Oculo-zygomatic nerve transfer for facial synkinesis: An anatomical feasibility study.

BACKGROUND: Patients with severe oro-ocular synkinesis often present with concomitant inefficient smile excursion on the affected site. In theory, oculo-zygomatic nerve transfer may decrease synkinesis and improve smile by redirecting nerve fibers to their target muscle. The aim of this study was to explore the feasibility of nerve transfer in human cadavers between a caudal branch innervating the orbicularis oculi to a cephalad branch innervating the zygomaticus major muscles. METHODS: Eighteen hemi-faces were dissected. Reach for direct coaptation of a caudal nerve branch innervating the orbicularis oculi muscle to a cephalad nerve branch innervating the zygomaticus major muscle was assessed. Measurements included total number of nerve branches as well as maximum dissection length. Nerve samples were taken from both branches at the site of coaptation and histomorphometric analysis for axonal count was performed. RESULTS: The number of sub-branches to the orbicularis oculi muscle was 3.1 ± 1.0 and to the zygomaticus major muscle 4.7 ± 1.2. The maximal length of dissection of the caudal nerve branch to the orbicularis oculi muscle was 28.3 ± 7.3 mm and for the cranial nerve branch to the zygomaticus major muscle 23.8 ± 6.5 mm. Transection and tension-free coaptation was possible in all cases but one. The average myelinated fiber counts per mm2 was of 5,173 ± 2,293 for the caudal orbicularis oculi branch and 5,256 ± 1,774 for the cephalad zygomaticus major branch. CONCLUSION: Oculo-zygomatic nerve transfer is an anatomically feasible procedure. The clinical value of this procedure, however, remains to be proven.

The Spinal Accessory Nerve for Functional Muscle Innervation in Facial Reanimation Surgery: An Anatomical and Histomorphometric Study.

INTRODUCTION: Facial reanimation surgery is performed in severe cases of facial palsy to restore facial function. In a 1-stage procedure, the spinal accessory nerve can be used as a donor nerve to power a free gracilis muscle transplant for the reanimation of the mouth. The aim of this study was to describe the surgical anatomy of the spinal accessory nerve, provide a guide for reliable donor nerve dissection, and analyze the available donor axon counts. METHODS: Dissections were performed on 10 nonembalmed cadavers (measurements of 20 nerves). Surgical anatomy of the spinal accessory nerve was described and distances to important landmarks were measured. Nerve biopsies were obtained of the main nerve trunk distal to the skull base, caudoposterior to the sternocleidomastoid muscle, proximal to the trapezius muscle and at the level of donor nerve harvest to analyze the myelinated axon count throughout the course of the spinal accessory nerve. The donor nerve length and available donor nerve axon count were the primary outcome parameters in this study. RESULTS: The mean donor nerve length was 11.6 cm. The spinal accessory nerve was transferred to the mandibular angle without tension for ideal coaptation to the free muscle transplant. After retraction of the trapezius muscle, a small distal nerve branch that leaves the main nerve trunk at a 90-degree angle medially was used as a landmark to indicate the level of donor nerve transection. On average, 1400 myelinated donor axons were available for innervation of the gracilis muscle transplant. CONCLUSIONS: This study gives a practical guide for spinal accessory nerve dissection for its application in facial reanimation as a motor source for the innervation of a free muscle transplant.

Botulinum Toxin A: Dose-dependent Effect on Reepithelialization and Angiogenesis.

BACKGROUND: Botulinum (neuro)toxin A (BoNT) is widely used in the field of plastic and reconstructive surgery. Among treatment of pain, hyperhidrosis, or aesthetic purposes, it is also used to enhance wound healing and prevent excessive scar formation. Some clinical data already exist, but only little is known on a cellular level. The aim of this study was to evaluate the effect of BoNT on cells essential for wound healing in vitro. Therefore, primary human keratinocytes and endothelial cells were treated with different concentrations of BoNT and tested on proliferation, migration, and angiogenic behavior. METHODS: BoNT was exposed to human keratinocytes and endothelial cells in a low (1 IU/mL), medium (10 IU/mL), and high (20 IU/mL) concentrations in cell culture. Proliferation and migration of the 2 cell types were observed and also the angiogenic potential of endothelial cells in vitro. RESULTS: BoNT 20 IU/mL negatively influenced proliferation and migration of keratinocytes but not those of endothelial cells. Angiogenesis in vitro was less effective with the highest BoNT concentrations tested. Low concentrations of BoNT supported sprouting of endothelial cells. CONCLUSIONS: High concentrations of botulinum toxin interfered with wound closure as keratinocytes' proliferation and migration were deteriorated. Furthermore, BoNT concentrations of 20 IU/mL constrain in vitro vessel formation but do not influence proliferation or migration of endothelial cells.

