Detection of Rare Mutations in CtDNA Using Next Generation Sequencing.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter's ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

Expression of MicroRNA-301a and its Functional Roles in Malignant Melanoma.

BACKGROUND/AIMS: Although microRNA-301a has been reported to function as an oncogene in many human cancers, the roles of miR-301a in malignant melanoma (MM) is unclear. The present study aims to investigate the functional roles of miR-301a in MM and its possible molecular mechanisms. METHODS: Quantitative real-time PCR (qRT-PCR) assay was performed to detect the expression of miR-301a in MM tissues, and analyze its correlation with metastasis and prognosis of MM patients. In vitro, miR-301a was ectopically expressed using overexpression and knock-down strategies, and the effects of miR-301a expression on growth, apoptosis, migration, invasion and chemosensitivity of MM cells were further investigated. Furthermore, the potential and functional target gene was identified by luciferase reporter, qRT-PCR, Western blot assays. RESULTS: We showed that the expression of miR-301a was significantly upregulated in MM tissues, and upregulation of miR-301a correlated with metastasis and poor prognosis of MM patients. Transfection of miR-301a/inhibitor significantly inhibited growth, colony formation, migration, invasion and enhanced apoptosis and chemosensitivity in MM cells, while transfection of miR-301a/mimic could induce the inverse effects on phenotypes of MM cells. Luciferase reporter, qRT-PCR and Western blot assays showed that phosphatase and tensin homolog (PTEN) was a direct and functional target of miR-301a. It was also observed that the Akt and FAK signaling pathways were involved in miR-301/PTEN-promoting MM progression. CONCLUSION: Taken together, our study suggests that miR-301a may be used as a potential therapeutic target in the treatment of human MM.

Lumican alleviates hypertrophic scarring by suppressing integrin-FAK signaling.

Hypertrophic scarring (HS) is an overcompensation of wound healing that increases the risk of cosmetic disfigurement and functional impairment. No gold standard has been established for the treatment or prevention of HS. Our study aims to elucidate the expression and function of lumican in the pathogenesis of HS as well as the underlying mechanism involved in this procedure. An animal model of HS (rabbit ear) was established, and the Ad-lumican vectors were locally injected. Primary fibroblasts isolated from patients with hypertrophic burn scars were used in vitro. Histological and molecular changes in HS pathogenesis were evaluated. The results showed that lumican is significantly reduced in HS tissues and fibroblasts from HS patients as compared to normal skin or cells. Lumican levels were further suppressed in response to TGF-ß stimulation. However, lumican upregulation effectively thinned the scar area and inhibited fibroblast proliferation and the cell cycle. Meanwhile, Ad-lumican administration suppressed the deposition of extracellular matrix, such as collagen and CTGF. Ad-lumican injected animals or fibroblasts presented comparable integrin α2ß1 expression while greatly reduced phosphorylation of FAK compared to the negative control. Moreover, Ad-lumican administration largely enhanced the binding of lumican to integrin α2ß1 and may thus inhibit the signaling propagation of collagen-integrin α2ß1. Overall, the restoration of lumican levels contributed to suppressing the HS progression by inhibiting collagen-integrin α2ß1-FAK signaling.

Wound-healing improvement by resurfacing split-thickness skin donor sites with thin split-thickness grafting.

INTRODUCTION: Split-thickness skin graft (STSG) donor site dressing has been controversial until now. Our study aimed to assess the patient comfort and wound-healing efficacy with the application of thin split-thickness skin grafts regrafting on STSG donor sites. METHODS: One hundred ninety-two consecutive patients undergoing split-thickness skin grafting were included in the study, and the participants were randomly divided into the following three groups: group A was regrafted with thin STSGs and groups B and C were covered with occlusive hydrocellular dressing and paraffin gauze, respectively. The participants were compared according to the epithelialization time, pain and scar formation. RESULTS: The average time of epithelialization was 6.2 ± 1.1 days in group A, 11.1 ± 2.1 days in group B and 13.5 ± 2.5 days in group C. The pain scores on days 2 and 5 after operation were 2.3 ± 0.8 and 1.9 ± 0.8 in group A, 2.5 ± 1.1 and 3.9 ± 1.3 in group B, and 3.8 ± 1.4 and 5.9 ± 2.1 in group C. The scar scores at half a year and one year after operation were 4.3 ± 0.6 and 2.50 ± 0.6 in group A, 7.4 ± 0.6 and 6.2 ± 0.6 in group B, and 11.8 ± 0.4 and 10.9 ± 1.0 in group C, separately. The difference in the three groups was significant. CONCLUSION: Utilizing thin STSGs regrafting on donor sites could significantly shorten the epithelialization time, reduce pain and prevent hyperplastic scar formulation.

