C9orf72-Associated Dipeptide Repeat Expansions Perturb ER-Golgi Vesicular Trafficking, Inducing Golgi Fragmentation and ER Stress, in ALS/FTD.

Hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are debilitating neurodegenerative conditions affecting either motor neurons (ALS) in the brain and spinal cord or neurons in the frontal and/or temporal cortical lobes (FTD). HREs undergo repeat-associated non-ATG (RAN) translation on both sense and anti-sense strands, generating five distinct dipeptide repeat proteins (DPRs), poly-GA, -GR, -GP, -PA and -PR. Perturbed proteostasis is well-recognised in ALS pathogenesis, including processes affecting the endoplasmic reticulum (ER) and Golgi compartments. However, these mechanisms have not been well characterised for C9orf72-mediated ALS/FTD. In this study we demonstrate that C9orf72 DPRs polyGA, polyGR and polyGP (× 40 repeats) disrupt secretory protein transport from the ER to the Golgi apparatus in neuronal cells. Consistent with this finding, these DPRs also induce fragmentation of the Golgi apparatus, activate ER stress, and inhibit the formation of the omegasome, the precursor of the autophagosome that originates from ER membranes. We also demonstrate Golgi fragmentation in cells undergoing RAN translation that express polyGP. Furthermore, dysregulated ER-Golgi transport was confirmed in C9orf72 patient dermal fibroblasts. Evidence of aberrant ER-derived vesicles in spinal cord motor neurons from C9orf72 ALS patients compared to controls was also obtained. These data thus confirm that ER proteostasis and ER-Golgi transport is perturbed in C9orf72-ALS in the absence of protein over-expression. Hence this study identifies novel molecular mechanisms associated with the ER and Golgi compartments induced by the C9orf72 HRE.

Preventing Bacterial Contamination of Breast Implants Using Infection Mitigation Techniques: An In Vitro Study.

BACKGROUND: Bacterial contamination of implants has been linked to biofilm formation and subsequent infection, capsular contracture, and breast implant-associated anaplastic large cell lymphoma. Reducing contamination during implant insertion should therefore reduce biofilm formation disease sequelae. OBJECTIVES: The aim of this study was to compare levels of contamination between preventative techniques. METHODS: A model to simulate the passage of implants through a skin incision was designed that utilized a sterile textured polyvinyl plastic sheet contaminated with Staphylococcus epidermidis. In the first stage of the polyvinyl contamination model, implants were subject to infection-mitigation techniques and passed through the incision, then placed onto horse blood agar plates and incubated for 24 hours. In the second stage of the study the same contamination was applied to human abdominal wall specimens. A 5 cm incision was made through skin and fat, then implants were passed through and levels of contamination were measured as described. RESULTS: Smooth implants grew a mean of 95 colony-forming units (CFUs; approximately 1 CFU/cm2) and textured implants grew 86 CFUs (also approximately 1 CFU/cm2). CFU counts were analyzed by the Mann-Whitney U-test which showed no significant difference between implant types (P < .05); independent-sample t-tests showed a significant difference. The dependent-variable techniques were then compared as groups by one-way analysis of variance, which also showed a significant reduction compared with the control group (P < .01). CONCLUSIONS: This in vitro study has shown the effectiveness of antiseptic rinse and skin/implant barrier techniques for reducing bacterial contamination of breast implants at the time of insertion.

CD30 Lateral Flow and Enzyme-Linked Immunosorbent Assays for Detection of BIA-ALCL: A Pilot Study.