Platysma Motor Nerve Transfer for Restoring Marginal Mandibular Nerve Function.

BACKGROUND: Injuries of the marginal mandibular nerve (MMN) of the facial nerve result in paralysis of the lower lip muscle depressors and an asymmetrical smile. Nerve reconstruction, when possible, is the method of choice; however, in cases of long nerve gaps or delayed nerve reconstruction, conventional nerve repairs may be difficult to perform or may provide suboptimal outcomes. Herein, we investigate the anatomical technical feasibility of transfer of the platysma motor nerve (PMN) to the MMN for restoration of lower lip function, and we present a clinical case where this nerve transfer was successfully performed. METHODS: Ten adult fresh cadavers were dissected. Measurements included the number of MMN and PMN branches, the maximal length of dissection of the PMN from the parotid, and the distance from the anterior border of the parotid to the facial artery. The PMN reach for direct coaptation to the MMN at the level of the crossing with the facial artery was assessed. We performed histomorphometric analysis of the MMN and PMN branches. RESULTS: The anatomy of the MMN and PMN was consistent in all dissections, with an average number of subbranches of 1.5 for the MMN and 1.2 for the PMN. The average maximal length of dissection of the PMN was 46.5 mm, and in every case, tension-free coaptation with the MMN was possible. Histomorphometric analysis demonstrated that the MMN contained an average of 3,866 myelinated fiber counts per millimeter, and the PMN contained 5,025. After a 3-year follow-up of the clinical case, complete recovery of MMN function was observed, without the need of central relearning and without functional or aesthetic impairment resulting from denervation of the platysma muscle. CONCLUSIONS: PMN to MMN transfer is an anatomically feasible procedure for reconstruction of isolated MMN injuries. In our patient, by direct nerve coaptation, a faster and full recovery of lower lip muscle depressors was achieved without the need of central relearning because of the synergistic functions of the PMN and MMN functions and minimal donor-site morbidity.

Wound Healing Effect of Conditioned Media Obtained From Adipose Tissue on Human Skin Cells: A Comparative in Vitro Study.

BACKGROUND: Split-thickness skin grafting is the gold standard to cover extensive acute and chronic wounds with a well-vascularized wound bed. Although some headway has been made in developing biological agents to speed up healing, there is still no treatment that sufficiently replaces skin grafts to date. The use of secretory factors of adipose tissue may be a feasible approach to developing topical wound applications for faster wound healing. METHODS: In this study, the effect of conditioned media (CMs) of human adipose-derived stem cells (ASCs), adipocytes, or adipose tissue on human skin cells was evaluated for viability, proliferation, and migration in vitro. Differentiation potential of stem cells treated with CM was monitored by AdipoRed staining and qualitative real-time polymerase chain reaction. Angiogenic potential of human endothelial cells treated with CM was tested via sprouting assay. RESULTS: The CM of adipose tissue significantly enhanced ASC proliferation (P < 0.01). Treatment with CM showed no inductive effect on ASC differentiation into adipocytes but, at the same time, significantly induced cell sprouting of endothelial cells (P < 0.001). We show for the first time that CM of adipose tissue is a potent inducer of proliferation of ASCs and angiogenesis, with comparable effects with those of stem cell-enriched CM. CONCLUSIONS: We suggest the use of the secretome of adipose tissue to produce CM for topical application on wounds, rather than working with adipose tissue or including the difficult process of enriching the patients' stem cells in vitro.

Generation of a Fibrin Based Three-Layered Skin Substitute.

A variety of skin substitutes that restore epidermal and dermal structures are currently available on the market. However, the main focus in research and clinical application lies on dermal and epidermal substitutes whereas the development of a subcutaneous replacement (hypodermis) is often disregarded. In this study we used fibrin sealant as hydrogel scaffold to generate a three-layered skin substitute. For the hypodermal layer adipose-derived stem cells (ASCs) and mature adipocytes were embedded in the fibrin hydrogel and were combined with another fibrin clot with fibroblasts for the construction of the dermal layer. Keratinocytes were added on top of the two-layered construct to form the epidermal layer. The three-layered construct was cultivated for up to 3 weeks. Our results show that ASCs and fibroblasts were viable, proliferated normally, and showed physiological morphology in the skin substitute. ASCs were able to differentiate into mature adipocytes during the course of four weeks and showed morphological resemblance to native adipose tissue. On the surface keratinocytes formed an epithelial-like layer. For the first time we were able to generate a three-layered skin substitute based on a fibrin hydrogel not only serving as a dermal and epidermal substitute but also including the hypodermis.

