenothiazinium dyes: methylene blue, toluidine blue O, new methylene blue, dimethylmethylene blue, azure A, and azure B. We found both significant potentiation (up to 2 logs) and also significant inhibition (>3 logs) of killing by adding 10 mM azide depending on Gram classification, washing the dye from the cells, and dye structure. Killing of E. coli was potentiated with all 6 dyes after a wash, while S. aureus killing was only potentiated by MB and TBO with a wash and DMMB with no wash. More lipophilic dyes (higher log P value, such as DMMB) were more likely to show potentiation. We conclude that the Type I photochemical mechanism (potentiation with azide) likely depends on the microenvironment, i.e. higher binding of dye to bacteria. Bacterial dye-binding is thought to be higher with Gram-negative compared to Gram-positive bacteria, when unbound dye has been washed away, and with more lipophilic dyes.

Physical energy for drug delivery; poration, concentration and activation.

Techniques for controlling the rate and duration of drug delivery, while targeting specific locations of the body for treatment, to deliver the cargo (drugs or DNA) to particular parts of the body by what are becoming called "smart drug carriers" have gained increased attention during recent years. Using such smart carriers, researchers have also been investigating a number of physical energy forces including: magnetic fields, ultrasound, electric fields, temperature gradients, photoactivation or photorelease mechanisms, and mechanical forces to enhance drug delivery within the targeted cells or tissues and also to activate the drugs using a similar or a different type of external trigger. This review aims to cover a number of such physical energy modalities. Various advanced techniques such as magnetoporation, electroporation, iontophoresis, sonoporation/mechnoporation, phonophoresis, optoporation and thermoporation will be covered in the review. Special emphasis will be placed on photodynamic therapy owing to the experience of the authors' laboratory in this area, but other types of drug cargo and DNA vectors will also be covered. Photothermal therapy and theranostics will also be discussed.

Self-assembled liposomal nanoparticles in photodynamic therapy.

Photodynamic therapy (PDT) employs the combination of non-toxic photosensitizers (PS) together with harmless visible light of the appropriate wavelength to produce reactive oxygen species that kill unwanted cells. Because many PS are hydrophobic molecules prone to aggregation, numerous drug delivery vehicles have been tested to solubilize these molecules, render them biocompatible and enhance the ease of administration after intravenous injection. The recent rise in nanotechnology has markedly expanded the range of these nanoparticulate delivery vehicles beyond the well-established liposomes and micelles. Self-assembled nanoparticles are formed by judicious choice of monomer building blocks that spontaneously form a well-oriented 3-dimensional structure that incorporates the PS when subjected to the appropriate conditions. This self-assembly process is governed by a subtle interplay of forces on the molecular level. This review will cover the state of the art in the preparation and use of self-assembled liposomal nanoparticles within the context of PDT.

Low-level laser (light) therapy (LLLT) in skin: stimulating, healing, restoring.

Low-level laser (light) therapy (LLLT) is a fast-growing technology used to treat a multitude of conditions that require stimulation of healing, relief of pain and inflammation, and restoration of function. Although skin is naturally exposed to light more than any other organ, it still responds well to red and near-infrared wavelengths. The photons are absorbed by mitochondrial chromophores in skin cells. Consequently, electron transport, adenosine triphosphate nitric oxide release, blood flow, reactive oxygen species increase, and diverse signaling pathways are activated. Stem cells can be activated, allowing increased tissue repair and healing. In dermatology, LLLT has beneficial effects on wrinkles, acne scars, hypertrophic scars, and healing of burns. LLLT can reduce UV damage both as a treatment and as a prophylactic measure. In pigmentary disorders such as vitiligo, LLLT can increase pigmentation by stimulating melanocyte proliferation and reduce depigmentation by inhibiting autoimmunity. Inflammatory diseases such as psoriasis and acne can also be managed. The noninvasive nature and almost complete absence of side effects encourage further testing in dermatology.

Thiocyanate potentiates antimicrobial photodynamic therapy: in situ generation of the sulfur trioxide radical anion by singlet oxygen.

Antimicrobial photodynamic therapy (PDT) is used for the eradication of pathogenic microbial cells and involves the light excitation of dyes in the presence of O2, yielding reactive oxygen species including the hydroxyl radical (OH) and singlet oxygen ((1)O2). In order to chemically enhance PDT by the formation of longer-lived radical species, we asked whether thiocyanate (SCN(-)) could potentiate the methylene blue (MB) and light-mediated killing of the gram-positive Staphylococcus aureus and the gram-negative Escherichia coli. SCN(-) enhanced PDT (10 µM MB, 5 J/cm(2) 660 nm hv) killing in a concentration-dependent manner of S. aureus by 2.5 log10 to a maximum of 4.2 log10 at 10mM (P<0.001) and increased killing of E. coli by 3.6 log10 to a maximum of 5.0 log10 at 10mM (P<0.01). We determined that SCN(-) rapidly depleted O2 from an irradiated MB system, reacting exclusively with (1)O2, without quenching the MB excited triplet state. SCN(-) reacted with (1)O2, producing a sulfur trioxide radical anion (a sulfur-centered radical demonstrated by EPR spin trapping). We found that MB-PDT of SCN(-) in solution produced both sulfite and cyanide anions, and that addition of each of these salts separately enhanced MB-PDT killing of bacteria. We were unable to detect EPR signals of OH, which, together with kinetic data, strongly suggests that MB, known to produce OH and (1)O2, may, under the conditions used, preferentially form (1)O2.

Photodynamic inactivation of biofilm: taking a lightly colored approach to stubborn infection.

Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents.

Antimicrobial strategies centered around reactive oxygen species--bactericidal antibiotics, photodynamic therapy, and beyond.

Reactive oxygen species (ROS) can attack a diverse range of targets to exert antimicrobial activity, which accounts for their versatility in mediating host defense against a broad range of pathogens. Most ROS are formed by the partial reduction in molecular oxygen. Four major ROS are recognized comprising superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen ((1)O2), but they display very different kinetics and levels of activity. The effects of O2•- and H2O2 are less acute than those of •OH and (1)O2, because the former are much less reactive and can be detoxified by endogenous antioxidants (both enzymatic and nonenzymatic) that are induced by oxidative stress. In contrast, no enzyme can detoxify •OH or (1)O2, making them extremely toxic and acutely lethal. The present review will highlight the various methods of ROS formation and their mechanism of action. Antioxidant defenses against ROS in microbial cells and the use of ROS by antimicrobial host defense systems are covered. Antimicrobial approaches primarily utilizing ROS comprise both bactericidal antibiotics and nonpharmacological methods such as photodynamic therapy, titanium dioxide photocatalysis, cold plasma, and medicinal honey. A brief final section covers reactive nitrogen species and related therapeutics, such as acidified nitrite and nitric oxide-releasing nanoparticles.

Low-level laser therapy for fat layer reduction: a comprehensive review.

BACKGROUND AND OBJECTIVE: Low-level laser (light) therapy (LLLT) is a noninvasive, nonthermal approach to disorders requiring reduction of pain and inflammation and stimulation of healing and tissue regeneration. Within the last decade, LLLT started being investigated as an adjuvant to liposuction, for noninvasive body contouring, reduction of cellulite, and improvement of blood lipid profile. LLLT may also aid autologous fat transfer procedures by enhancing the viability of adipocytes. However the underlying mechanism of actions for such effects still seems to be unclear. It is important, therefore, to understand the potential efficacy and proposed mechanism of actions of this new procedure for fat reduction. MATERIALS AND METHODS: A review of the literature associated with applications of LLLT related to fat layer reduction was performed to evaluate the findings from pre-clinical and clinical studies with respect to the mechanism of action, efficacy, and safety. RESULTS: The studies as of today suggest that LLLT has a potential to be used in fat and cellulite reduction as well as in improvement of blood lipid profile without any significant side effects. One of the main proposed mechanism of actions is based upon production of transient pores in adipocytes, allowing lipids to leak out. Another is through activation of the complement cascade which could cause induction of adipocyte apoptosis and subsequent release of lipids. CONCLUSION: Although the present studies have demonstrated safety and efficacy of LLLT in fat layer reduction, studies demonstrating the efficacy of LLLT as a stand-alone procedure are still inadequate. Moreover, further studies are necessary to identify the mechanism of action.

Animal models of skin disease for drug discovery.

INTRODUCTION: Discovery of novel drugs, treatments, and testing of consumer products in the field of dermatology is a multi-billion dollar business. Due to the distressing nature of many dermatological diseases, and the enormous consumer demand for products to reverse the effects of skin photodamage, aging, and hair loss, this is a very active field. AREAS COVERED: In this paper, we will cover the use of animal models that have been reported to recapitulate to a greater or lesser extent the features of human dermatological disease. There has been a remarkable increase in the number and variety of transgenic mouse models in recent years, and the basic strategy for constructing them is outlined. EXPERT OPINION: Inflammatory and autoimmune skin diseases are all represented by a range of mouse models both transgenic and normal. Skin cancer is mainly studied in mice and fish. Wound healing is studied in a wider range of animal species, and skin infections such as acne and leprosy also have been studied in animal models. Moving to the more consumer-oriented area of dermatology, there are models for studying the harmful effect of sunlight on the skin, and testing of sunscreens, and several different animal models of hair loss or alopecia.

Shining light on nanotechnology to help repair and regeneration.

Phototherapy can be used in two completely different but complementary therapeutic applications. While low level laser (or light) therapy (LLLT) uses red or near-infrared light alone to reduce inflammation, pain and stimulate tissue repair and regeneration, photodynamic therapy (PDT) uses the combination of light plus non-toxic dyes (called photosensitizers) to produce reactive oxygen species that can kill infectious microorganisms and cancer cells or destroy unwanted tissue (neo-vascularization in the choroid, atherosclerotic plaques in the arteries). The recent development of nanotechnology applied to medicine (nanomedicine) has opened a new front of advancement in the field of phototherapy and has provided hope for the development of nanoscale drug delivery platforms for effective killing of pathological cells and to promote repair and regeneration. Despite the well-known beneficial effects of phototherapy and nanomaterials in producing the killing of unwanted cells and promoting repair and regeneration, there are few reports that combine all three elements i.e. phototherapy, nanotechnology and, tissue repair and regeneration. However, these areas in all possible binary combinations have been addressed by many workers. The present review aims at highlighting the combined multi-model applications of phototherapy, nanotechnology and, reparative and regeneration medicine and outlines current strategies, future applications and limitations of nanoscale-assisted phototherapy for the management of cancers, microbial infections and other diseases, and to promote tissue repair and regeneration.