The Effect of Vascular Endothelial Growth Factor C and Adipose-Derived Stem Cells on Lymphatic Regeneration in a Rat Vascularized Lymph Node Transfer Model.

BACKGROUND: Lymphedema is a chronic condition characterized by progressive edema with complicated treatment. Recently, new treatment strategies inducing lymphangiogenesis were proposed. The aim of our study was to examine the effect of vascular endothelial growth factor C (VEGF-C) and adipose-derived stem cells (ADSCs) on lymphatic regeneration and drainage re-establishment in vascularized lymph node transfer (VLNT) model using a pedicled vascularized lymph node (VLN) groin flap. METHODS: Female Lewis rats with groin VLN flaps were utilized as a lymphedema model. Group A served as the control. Group B received VEGF-C. Group C received both VEGF-C and ADSCs. Group D received ADSCs only. Lymphatic drainage re-establishment was evaluated by ultrasound-photoacoustic imaging (US-PAI) after indocyanine green (ICG) injection. RESULTS: The fastest regeneration of elevated flaps was observed in Groups B and C in all monitored periods. After the first month, ICG positivity was detected in 14.3% of animals in Group A, 71.43% of animals in Group B (odds ratio [OR] = 15; p = 0.048), and 83.33% in Group C (OR = 30; p = 0.027). On the contrary, the difference between control group and Group D (16.67%; p = 0.905) was statistically insignificant. Administration of VEGF-C, ADSC + VEGF-C, and ADSC led to full flap regeneration after 6 months. The control group had the lowest percentage of ICG positivity at all monitored time points. CONCLUSION: We found that the fastest regeneration occurred with the combination of the VLN flap and VEGF-C. The addition of ADSC had an insignificant effect in our study. Furthermore, we proved the feasibility of PAI as an assessment tool of the lymphatic drainage recovery in a VLNT model.

Animal Models Used in the Research of Vascularized Lymph Node Transfer: A Systematic Review.

BACKGROUND: Lymphedema is a common adverse consequence of breast cancer therapy, while still relatively little is known about its pathophysiology. Several treatment options emerged over the past decades, and among them, vascularized lymph node transfer (VLNT) seems to be particularly promising. Animal models are indispensable to improve our understanding of the underlying processes surrounding the transplantation of a vascularized lymph node. This review aimed to systematically evaluate animal models of VLNT and compare their advantages and disadvantages. MATERIALS AND METHODS: A systematic review of literature in the Scopus, Web of Science, and Ovid MEDLINE databases was conducted according to the PRISMA guidelines to identify all studies on animal models used for the research of VLNT. The algorithm used in search of articles was "Vascularized Lymph Node Transfer" AND "Model". Articles were manually verified for relevance to the topic. The resulting models were assessed for their suitability for VLNT research. RESULTS: The literature search yielded a total of 233 studies after duplicates removal. Of those, 217 were excluded based on title and abstract review. Another study was excluded after reviewing the full-text article leaving 15 eligible studies to be included in this review article. CONCLUSIONS: Rats were found to be the most dominantly used animal model in the VLNT research, although other models had their benefits. The main areas of study were the functionality of VLNT within or without a preinduced lymphedema, its response to ischemia, and clarification of lymphatic pathways reestablishment following VLNT.

Evaluation of the Number of Follow-up Surgical Procedures and Time Required for Delayed Breast Reconstruction by Clinical Risk Factors, Type of Oncological Therapy, and Reconstruction Approach.

BACKGROUND: Breast reconstructive surgery is often a multistage process. The aim was to understand which factors might increase the number of follow-up surgeries and the length of time required to complete the reconstruction process. METHODS: A cross-sectional analysis was performed. Clinical data of 110 patients who underwent delayed postmastectomy breast reconstruction with a 5-year examination period were reviewed retrospectively. Impact of clinical risk factors, oncological therapy, and reconstruction approach on the number of surgeries and the length of required time was analyzed. Mann-Whitney U test and Kruskal-Wallis rank sum test were used. RESULTS: In patients undergoing perioperative hormone therapy, an average of 2.9 surgeries and length of 20.2 months were required compared to 2.3 surgeries and 14.0 months in patients without hormone therapy (P = 0.003; P = 0.005). Previous abdominal surgery was associated with an increased number of breast reconstruction stages of 3.1 per patient (P = 0.056) and a longer reconstruction time of 23.0 months (P = 0.050). Patients undergoing nipple reconstruction or implant revision required an increased number of surgeries (P < 0.001; P = 0.012) and a longer reconstruction time (P = 0.002; P < 0.001). Contralateral breast surgery and flap revision were associated only with an increased number of surgeries (P < 0.001; P < 0.001). CONCLUSION: Perioperative hormone therapy was associated with a significantly higher number of surgeries and duration of time required to complete the reconstruction process. The highest increase in the number of surgeries was in patients with flap revision including anastomosis revision and necrectomy. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Bilateral Prophylactic Nipple-Sparing Mastectomy: Analysis of the Risk-Reducing Effect in BRCA1/2 Mutation Carriers.