Association of CRR9 locus with elevated risk of squamous cell carcinoma and basal cell carcinoma.

BACKGROUND: Published studies have generated inconsistent results related to the contribution of CRR9 rs401681 C allele to the risk of developing non-melanoma skin cancer (NMSC), and it is the inconsistency that promoted us to undertake a meta-analysis to identify the degree of impact the C allele has on NMSC. METHOD: The PubMed, Science Direct, Embase and Cochrane Library were thoroughly searched from the start of November 2013 to the end of April 2014 by using CRR9, polymorphism, skin cancer and their synonyms. Based on a total of 44,036 subjects, we calculated ORs and 95% CIs to measure the influence of the C allele on NMSC predisposition. RESULTS: Overall, individuals carrying the risk C allele at rs401681 had 1.16 times (OR = 1.16, 95% CI: 1.10-1.21, heterogeneity: P = 0.298 and I2 = 0.166, Figure 2) greater risk of NMSC compared to the common T allele. In the further stratified analyses, we found a significant association between the C allele and BCC, Icelanders, and non-Icelanders. CONCLUSION: The results of this meta-analysis suggest that the C allele at rs401681 is likely to modify the genetic predisposition to NMSC.

Biomimetic LBL structured nanofibrous matrices assembled by chitosan/collagen for promoting wound healing.

This paper reports the fabrication of biomimetic nanofibrous matrices via co-electrospinning of polycaprolactone (PCL)/cellulose acetate (CA) and layer-by-layer self-assembly (LBL) of positively charged chitosan (CS) and negatively charged Type Ⅰ collagen on the nanofibrous matrix. FE-SEM images indicate that the average fiber diameter increased from 392 to 541 nm when the coating bilayers varied from 5 to 20.5. Besides, the excellent biocompatibility and enhanced attachment and spreading of normal human dermal fibroblasts (NHDFs) of prepared nanofibrous mats are confirmed by MTT and SEM results. Furthermore, the LBL structured (CS/collagen)n nanofibrous mats greatly improve the cell migration in vitro, promote re-epithelialization and vascularization in vivo, and up-regulate the expression of collagen Ⅳ and α-tubulin, as well as the Integrin ß1 and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. The levels of expressed protein are significantly enhanced with increasing coating bilayers via immunohistochemistry and western blotting analyses. Collectively, these results suggest that the LBL structured biomimetic nanofibrous matrices may enhance cell migration and further promote the skin regeneration by up-regulating the secretion of ECM protein and triggering Integrin/FAK signaling pathway, which demonstrate the potential use of the nanofibrous mats to rapidly restore the structural and functional properties of wounded skin.

Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns.

The potential of acellular dermal matrix (ADM) to improve cosmetic and functional outcomes has been demonstrated; however, there have been few clinical comparative studies assessing the long-term morphological, histological and functional changes after ADM placement. This study was designed to retrospectively evaluate the long-term outcomes of the cograft acellular dermal matrix with autologous thin split-thickness skin for the coverage of wounds in extensively burned patients. Thirty burn patients treated with a composite graft of ADM with autologous split-thickness skin from January 2007 to December 2009 were enrolled in this study. Another group of thirty patients who received only an autogenous split-thickness skin implant served as the control. Our study revealed that the collagen in the dermis treated with ADM were ordered, and the proportion of collagen III/I was much higher in the control group than in the ADM group. The basement membrane was prominent and continuous. Meanwhile, the VBSS (Vancouver Burn Skin Score) was used to evaluate skin quality, which shows a significant differences between the two group (P<0.001). Then the functional level was evaluated by the BI (Barthel Index), and the ADM group was much better than the control group (P=0.005). Based on these results, we concluded that the composite graft of ADM with autologous thin split-thickness skin was suitable for repairing the defects in functional areas after a burn. This technique might facilitate wound management with acceptable esthetic outcomes, good functional recovery and less scar hyperplasia at the donor site.

Zeolite-based hemostat QuikClot releases calcium into blood and promotes blood coagulation in vitro.