INTRODUCTION: Breast Implant-Associated Anaplastic Large Cell Lymphoma (BIA-ALCL) commonly presents as a peri-implant effusion (seroma). CD30 (TNFRSF8) is a consistent marker of tumor cells but also can be expressed by activated lymphocytes in benign seromas. Diagnosis of BIA-ALCL currently includes cytology and detection of CD30 by immunohistochemistry or flow cytometry, but these studies require specialized equipment and pathologists' interpretation. We hypothesized that a CD30 lateral flow assay (LFA) could provide a less costly rapid test for soluble CD30 that eventually could be used by non-specialized personnel for point-of-care diagnosis of BIA-ALCL. METHODS: We performed LFA for CD30 and enzyme-linked immunosorbent assay (ELISA) for 15 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. To determine the dynamic range of CD30 detection by LFA, we added recombinant CD30 protein to universal buffer at seven different concentrations ranging from 125 pg/mL to 10,000 pg/mL. We then performed LFA for CD30 on cryopreserved seromas of 10 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. RESULTS: Recombinant CD30 protein added to universal buffer produced a distinct test line at concentrations higher than 1000 pg/mL and faint test lines at 250-500 pg/mL. LFA produced a positive test line for all BIA-ALCL seromas undiluted and for 8 of 10 malignant seromas at 1:10 dilution, whereas 3 of 10 benign seromas were positive undiluted but all were negative at 1:10 dilution. Undiluted CD30 LFA had a sensitivity of 100.00%, specificity of 70.00%, positive predictive value of 76.92%, and negative predictive value of 100.00% for BIA-ALCL. When specimens were diluted 1:10, sensitivity was reduced to 80.00% but specificity and positive predictive values increased to 100.00%, while negative predictive value was reduced to 88.33%. When measured by ELISA, CD30 was below 1200 pg/mL in each of six benign seromas, whereas seven BIA-ALCL seromas contained CD30 levels > 2300 pg/mL, in all but one case calculated from dilutions of 1:10 or 1:50. CONCLUSIONS: BIA-ALCL seromas can be distinguished from benign seromas by CD30 ELISA and LFA, but LFA requires less time (<20 min) and can be performed without special equipment by non-specialized personnel, suggesting future point-of-care testing for BIA-ALCL may be feasible.

Developing an International Framework for Informed Consent in Plastic Surgery: A Focus on Cosmetic Breast Augmentation.

Background: Informed consent is a fundamental pillar of patient rights and is an essential part of good clinical practice. In 2019, the International Confederation of Plastic Surgery Societies launched a survey to collect feedback on informed consent practices, with an aim to develop an international guideline for cosmetic surgery. Methods: A 15-question survey was sent to delegates of the International Confederation of Plastic Surgery Societies for dissemination to their national society members. The survey comprised a range of quantitative and qualitative questions. Descriptive and thematic analysis was performed. Results: There were 364 respondents. Over half of the respondents reported no local informed consent policy, whereas others noted national society, specialist college, or government policies. The majority of respondents believed that the performing surgeon should be responsible for obtaining informed consent with at least two face-to-face consultations. Most respondents agreed with a cooling-off period (duration based on procedure type and use of high-risk devices). Regarding cosmetic breast augmentation, the majority of respondents felt that the performing surgeon should be responsible for postoperative management, including cases that occur as part of surgical tourism. Some respondents incorporate financial consent as part of their informed consent practice. Most supported the development of an international informed consent guideline. Conclusions: Informed consent should result from face-to-face consultations with the performing surgeon. There should be a minimum cooling-off period. Postoperative surveillance should be available in all settings. The findings of this survey will help inform an international standardized informed consent guideline for cosmetic surgery.

Diagnosis of breast implant associated anaplastic large cell lymphoma by analysis of cytokines in peri-implant seromas.

More than 1300 women with breast implants have developed an anaplastic large cell lymphoma (ALCL) in fluid (seroma) around their implant. More often, seromas are due to benign causes, for example, capsule contracture, leakage, or trauma. Our report in American Journal of Hematology identified several cytokines (IL-9, IL-10, IL-13) as significantly elevated only in seromas due to ALCL. We further showed that the most robust biomarker, IL-10, could be detected by a lateral flow assay (similar to COVID detection) within minutes allowing physicians to quickly plan management, eliminate or reduce costly testing and patient time away from family. Early detection of ALCL in seromas before infiltration may avoid need for cytotoxic or immunotherapy and is possibly life-saving.

Topical Tranexamic Acid in Primary Breast Augmentation Surgery: Short- and Long-term Outcomes.

BACKGROUND: Breast augmentation mammaplasty (BAM) remains the most popular cosmetic procedure done worldwide. Bleeding in this procedure increases the chance of capsular contracture. Tranexamic acid (TXA), an antifibrinolytic, has been widely used by other surgical specialties to reduce bleeding. OBJECTIVES: We aimed to evaluate the use of TXA in BAM surgery. METHODS: This was a single-surgeon case series of all patients who underwent primary BAM from March 2017 to March 2018 and received topical TXA spray to the implant pocket before implant insertion. Early postoperative complications and long-term outcomes, such as capsular contracture and revisional surgery, were recorded and described. RESULTS: Two hundred and eighty-eight patients were included in the study with an overall complication rate of 2.8% over 5 years. No patients had postoperative bleeding or hematoma formation. One patient had a seroma, managed with ultrasound drainage. Complications requiring reoperation included rippling (3, 1.0%), pocket revision (2, 0.7%), capsule contracture (1, 0.3%) and rupture (1, 0.3%). CONCLUSIONS: This study highlights the safety and potential benefits of the use of topical TXA in breast augmentation, with low bleeding and capsular contracture rates.

Disease Focused Integrated Care - a New Model of Healthcare Delivery for the Treatment of Skin Cancer.

Introduction: As the most common cancer in Australia, skin cancer generates a considerable health burden. This study outlines the establishment of a new model of integrated care for the diagnosis and management of skin cancer. Methods: A new model of integrated care was established to provide access to all aspects of skin cancer management. General practitioners (GPs) were upskilled through hands-on training and a 6-month skin cancer education program and partnered with specialist Dermatologists and Plastic Surgeons co-located in the same clinic. Data including median wait times between the initial consultation and treatment were prospectively collected and compared patients seen through the integrated pathway to patients referred from their primary GP to specialist Dermatologists and Plastic Surgeons directly (non-integrated pathway). The percentage of patients needing co-consultation with a specialist in the integrated pathway was also measured over time. Results: A total of 25341 patients were seen from the commencement of the clinic in August 2015 to June 2021. In 2017 and 2018 the median wait time to be treated was 7 days for the integrated model compared to 54 days (2017) and 46 days (2018) for non-integrated care (p < 0.0001). The percentage of GPs requesting specialist co-consultations for assessment of skin cancer fell from 98% in 2015, to 5.6% in 2021. Histopathology shows that 66% of lesions excised by GPs in this model were malignant or pre-malignant. Conclusions: This study firstly shows a significant reduction in time to treatment in an integrated skin cancer model over traditional models of health. Secondly it demonstrates GP upskilling over time in the integrated program. Integrating GP and specialist medical practitioners in the treatment of skin cancer offers potential for more efficient, accessible, and affordable care. This cooperative, co-located model may provide a template for the integrating the management of other conditions.

Staphylococcus aureus Cell Wall Phenotypic Changes Associated with Biofilm Maturation and Water Availability: A Key Contributing Factor for Chlorine Resistance.

Staphylococcus aureus biofilms are resistant to both antibiotics and disinfectants. As Staphylococci cell walls are an important defence mechanism, we sought to examine changes to the bacterial cell wall under different growth conditions. Cell walls of S. aureus grown as 3-day hydrated biofilm, 12-day hydrated biofilm, and 12-day dry surface biofilm (DSB) were compared to cell walls of planktonic organisms. Additionally, proteomic analysis using high-throughput tandem mass tag-based mass spectrometry was performed. Proteins involved in cell wall synthesis in biofilms were upregulated in comparison to planktonic growth. Bacterial cell wall width (measured by transmission electron microscopy) and peptidoglycan production (detected using a silkworm larva plasma system) increased with biofilm culture duration (p < 0.001) and dehydration (p = 0.002). Similarly, disinfectant tolerance was greatest in DSB, followed by 12-day hydrated biofilm and then 3-day biofilm, and it was least in the planktonic bacteria--suggesting that changes to the cell wall may be a key factor for S. aureus biofilm biocide resistance. Our findings shed light on possible new targets to combat biofilm-related infections and hospital dry surface biofilms.

Enhanced Skin Penetration of Cannabidiol Using Organosilane Particles as Transdermal Delivery Vehicles.

There is potential for cannabidiol to act as an analgesic, anxiolytic and antipsychotic active ingredient; however, there is a need to find alternate administration routes to overcome its low oral bioavailability. In this work, we propose a new delivery vehicle based on encapsulation of cannabidiol within organosilica particles as drug delivery vehicles, which are subsequently incorporated within polyvinyl alcohol films. We investigated the long-term stability of the encapsulated cannabidiol, as well as its release rate, in a range of simulated fluids with different characterization techniques, including Fourier Transform Infrared (FT-IR) and High-performance Liquid Chromatography (HPLC). Finally, we determined the transdermal penetration in an ex vivo skin model. Our results show that cannabidiol is stable for up to 14 weeks within polyvinyl alcohol films at a range of temperatures and humidity. Release profiles are first-order, consistent with a mechanism involving diffusion of the cannabidiol (CBD) out of the silica matrix. The silica particles do not penetrate beyond the stratum corneum in the skin. However, cannabidiol penetration is enhanced and is detected in the lower epidermis, which was 0.41% of the total CBD in a PVA formulation compared with 0.27% for pure CBD. This is partly due to an improvement of its solubility profile as it is released from the silica particles, but we cannot rule out effects of the polyvinyl alcohol. Our design opens a route for new membrane technologies for cannabidiol and other cannabinoid products, where administration via non-oral or pulmonary routes can lead to better outcomes for patient cohorts in a range of therapeutics.

Making Sense of Late Tissue Nodules Associated With Hyaluronic Acid Injections.

BACKGROUND: The pathogenesis of delayed-onset tissue nodules (DTNs) due to hyaluronic acid (HA) injections is uncertain. OBJECTIVES: To formulate a rational theory for DTN development and their avoidance and treatment. METHODS: A multidisciplinary and multicountry DTN consensus panel was established, with 20 questions posed and consensus sought. Consensus was set at 75% agreement. RESULTS: Consensus was reached in 16 of 20 questions regarding the pathogenesis of DTNs, forming the basis for a classification and treatment guide. CONCLUSIONS: The group believes that filler, pathogens, and inflammation are all involved in DTNs and that DTNs most likely are infection initiated with a variable immune response. Injected filler may incorporate surface bacteria, either a commensal or a true pathogen, if the skin barrier is altered. The initially high molecular weight HA filler is degraded to low molecular weight HA (LMWHA) at the edge of the filler. Commensals positioned within the filler bolus may be well tolerated until the filler is degraded and the commensal becomes visible to the immune system. LMWHA is particularly inflammatory in the presence of any local bacteria. Commensals may still be tolerated unless the immune system is generally heightened by viremia or vaccination. Systemic pathogenic bacteremia may also interact with the filler peripheral LMWHA, activating Toll-like receptors that induce DTN formation. Given this scenario, attention to practitioner and patient hygiene and early systemic infection treatment deserve attention. Classification and treatment systems were devised by considering each of the 3 factors-filler, inflammation, and infection-separately.

Prospective Study of Clinical Outcomes From a Breast Implant Assessment Service.

BACKGROUND: Breast augmentation remains the commonest cosmetic surgical procedure worldwide, in spite of recent regulatory action. OBJECTIVES: The aim of this study was to evaluate women with breast implants attending a breast implant assessment clinic and to capture clinical and implant data in women presenting to the service. METHODS: Patients were enrolled prospectively between January 2018 and December 2021. Clinical, implant, and practitioner data were recorded. Patients reported satisfaction on size, shape, and overall outcome as well as the presence or pain. Radiological evaluation, where indicated, was performed and data were included on these findings. RESULTS: A total of 603 patients were assessed. Their mean age was 42.7 years and mean age at implantation was 29.1 years. The most common complications were capsular contracture followed by pain, waterfall deformity, and double bubble, with rupture/contracture rates increasing after the 10-year mark. The risk of double bubble was significantly lower if patients were operated on by certified practitioners (odds ratio = 0.49, P = 0.011). There was almost universally poor awareness of the risks of breast implants in patients presenting for evaluation. CONCLUSIONS: This study has shown benefit in a breast implant assessment clinic to gather information on adverse events and patient-reported outcomes following breast implant surgery. Having appropriately trained and certified practitioners perform cosmetic augmentation significantly lowers the risk of implant malposition and deformity. Any adverse event occurring within 5 years of initial surgery should be flagged as a mandatory reportable clinical indicator and trigger further investigation.

One Step Forward with Dry Surface Biofilm (DSB) of Staphylococcus aureus: TMT-Based Quantitative Proteomic Analysis Reveals Proteomic Shifts between DSB and Hydrated Biofilm.

The Gram-positive bacterium Staphylococcus aureus is responsible for serious acute and chronic infections worldwide and is well-known for its biofilm formation ability. Recent findings of biofilms on dry hospital surfaces emphasise the failures in current cleaning practices and disinfection and the difficulty in removing these dry surface biofilms (DSBs). Many aspects of the formation of complex DSB biology on environmental surfaces in healthcare settings remains limited. In the present study, we aimed to determine how the protein component varied between DSBs and traditional hydrated biofilm. To do this, biofilms were grown in tryptic soy broth (TSB) on removable polycarbonate coupons in the CDC biofilm reactor over 12 days. Hydrated biofilm (50% TSB for 48 h, the media was then changed every 48 h with 20% TSB, at 37 °C with 130 rpm). DSB biofilm was produced in 5% TSB for 48 h at 35 °C followed by extended periods of dehydration (48, 66, 42 and 66 h at room temperature) interspersed with 6 h of 5% TSB at 35 °C. Then, we constructed a comprehensive reference map of 12-day DSB and 12-day hydrated biofilm associated proteins of S. aureus using a high-throughput tandem mass tag (TMT)-based mass spectrometry. Further pathway analysis of significantly differentially expressed identified proteins revealed that proteins significantly upregulated in 12-day DSB include PTS glucose transporter subunit IIBC (PtaA), UDP-N-acetylmuramate-L-alanine ligase (MurC) and UDP-N-acetylenolpyruvoylglucosamine (MurB) compared to 12-day hydrated biofilm. These three proteins are all linked with peptidoglycan biosynthesis pathway and are responsible for cell-wall formation and thicker EPS matrix deposition. Increased cell-wall formation may contribute to the persistence of DSB on dry surfaces. In contrast, proteins associated with energy metabolisms such as phosphoribosyl transferase (PyrR), glucosamine--fructose-6-phosphate aminotransferase (GlmS), galactose-6-phosphate isomerase (LacA), and argininosuccinate synthase (ArgG) were significantly upregulated whereas ribosomal and ABC transporters were significantly downregulated in the 12-day hydrated biofilm compared to DSB. However, validation by qPCR analysis showed that the levels of gene expression identified were only partially in line with our TMT-MS quantitation analysis. For the first time, a TMT-based proteomics study with DSB has shed novel insights and provided a basis for the identification and study of significant pathways vital for biofilm biology in this reference microorganism.

Mild Positive Pressure Improves the Efficacy of Benzalkonium Chloride against Staphylococcus aureus Biofilm.

Current protocols using liquid disinfectants to disinfect heat-sensitive hospital items frequently fail, as evidenced by the continued isolation of bacteria following decontamination. The contamination is, in part, due to biofilm formation. We hypothesize that mild positive pressure (PP) will disrupt this biofilm structure and improve liquid disinfectant/detergent penetration to biofilm bacteria for improved killing. Staphylococcus aureus biofilm, grown on polycarbonate coupons in the biofilm reactor under shear at 35 °C for 3 days, was treated for 10 min and 60 min with various dilutions of benzalkonium chloride without PP at 1 atmosphere (atm), and with PP at 3, 5, 7, and 10 atm. The effect on biofilm and residual bacterial viability was determined by standard plate counts, confocal laser scanning microscopy, and scanning electron microscopy. Combined use of benzalkonium chloride and PP up to 10 atm significantly increased biofilm killing up to 4.27 logs, as compared to the treatment using disinfectant alone. Microscopy results were consistent with the viability plate count results. PP improved disinfectant efficacy against bacterial biofilm. The use of mild PP is possible in many flow situations or if equipment/contaminated surfaces can be placed in a pressure chamber.

Proteome of Staphylococcus aureus Biofilm Changes Significantly with Aging.

Staphylococcus aureus is a notorious biofilm-producing pathogen that is frequently isolated from implantable medical device infections. As biofilm ages, it becomes more tolerant to antimicrobial treatment leading to treatment failure and necessitating the costly removal of infected devices. In this study, we performed in-solution digestion followed by TMT-based high-throughput mass spectrometry and investigated what changes occur in the proteome of S. aureus biofilm grown for 3-days and 12-days in comparison with 24 h planktonic. It showed that proteins associated with biosynthetic processes, ABC transporter pathway, virulence proteins, and shikimate kinase pathway were significantly upregulated in a 3-day biofilm, while proteins associated with sugar transporter, degradation, and stress response were downregulated. Interestingly, in a 3-day biofilm, we observed numerous proteins involved in the central metabolism pathways which could lead to biofilm growth under diverse environments by providing an alternative metabolic route to utilize energy. In 12-day biofilms, proteins associated with peptidoglycan biosynthesis, sugar transporters, and stress responses were upregulated, whereas proteins associated with ABC transporters, DNA replication, and adhesion proteins were downregulated. Gene Ontology analysis revealed that more proteins are involved in metabolic processes in 3dwb compared with 12dwb. Furthermore, we observed significant variations in the formation of biofilms resulting from changes in the level of metabolic activity in the different growth modes of biofilms that could be a significant factor in S. aureus biofilm maturation and persistence. Collectively, potential marker proteins were identified and further characterized to understand their exact role in S. aureus biofilm development, which may shed light on possible new therapeutic regimes in the treatment of biofilm-related implant-associated infections.

Efficacy of Surgical/Wound Washes against Bacteria: Effect of Different In Vitro Models.

Topical antiseptics are often used to treat chronic wounds with biofilm infections and during salvage of biofilm contaminated implants, but their antibacterial efficacy is frequently only tested against non-aggregated planktonic or free-swimming organisms. This study evaluated the antibacterial and antibiofilm efficacy of four commercial surgical washes Bactisure, TorrenTX, minimally invasive lavage (MIS), and Betadine against six bacterial species: Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pyogenes, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli, which are commonly isolated from surgical site infections and chronic wound infections using different in vitro models. We determined minimum planktonic inhibitory and eradication concentration and minimum 1-day-old biofilm inhibition and eradication concentration of antiseptics in 96-well plates format with 24 h contact time. We also tested the efficacy of antiseptics at in-use concentration and contact time in the presence of biological soil against 3-day-old biofilm grown on coupons with shear in a bioreactor, such that the results are more applicable to the clinical biofilm situations. In the 96-well plate model, the minimum concentration required to inhibit or kill planktonic and biofilm bacteria was lower for Bactisure and TorrenTX than for MIS and Betadine. However, Betadine and Bactisure showed better antibiofilm efficacy than TorrenTX and MIS in the 3-day-old biofilm bioreactor model at in-use concentration. The minimal concentration of surgical washes required to inhibit or kill planktonic bacterial cells and biofilms varies, suggesting the need for the development and use of biofilm-based assays to assess antimicrobial therapies, such as topical antiseptics and their effective concentrations. The antibiofilm efficacy of surgical washes against different bacterial species also varies, highlighting the importance of testing against various bacterial species to achieve a thorough understanding of their efficacy.