BACKGROUND: Mutations in the BRCA1 or BRCA2 genes increase the lifetime risk of developing breast cancer to 68-72% by the age of 80. One of the modalities to manage the risk is a prophylactic mastectomy. Bilateral nipple-sparing mastectomy specifically offers the most favorable esthetic outcomes but the evidence for its oncological safety remains limited. Thus, we aimed to compare the occurrence of breast cancer between nipple-sparing mastectomy and surveillance groups of BRCA1 or BRCA 2 mutations carriers. MATERIALS AND METHODS: BRCA1 or BRCA2-positive patients undergoing bilateral prophylactic nipple-sparing mastectomy at our department were identified. Only those unaffected by breast cancer were eligible. Each patient was pair-matched with a BRCA1 or BRCA2-positive patient of equal age from the surveillance group. Breast cancer incidence in both groups was recorded and the results were compared. RESULTS: None of 105 patients who underwent NSM between 2009 and 2019 at a single institution with a mean follow-up time of 50 months developed breast cancer over this time period. One patient in this group died of an unrelated cause. Nine patients from 105 in the match-paired surveillance group were diagnosed with breast cancer during a mean follow-up time of 58.3 months, however, none of them died. CONCLUSION: To the best of our knowledge, this is the largest single-center study of risk-reducing bilateral NSM in healthy BRCA1 or BRCA2 mutation carriers. Based on our results and those of other series, we conclude that NSM in its current form appears to be at least equally as safe as other types of mastectomy for preventing breast cancer in BRCA1 or BRCA2 mutation carriers. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The Influence of Low- and High-Negative-Pressure Liposuction and Different Harvesting Sites on the Viability and Yield of Adipocytes and Other Nucleated Cells.

BACKGROUND: The volume effect of fat grafting is highly dependent on the presence of viable adipocytes and other nucleated cells within the lipoaspirate. We suspected that one of the crucial factors influencing cell viability is the negative pressure applied during the fat graft harvesting and the suitability of various harvest sites when compared to others. Despite much discussion, there is no consensus on the optimal negative pressure or the best site for harvesting so we designed an experiment to test this. METHODS: Fat graft taken under low negative pressure (- 200 mmHg) or high negative pressure (- 700 mmHg) from the thigh or abdominal regions from 21 healthy human donors was evaluated. The principal variables studied were: a) total number and viability of nucleated cells, b) liposuction duration and c) blood admixture. Other variables studied were body mass index, the impact of age and enzymatic digestion. RESULTS: The absolute number and viability of nucleated cells and the blood admixture did not differ significantly between lipoaspirates obtained under different vacuum conditions or from different regions. The time taken to acquire the same volume of lipoaspirate was significantly increased using low negative pressure. The time taken to collect cells in the thigh region significantly increased with increasing BMI but this correlation was not found when harvesting in the abdominal region. The BMI and age did not impact the results in any of the measured variables. The enzymatic digestion rate was independent of the negative pressure used to harvest. CONCLUSION: Our results indicate that neither the negative pressure used nor the area chosen has any significant influence on the viability and yield of harvested cells. The time taken to obtain lipoaspirate using low pressure is significantly longer than when using high pressure. No significant difference was found in the value of blood admixture using different vacuum pressures, and no correlation exists between the body mass index and the cell viability or age of the patients and the time of liposuction. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine Ratings, please refer to Table of Contents or online Instructions to Authors www.springer.com/00266 .

The Influence of High and Low Negative Pressure Liposuction and Various Harvesting Techniques on the Viability and Function of Harvested Cells-a Systematic Review of Animal and Human Studies.