Computational tools for modeling xenometabolism of the human gut microbiota.

The gut microbiota is increasingly being recognized as a key site of metabolism for drugs and other xenobiotic compounds that are relevant to human health. The molecular complexity of the gut microbiota revealed by recent metagenomics studies has highlighted the need as well as the challenges for system-level modeling of xenobiotic metabolism in the gut. Here, we outline the possible pathways through which the gut microbiota can modify xenobiotics and review the available computational tools towards modeling complex xenometabolic processes. We put these diverse computational tools and relevant experimental findings into a unified perspective towards building holistic models of xenobiotic metabolism in the gut.

DNA virus replication compartments.

Viruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication.

Amino acid exchanges in the putative nuclear export signal of adenovirus type 5 L4-100K severely reduce viral progeny due to effects on hexon biogenesis.

The adenovirus type 5 nonstructural L4-100K protein is indispensable for efficient lytic infection. During the late phase, L4-100K promotes selective translation of viral late transcripts and mediates the trimerization of the major capsid protein hexon. In the present study, the role of a potential nuclear export signal in L4-100K was investigated. Intriguingly, amino acid substitutions in this sequence resulted in severely diminished progeny virus production, seemingly by precluding proper hexon biogenesis.

Functional cooperation between human adenovirus type 5 early region 4, open reading frame 6 protein, and cellular homeobox protein HoxB7.

Human adenovirus type 5 (HAdV5) E4orf6 (early region 4 open reading frame 6 protein) is a multifunctional early viral protein promoting efficient replication and progeny production. E4orf6 complexes with E1B-55K to assemble cellular proteins into a functional E3 ubiquitin ligase complex that not only mediates proteasomal degradation of host cell substrates but also facilitates export of viral late mRNA to promote efficient viral protein expression and host cell shutoff. Recent findings defined the role of E4orf6 in RNA splicing independent of E1B-55K binding. To reveal further functions of the early viral protein in infected cells, we used a yeast two-hybrid system and identified the homeobox transcription factor HoxB7 as a novel E4orf6-associated protein. Using a HoxB7 knockdown cell line, we observed a positive role of HoxB7 in adenoviral replication. Our experiments demonstrate that the absence of HoxB7 leads to inefficient viral progeny production, as HAdV5 gene expression is highly regulated by HoxB7-mediated activation of various adenoviral promoters. We have thus identified a novel role of E4orf6 in HAdV5 gene transcription via regulation of homeobox protein-dependent modulation of viral promoter activity.

CRM1-dependent transport supports cytoplasmic accumulation of adenoviral early transcripts.

The life cycle of adenoviruses is divided by convention into early and late phases, separated by the onset of viral genome replication. Early events include virus adsorption, transport of the genome into the nucleus, and the expression of early genes. After the onset of viral DNA replication, transcription of the major late transcription unit (MLTU) and thereby synthesis of late proteins is induced. These steps are controlled by an orchestra of regulatory processes and require import of the genome and numerous viral proteins into the nucleus, as well as active transport of viral transcripts and proteins from the nucleus to the cytoplasm. The latter is achieved by exploiting the shuttling functions of cellular transport receptors, which normally stimulate the nuclear export of cellular mRNA and protein cargos. A set of adenoviral early and late proteins contains a leucine-rich nuclear export signal of the HIV-1 Rev type, known to be recognized by the cellular export receptor CRM1. However, a role for CRM1-dependent export in supporting adenoviral replication has not been established. To address this issue in detail, we investigated the impact of two different CRM1 inhibitors on several steps of the adenoviral life cycle. Inhibition of CRM1 led to a reduction in viral early and late gene expression, viral genome replication, and progeny virus production. For the first time, our findings indicate that CRM1-dependent shuttling is required for the efficient export of adenoviral early mRNA.

The E3 ubiquitin ligase activity associated with the adenoviral E1B-55K-E4orf6 complex does not require CRM1-dependent export.

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase.