Ayman Grada

Next-Generation Sequencing: Methodology and Application

inTrOducTiOn Nucleic acid sequencing is a method for determining the exact order of nucleotides present in a given DNA or RNA molecule. In the past decade, the use of nucleic acid sequencing has increased exponentially as the ability to sequence has become accessible to research and clinical labs all over the world. The first major foray into DNA sequencing was the Human Genome Project, a

Lymphedema: Diagnostic workup and management

3 billion, 13-year-long endeavor, completed in 2003. The Human Genome Project was accomplished with first-generation sequencing, known as Sanger sequencing. Sanger sequencing (the chain-termination method), developed in 1975 by Edward Sanger, was considered the gold standard for nucleic acid sequencing for the subsequent two and a half decades (Sanger et al., 1977). Since completion of the first human genome sequence, demand for cheaper and faster sequencing methods has increased greatly. This demand has driven the development of second-generation sequencing methods, or nextgeneration sequencing (NGS). NGS platforms perform massively parallel sequencing, during which millions of fragments of DNA from a single sample are sequenced in unison. Massively parallel sequencing technology facilitates high-throughput sequencing, which allows an entire genome to be sequenced in less than one day. In the past decade, several NGS platforms have been developed that provide low-cost, high-throughput sequencing. Here we highlight two of the most commonly used platforms in research and clinical labs today: the LifeTechnologies Ion Torrent Personal Genome Machine (PGM) and the Illumina MiSeq. The creation of these and other NGS platforms has made sequencing accessible to more labs, rapidly increasing the amount of research and clinical diagnostics being performed with nucleic acid sequencing.

Cryofibrinogenemia-Induced Cutaneous Ulcers: A Review and Diagnostic Criteria

Lymphedema is a localized form of tissue swelling resulting from excessive retention of lymphatic fluid in the interstitial compartment. It is caused by impaired lymphatic drainage. Lymphedema is a chronic progressive disease with serious physical and psychosocial implications. It can be challenging to diagnose, especially in obese patients and in those with coexisting venous disease. We performed PubMed and Google Scholar searches of the English-language literature (1966-2017) using the terms lymphedema, lymphedema management, and lymphatic complications. Relevant publications were manually reviewed for additional resources. There are currently no standard guidelines for the diagnosis of lymphedema. There is no cure yet for lymphedema, and the objective for management is to limit disease progression and prevent complications.

Stem Cell Therapies for Wound Healing

Chronic skin ulcers are frequently encountered in clinical practice and are often due to very heterogeneous etiologies. Cryofibrinogenemia is an unusual cause of non-healing skin ulcers. It is a small-vessel occlusive vascular disorder that results from the precipitation of cryofibrinogens in plasma. The lack of definitive diagnostic criteria means cryofibrinogenemia remains an under-diagnosed entity that causes significant morbidity. One of the most common manifestations of cryofibrinogenemia is skin ulceration. The presence of non-healing ulcers in otherwise healthy patients with no evidence of large-vessel disease should raise the suspicion of essential cryofibrinogenemia. An important clinical feature is the presence of microlivedo, which represents short hyperpigmented linear streaks around the ulcer or even distally about the foot. Histopathologic findings are microthrombi in the dermis and not confined exclusively to the ulcerated area. Cryofibrinogenemia can be secondary to an underlying disorder, so careful investigation to exclude other etiologies is always necessary.

Research Techniques Made Simple: Animal Models of Wound Healing

The number of individuals suffering from chronic cutaneous wounds has been increasing worldwide due to an increase in comorbidities such as vascular disease, diabetes, obesity, and aging population. In the USA, almost seven million Americans have chronic cutaneous ulcers. Moreover, chronic wound management is costly and present a substantial economic burden to the healthcare system. Various therapeutic modalities have been used. However, the treatment outcomes are not always satisfactory because of failure to achieve complete wound closure in around 60% of cases, high rate of recurrence, and scarring. Therefore, there is a need for more effective therapies. Stem cells offer promising possibilities. Preclinical studies have shown that mesenchymal stem cells (MSCs) have a competitive advantage over other types of stem cells due to their better defined potency, paracrine effects, immunomodulatory properties, and safety. For now, multipotent stem cells have a definite advantage in being used for the acceleration of healing. This has to do with their generally favorable risk/benefit ratio. Still, we do not reject the notion that pluripotent stem cells may find an extraordinary role in wound healing. When properly handled and controlled, such cells could be directed toward diverse differentiation pathways that might bring the field of tissue repair closer to regeneration or true wound healing. Large well-controlled clinical trials are needed to examine the capabilities of stem cells in humans and assess their safety profile. Herein, we highlight emerging treatments in tissue regeneration and repair and provide some perspectives on how to translate current knowledge about stem cells, both multipotent and pluripotent, into viable clinical approaches for treating patients with difficult to heal wounds.

Research Techniques Made Simple: Analysis of Collective Cell Migration Using the Wound Healing Assay

Animal models have been developed to study the complex cellular and biochemical processes of wound repair and to evaluate the efficacy and safety of potential therapeutic agents. Several factors can influence wound healing. These include aging, infection, medications, nutrition, obesity, diabetes, venous insufficiency, and peripheral arterial disease. Lack of optimal preclinical models that are capable of properly recapitulating human wounds remains a significant translational challenge. Animal models should strive for reproducibility, quantitative interpretation, clinical relevance, and successful translation into clinical use. In this concise review, we discuss animal models used in wound experiments including mouse, rat, rabbit, pig, and zebrafish, with a special emphasis on impaired wound healing models.