BACKGROUND: An understanding of fat grafting methodology, techniques and patient-related factors is crucial when considering fat grafting. Multiple factors can influence the success of a fat graft and consequently the outcome of the procedure. The aim of this systematic review is to elucidate the influence of negative pressure and various techniques of fat harvesting on the viability and function of cells, particularly adipocytes and adipose-derived stem cells. METHODS: We conducted a literature search from 1975 to 2020 using the PubMed bibliography, ScienceDirect, SCOPUS and the Google Scholar databases which produced 168,628 articles on the first pass. After applying all the exclusion criteria by two independent reviewers, we were left with 21 articles (level IV of Oxford Centre for Evidence-Based Studies and Grade C of Grade Practice Recommendation from the American Society of Plastic Surgeons) on which this review is based. RESULTS: From 11 studies focused on different negative pressures, no one found using high negative pressure advantageous. Summarising 13 studies focused on various harvesting techniques (excision, syringe, and pump-machine), most often equal results were reported, followed by excision being better than either syringe or liposuction. CONCLUSION: From our systematic review, we can conclude that the low negative pressure seems to yield better results and that the excision seems to be the most sparing method for fat graft harvesting. However, we have to point out that this conclusion is based on a very limited number of statistically challengeable articles and we recommend well-conducted further research. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Patient-Reported Outcomes of Three Different Types of Breast Reconstruction with Correlation to the Clinical Data 5 Years Postoperatively.

BACKGROUND: The aim of this study was to compare long-term clinical and patient-reported outcomes in terms of satisfaction and health-related quality of life (HRQoL) following postmastectomy breast reconstruction using BREAST-Q in patients undergoing implant-based reconstruction, abdominal-based autologous reconstruction, and combined reconstruction (with implant and LD flap or implant and TDAP flap). METHODS: A cross-sectional study was conducted. Patients had undergone delayed postmastectomy breast reconstruction and completed the BREAST-Q reconstruction module. The results were related to the clinical data obtained from the clinic's patient record system. A 5-year examination was included. Mean scores and standard deviations were calculated. Kruskal-Wallis test, Chi-square goodness of fit test and Chi-square test were used for the statistical analysis. RESULTS: Overall, 110 patients (n = 24 implant, n = 38 autologous, n = 48 combination) were included. Patients with autologous reconstruction reported greater postoperative satisfaction with breasts (p < 0.001), satisfaction with outcome (p < 0.001), psychosocial well-being (p = 0.001), and sexual well-being (p = 0.051). CONCLUSION: This study represents a comprehensive long-term examination of postmastectomy breast reconstruction. Autologous reconstruction patients had higher postoperative satisfaction and HRQoL than patients receiving other types of reconstruction despite having more intense oncological therapy and the highest mean number of follow-up surgical procedures. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Local application of adipose-derived mesenchymal stem cells supports the healing of fistula: prospective randomised study on rat model of fistulising Crohn's disease.

OBJECTIVE: Local application of adipose-derived mesenchymal stem cells (ADSC) represents a novel approach for the management of perianal fistula in patients with Crohn's disease. A randomised study on an animal model was performed to investigate the efficacy and to detect the distribution of implanted ADSCs by bioluminescence (BLI). MATERIALS AND METHODS: A caecostomy was used as a fistula model in 32 Lewis rats. The ADSCs were isolated from transgenic donor expressing firefly luciferase. Animals were randomly assigned to groups given injections of 4 × 106 cells (n = 16, group A) or placebo (n = 16, group B) in the perifistular tissue. Fistula drainage assessment was used to evaluate the fistula healing. After application of D-luciferin, cell viability and distribution was detected using an IVIS Lumina XR camera on days 0, 2, 7, 14 and 30. RESULTS: The fistula was identified as healed in 6 (38%) animals in group A vs. 1 case (6.3%) in group B (p = .033). The BLI was strongest immediately after administration of ADSCs 31.2 × 104 (6.09-111 × 104) p/s/cm2/sr. The fastest decrease was observed within the first 2 days when values fell by 50.2%. The BLI 30 days after injection was significantly higher in animals with healed fistulas - 8.23 × 104 (1.18-16.9 × 104) vs. 1.74 × 104 (0.156-6.88 × 104); p = .0393. CONCLUSIONS: Local application of ADSCs resulted in significantly higher fistula closure rate on an animal model. BLI monitoring was proved to be feasible and showed rapid reduction of the ADSC mass after application. More viable cells were detected in animals with healed fistula at the end of the follow-up.

Evaluation of Oncological Safety of Fat Grafting After Breast-Conserving Therapy: A Prospective Study.

INTRODUCTION: Breast-conserving treatment (BCT) consisting of a lumpectomy followed by radiation is considered the standard of care in early-stage breast cancer, and breast reconstruction using fat transfer has become the standard of care in these patients. Immediately following BCT, patients are theoretically the most prone for cancer recurrence caused by the remaining mass of glandular tissue. Therefore, we conducted a prospective study to evaluate the oncological safety of fat grafting in patients after BCT. METHODS: We analyzed patients who underwent breast reconstruction after BCT between April 2011 and February 2014. The control subjects were matched from a prospective database of women treated for breast cancer who did not undergo fat grafting, and each control was matched for the following variables: date of primary cancer surgery, date of fat grafting, histology, estrogen and progesterone receptors (ER+PR), adjuvant hormone therapy, disease-free interval from primary operation (BCT), and disease-free interval from breast reconstruction involving fat grafting. The primary endpoint of this study was tumor recurrence. RESULTS: The study group consisted of 32 patients, while the control group consisted of 45 patients. Breast tumor recurrence was observed in 2 of 32 cases (6.25%) in the reconstruction group, and distant metastases were detected in both cases. In the control group without reconstruction, we found cancer recurrence in 2 of 41 cases (4.88%), and locoregional recurrence was observed in both cases. The difference in cancer recurrence after BCT was insignificant between groups (p = 0.593). CONCLUSION: The recurrence rate in patients reconstructed with fat grafts after BCT was not significantly different from the recurrence rate of control BCT patients.

Mesenchymal stem cells seeded on cross-linked and noncross-linked acellular porcine dermal scaffolds for long-term full-thickness hernia repair in a small animal model.

Biological meshes are biomaterials consisting of extracellular matrix that are used in surgery particularly for hernia treatment, thoracic wall reconstruction, or silicone implant-based breast reconstruction. We hypothesized that combination of extracellular matrices with autologous mesenchymal stem cells used for hernia repair would result in increased vascularization and increased strength of incorporation. We cultured autologous adipose-derived stem cells harvested from the inguinal region of Wistar rats on cross-linked and noncross-linked porcine extracellular matrices. In 24 Wistar rats, a standardized 2×4 cm fascial defect was created and repaired with either cross-linked or noncross-linked grafts enriched with stem cells. Non-MSC-enriched grafts were used as controls. The rats were sacrificed at 3 months of age. The specimens were examined for the strength of incorporation, vascularization, cell invasion, foreign body reaction, and capsule formation. Both materials showed cellular ingrowth and neovascularization. Comparison of both tested groups with the controls showed no significant differences in the capsule thickness, foreign body reaction, cellularization, or vascularization. The strength of incorporation of the stem cell-enriched cross-linked extracellular matrix specimens was higher than in acellular specimens, but this result was statistically nonsignificant. In the noncross-linked extracellular matrix, the strength of incorporation was significantly higher in the stem cell group than in the acellular group. Seeding of biological meshes with stem cells does not significantly contribute to their increased vascularization. In cross-linked materials, it does not ensure increased strength of incorporation, in contrast to noncross-linked materials. Owing to the fact that isolation and seeding of stem cells is a very complex procedure, we do not see sufficient benefits for its use in the clinical setting.

Centrifugation versus PureGraft for fatgrafting to the breast after breast-conserving therapy.

BACKGROUND: Breast-conserving treatment (BCT) leads to a progressive and deteriorating breast deformity. Fatgrafting is ideal for breast reconstruction after BCT. The most frequently utilized technique for fat processing is centrifugation. The PureGraft device (Cytori Therapeutics, San Diego, CA, USA) is a new method that involves washing and filtering the fat to prepare the graft. We compared the subjective and objective outcomes of two fat-processing methods, centrifugation and PureGraft filtration. METHODS: Thirty patients underwent breast reconstruction performed by a single surgeon (OM) after BCT in our department between April 2011 and September 2012. The patients were preoperatively divided into two groups randomly: 15 received fatgrafts processed by centrifugation, and 15 received fatgrafts processed by washing in PureGraft bags. The patients were followed up for 12 to 30 months. To measure the subjective outcome, we distributed the BREAST-Q questionnaire to all the patients both preoperatively and 1 year postoperatively. The BCCT.core software evaluated the objective outcome of breast reconstruction by fatgrafting. RESULTS: The Breast-Q results indicated a tremendous improvement in the modules "Satisfaction with Breast" and "Psychosocial Well-being". The "Sexual Well-being" scale also improved. Only the module "Satisfaction with Breasts" significantly differed between groups; patients treated with the PureGraft fat exhibited better outcomes. The BCCT.core results did not significantly differ between the groups. CONCLUSION: One year postoperatively, the outcomes of the use of PureGraft bags or centrifugation to process fat for breast reconstruction after BCT did not differ. The unpredictability of the results following fatgrafting procedures is likely due to interindividual differences with yet-undisclosed causes.

Unusual regression of severe recurrent lymphatic malformation of a face after contraception and pregnancy.

We report the case of a female who had suffered from progressive lymphatic malformation in the orbito-temporal region since childhood. Many surgical interventions were performed, including radical excision and shunt drainage. Despite aggressive surgical treatment, recurrence was observed after every intervention. Eventually, the condition regressed after the patient began taking a contraceptive. Moreover, it virtually disappeared after pregnancy.

Human keratinocyte growth and differentiation on acellular porcine dermal matrix in relation to wound healing potential.

A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecture in vitro as well as in vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes cultured in vitro on Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7-10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs), CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing.

Use of deepithelialized flap in mammoplasties: simple method with excellent results.

BACKGROUND: A deepithelialized flap is used in almost all surgical fields, particularly in plastic, reconstructive, and aesthetic surgery. This article describes several operating techniques using deepithelialized flaps that in specific cases can improve silicone breast implant coverage. METHODS: All the patients underwent surgery in our department. The operations described are subcutaneous mastectomies with immediate reconstruction using silicone implants, mastopexy with immediate augmentation using silicone implants in a patient with very thin skin, and reaugmentation with mastopexy and reimplantation of silicone implants in a patient with very thin skin and extremely thin pectoral muscles. In all the described operations, we used a superolaterally based deepithelialized flap from the lower part of the breast that we fixed to the thoracic wall to cover the inferior part of the implant. RESULTS: The authors have extensive experience using this flap in such specific cases. To date, they have performed more than 30 operations using this technique. Each patient was followed for 2-7 years, and the findings show excellent postoperative results. None of the patients had implant extrusions, flap extrusions, or infections. The long-term cosmetic results were outstanding. CONCLUSION: The use of deepithelialized flaps in patients with very thin skin or pectoral muscles is a safe and easy way to improve implant coverage and prevent implant extrusion. This technique provides an alternative surgical option that can be beneficial in certain mammary cases.

The use of sentinel skin islands for monitoring buried and semi-buried micro-vascular flaps. Part I: Summary and brief description of monitoring methods.

Micro-vascular flaps have been used for the repair of challenging defects for over 45 years. The risk of failure is reported to be around 5-10% which despite medical and technical advances in recent years remains essentially unchanged. Precise, continuous, sensitive and specific monitoring together with prompt notification of vascular compromise is crucial for the success of the procedure. In this review, we provide a classification and brief description of the reported methods for monitoring the micro-vascular flap and a summary of the benefits over direct visual monitoring. Over 40 different monitoring techniques have been reported but their comparative merits are not always obvious. One looks for early detection of a flap's compromise, improved flap salvage rate and a minimal false-positive or false-negative rate. The cost-effectiveness of any method should also be considered. Direct visualisation of the flap is the method most generally used and still seems to be the simplest, cheapest and most reliable method for flap monitoring. Considering the alternatives, only implantable Doppler ultrasound probes, near infrared spectroscopy and laser Doppler flowmetry have shown any evidence of improved flap salvage rates over direct visual monitoring.

The use of sentinel skin islands for monitoring buried and semi-buried micro-vascular flaps. Part II: Clinical application.

Despite the high success rate of micro-vascular flaps, anastomosis compromise occurs in 5-10% and that can lead to flap failure. Reliable monitoring of the flap is therefore of similar importance to that of the precise surgical procedure itself. Multiple methods have been reported for monitoring of the flap vitality, the first one being direct visual monitoring. In buried flaps direct visualisation is not feasible or is unreliable. In these cases we can extend the buried flap to expose a segment of it to act as a monitoring sentinel. For the purpose of this review we used our clinical experience as a starting point, and for the extended information and expertise we conducted a search of the PubMed database. Over 40 monitoring techniques have been reported to-date. Direct visual monitoring is still generally used method with a reliability of up to 100% and an overall success rate of up to 99%. Direct visualisation remains as the simplest, cheapest and yet a very reliable method of flap monitoring. In this review we provide a description of various possible techniques for externalising part of a buried flap, define the tissues that can be used for this purpose and we summarise the procedures that should be followed to achieve the best reliability and validity of monitoring the skin island.