AIM: To examine the changes in electrolyte concentrations after addition of zeolite-based hemostat QuikClot in blood and the effects of zeolite on blood coagulation in vitro. METHODS: Fresh blood was taken from healthy adult volunteers and sheep, and the electrolyte concentrations in blood were measured using a blood electrolyte analyzer. Zeolite Saline Solution (ZSS) was prepared by addition of 2 g zeolite to 0.9% NaCl solution (4, 8, or 16 mL). The electrolytes in ZSS were measured using inductively coupled plasma atomic emission spectroscopy. The prothrombin time (PT) and activated partial thromboplastin time (APTT) of blood were measured using the test tube method. The activated clotting time (ACT) and clotting rate (CR) of blood were measured with Sonoclot Coagulation and Platelet Function Analyzer. RESULTS: Addition of zeolite (50 and 100 mg) in 2 mL human blood significantly increased Ca(2+) concentration, while Na(+) and K(+) concentrations were significantly decreased. Addition of zeolite (50 and 100 mg) in 0.9% NaCl solution (2 mL) caused similar changes in Ca(2+) and Na(+) concentrations. Si(4+) (0.2434 g/L) and Al(3+) (0.2575 g/L) were detected in ZSS (2 g/8 mL). Addition of ZSS in sheep blood shortened APTT in a concentration dependent manner, without changing PT. ZSS or aqueous solution of CaCl2 that contained Ca(2+) concentration identical to that of ZSS significantly shortened ACT in human blood without significantly changing CR, and the effect of ZSS on ACT was not significantly different from that of CaCl2. CONCLUSION: Zeolite releases Ca(2+) into blood, thus accelerating the intrinsic pathway of blood coagulation and shortening the clot formation time.

The status of microRNA-21 expression and its clinical significance in human cutaneous malignant melanoma.

Dysregulation of microRNA-21 plays critical roles in tumor initiation and progression. The purpose of this study was to investigate the status of microRNA-21 expression in human cutaneous malignant melanoma and determine its clinical significance. TaqMan(®) real-time RT-PCR assay was performed to examine the expression of microRNA-21 in 10 cases of dysplastic nevi, 86 cases of primary cutaneous melanomas, 10 cases of melanoma metastases. The correlation of microRNA-21 expression with clinicopathological factors or prognosis of patients with cutaneous melanoma was statistically analyzed. Additionally, the effects of microRNA-21 expression on growth, apoptosis and chemo- or radiosensitivity of melanoma cells were also investigated by transfection of microRNA-21 inhibitor. We firstly showed that increased levels of microRNA-21 expression were shown from dysplastic nevi to primary cutaneous melanomas to melanoma metastases. Moreover, high miR-21 expression was found to be correlated with Breslow thickness and advanced clinical stage. Patients with high microRNA-21 expression showed shorter 5-year disease-free or overall survival than those with low microRNA-21 expression. Furthermore, multivariate regression analysis showed that the status of microRNA-21 expression was an independent prognostic factor for overall survival of patients. Antisense-mediated microRNA-21 inhibition could significantly suppress growth, increase apoptosis and enhance chemo- or radiosensitivity of human cutaneous melanoma cells by inducing the increased Bax/Bcl-2 ratio. Thus, the status of microRNA-21 might be an independent prognostic factor for patients with cutaneous melanoma, and microRNA-21 has the potential of being a novel molecular target for the treatment of human cutaneous melanoma.

Epidemiological studies of electrical injuries in Shaanxi province of China: a retrospective report of 383 cases.

A statistical survey was conducted at the Burn Unit of the Tangdu Hospital, Shaanxi, China, during the 10-year period from January 2000 to December 2009. In this retrospective study, 383 patients who admitted to our burn unit because of electrical trauma were included. Data including the patient's general condition, clinical presentation, complications and operation times was collected retrospectively and analyzed with epidemiological methods. Subjects in our collective were predominantly male (90.3%, n=346/383) and were composed by those who injured in work-related incidents (78.3%, n=300/383), rural individuals (58.2%, n=223/383) and students (9.4%, n=36/383). High voltage was directly correlated to severity clinical complications, and amputation. The percentage of myocardial impairment was 79.3% (n=92/116) among patients who suffered with electrical current through heart tissue. Along with the more developed east area of China, electrical injuries are becoming a growing concern of the developing West part in China as well. Electrical injuries induce serious tissue damage, need long hospital stay, and result in high rate of permanent disability and economic hardship for the afflicted families. A competent prevention program needs to be developed to address this problem.

Electrical injury alters ion channel expression levels and electrophysiological properties in rabbit dorsal root ganglia neurons.

The electrophysiological and morphological changes of nerve fibers induced by electrical injury have been widely addressed. However, the changes of ion channels in neurons after electrical shocks have not been systematically investigated yet. In this study, the sciatic nerves of rabbit were injured by 50 V 50 Hz, 110 V 50 Hz, and 220 V 50 Hz alternating current, respectively. One week later, the expression levels and electrophysiological changes of voltage-gated potassium (Kv) and sodium (Nav) channels in dorsal root ganglia (DRG) neurons were evaluated by RT-PCR, immunofluorescence staining and patch clamp technique. The Nav1.1 expression was decreased by 50V injury. The Kv1.2, Kv1.4, Nav1.1 and Nav1.7 expression levels and Kv current densities were reduced after 110 V injury. Under the 220 V injury circumstance, Kv1.2, Nav1.1, Nav1.7 expression levels, Kv current densities and TTX-R Na(+) current densities were significantly decreased, while TTX-S Na(+) current densities increased. These findings suggest that the expression levels, subunit compositions, and electrophysiological properties of Kv and Nav channels are altered after electrical injury, and the severity of injury gets worse as injury voltage increases.

[The influence of vacuum-assisted drainage on the growth of capillaries in the wound produced by explosion in pig].

OBJECTIVE: To investigate the influence of vacuum-assisted closure (VAC) technique on the growth of capillaries in the wound of the pig produced by explosion. METHODS: Four small white pigs were inflicted with 16 explosion wounds [(7.3 +/- 1.0) cm2 in area] on both sides of the buttocks, shoulders and hips by detonation of a specific type of explosive, and the wounds were randomly divided into 2 groups, i. e, control (C, with conventional treatment from 2 post-injury day (PID) on and treatment (T, with VAC treatment after debridement from 2 PID on) groups, with 8 wounds in each group. Wound tissues of 2mm x 2mm x 2mm in size were harvested for pathological examination before treatment and on 1 and 3 post-treatment day (PTU). The differentiation of adventitial cells were examined with light microscope, and the pixel value of desmin positive particles and the luminal area of newly formed capillaries were assessed with Image C software. RESULTS: Most of vessels in the wound of both groups were in elliptic shape when observed in longitudinal section. In C group, few newly formed capillaries vessels with lack of pericytes were observed before treatment and on 1, 3 PTD, then the number began to increase on 6 PTD. In T group, the number of newly formed capillaries with pericytes was increased on 1 PTD, and it continued to increase thereafter. The pixel values of desmin positive particles in C group on 1, 3, and 6 PTD were (91 +/- 54), (199 +/- 85), and (1552 +/- 298), respectively, which were obviously higher than those in T group [(2569 +/- 330), (3984 +/- 377), (9611 +/- 960), P < 0.01]. The area of vessel lumen in C group was (59 +/- 36), (250 +/- 70), and (938 +/- 287) microm2, respectively on 1, 3, and 6 PTD, which was also smaller than those in T group [(818 +/- 234), (4518 +/- 1080), and (9058 +/- 1656) microm2, P < 0.01]. CONCLUSION: Compared with conventional therapy, VAC can not only accelerate the formation of new capillaries, but also enhance the differentiation of pericytes and the process of enwrapping them around the vessels, and increase the luminal area of newly formed capillaries.

[The changes in blood flow in sciatic nerve after electrical injury in rabbit].

OBJECTIVE: To study the changes in sciatic nerve blood flow and the expression of collagen type I after electric injury of rabbit nerve with different voltages. METHODS: Thirty-six healty rabbits were randomized into 3 groups before receiving injury with electricity in voltages, i.e. 50 v, 75 v, and 100 v groups. The changes in blood flow of sciatic nerve were observed with Laser Doppler Flowmeter immediately after injury and 1, 4, 8 weeks after injury. The changes in the expression of collagen type I was observed by immunohistochemical method, and the positive expression rate was calculated. RESULTS: The sciatic nerve blood flow increased in all groups immediately after electric injury. In the 75 v and 100v groups, the nerve blood flow [(53 +/- 3 ), (48 +/- 5) PU] was obviously lower than that of normal value [(62 +/- 4) PU, P < 0.05]. There was little collagen type I deposition in 50 v group, while brown collagenous fibers in epineurium and perineurium were observed in 75 v and 100v groups 4 and 8 weeks after injury. The expression of collagen type I in all groups were obviously higher than that of normal value, and that in 75v and 100 v groups were higher than that in 50 v group at bachl time-point (P < 0.01). CONCLUSION: The restoration of sciatic nerve blood flow is postponed following by the injury with increase of the electrical voltage. The collagen deposition after electrical injury may be one of the reasons for nerve blood flow decrease.