Comparison of Patient-reported Outcomes after Implant Versus Autologous Tissue Breast Reconstruction Using the BREAST-Q.

BACKGROUND: The demand for reconstructive breast procedures of various types has accelerated in recent years. Coupled with increased patient expectations, it has fostered the development of oncoplastic and reconstructive techniques in breast surgery. In the setting of postmastectomy reconstruction, patient satisfaction and quality of life are the most significant outcome variables when evaluating surgical success. The aim of this study was to evaluate the quality of life after implant breast reconstruction compared with autologous breast reconstruction. MATERIALS AND METHODS: A cross-sectional study design was used. A total of 65 women who had completed postmastectomy implant-based or autologous reconstruction in the participating center were asked to complete the BREAST-Q (Reconstruction Module). RESULTS: Data analysis demonstrated that women with autologous breast reconstruction were significantly more satisfied with their breasts (P = 0.0003) and with the overall outcome (P = 0.0001) compared with women with implant breast reconstruction. All other BREAST-Q parameters that were considered and observed were not significantly different between the 2 patient groups. CONCLUSIONS: Through statistical analysis, our results showed that patients who underwent autologous tissue reconstruction had better satisfaction with the reconstructed breast and the outcome, while both techniques appear to equally improve psychosocial well-being, sexual well-being, and chest satisfaction.

Multiplex PCR and NGS-based identification of mRNA splicing variants: Analysis of BRCA1 splicing pattern as a model.

Alternative pre-mRNA splicing increases transcriptome plasticity by forming naturally-occurring alternative splicing variants (ASVs). Alterations of splicing processes, caused by DNA mutations, result in aberrant splicing and the formation of aberrant mRNA isoforms. Analyses of hereditary cancer predisposition genes reveal many DNA variants with unknown clinical significance (VUS) that potentially affect pre-mRNA splicing. Therefore, a comprehensive description of ASVs is an essential prerequisite for the interpretation of germline VUS in high-risk individuals. To identify ASVs in a gene of interest, we have proposed an approach based on multiplex PCR (mPCR) amplification of all theoretically possible exon-exon junctions and subsequent characterization of size-selected and pooled mPCR products by next-generation sequencing (NGS). The efficiency of this method is illustrated by a comprehensive analysis of BRCA1 ASVs in human leukocytes, normal mammary, and adipose tissues and stable cell lines. We revealed 94 BRCA1 ASVs, including 29 variants present in all tested samples. While differences in the qualitative expression of BRCA1 ASVs among the analyzed human tissues were minor, larger differences were detected between tissue and cell line samples. Compared with other ASV analysis methods, this approach represents a highly sensitive and rapid alternative for the identification of ASVs in any gene of interest.

In vitro and in vivo assessment of chitosan modified urocanic acid as gene carrier.

Chitosan nanoparticles modified with 10 and 30% urocanic acid (CUA) via carbodiimide crosslinking were examined for an efficient gene delivery carrier. The CUA gene carrier was characterized by FTIR, TEM, SEM and the in vitro transfection efficiency CUA polyplex was tested with HeLa and 3T3 cells. The loading efficiency of CUA complexes with DNA was assessed at different N/P ratio of 1, 2, 4, 6, 8, and 10. The DNA loading efficiency was found be to >85% for chitosan, CUA10 and CUA30% and the DNA protection ability of CUA10 and CUA30 nanoparticle complexes was confirmed upon incubation with NheI and HindIII. The cell toxicity and cell viability results have supported the non-toxic nature of CUA10 and CUA30 nanoparticles. In vitro transfection efficiency of CUA10 and CUA30 polyplex was tested for EGFP expression in 3T3 and HeLa cells and a relative maximum % transfection of about 10% was confirmed by CUA10 and CUA30 after 96h transfection. The feasibility and biocompatibility of CUA gene carrier in transgenic chickens was also demonstrated. The in vitro transfection and in vivo embryonic viability studies further confirmed the CUA as promising gene carrier because of the improved biocompatibility and DNA protection ability.