Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

Prediction, screening and characterization of novel bioactive tetrapeptide matrikines for skin rejuvenation.

BACKGROUND: Extracellular matrices play a critical role in tissue structure and function and aberrant remodelling of these matrices is a hallmark of many age-related diseases. In skin, loss of dermal collagens and disorganization of elastic fibre components are key features of photoageing. Although the application of some small matrix-derived peptides to aged skin has been shown to beneficially affect in vitro cell behaviour and, in vivo, molecular architecture and clinical appearance, the discovery of new peptides has lacked a guiding hypothesis. OBJECTIVES: To identify, using protease cleavage site prediction, novel putative matrikines with beneficial activities for skin composition and structure. METHODS: Here, we present an in silico (peptide cleavage prediction) to in vitro (proteomic and transcriptomic activity testing in cultured human dermal fibroblasts) to in vivo (short-term patch test and longer-term split-face clinical study) discovery pipeline, which enables the identification and characterization of peptides with differential activities. RESULTS: Using this pipeline we showed that cultured fibroblasts were responsive to all applied peptides, but their associated bioactivity was sequence-dependent. Based on bioactivity, toxicity and protein source, we further characterized a combination of two novel peptides, GPKG (glycine-proline-lysine-glycine) and LSVD (leucine-serine-valine-aspartate), that acted in vitro to enhance the transcription of matrix -organization and cell proliferation genes and in vivo (in a short-term patch test) to promote processes associated with epithelial and dermal maintenance and remodelling. Prolonged use of a formulation containing these peptides in a split-face clinical study led to significantly improved measures of crow's feet and firmness in a mixed population. CONCLUSIONS: This approach to peptide discovery and testing can identify new synthetic matrikines, providing insights into biological mechanisms of tissue homeostasis and repair and new pathways to clinical intervention.

Like other organs and tissues, the skin is composed of both cells and a complex network of molecules and proteins called an extracellular matrix. This matrix contains proteins such as collagen and elastin and undergoes many changes when the skin is damaged by the sun. We know from previous studies that small parts of matrix proteins (called peptide 'matrikines') can help to treat the signs of sun-related skin ageing. In this UK study, we show that new beneficial peptides (with matrikine activity) can be identified using machine learning (artificial intelligence) techniques that predict where common matrix proteins might be 'cut' by skin enzymes. Candidate peptides were first made in the laboratory and then applied to skin cells in culture. These cell culture screens demonstrated that, while all the peptides showed some matrikine activity, two were particularly promising. These two peptides were then tested in a short-term study on the forearm skin of volunteers and, in a longer-term study, on the face. We found that the combination of these two peptides can prompt forearm skin cells to express genes that are involved in many different aspect of skin health and, over the longer 6-month period, produce visible benefits in the appearance of fine lines and wrinkles and firmness on the face. Our findings suggest that this approach may be able to identify beneficial peptide treatments for not only skin ageing and diseases, but also unwanted changes in the extracellular matrix of other tissues and organs.

Proposed clinical operative synoptic report for ileocolic resection for Crohn's disease.

BACKGROUND: Crohn's disease patients may require multiple surgeries during their lifetime. Because operative reports are not standardized, information relevant to future management may not be documented. Synoptic reports used in other fields such as histopathology have proven to be effective and allow consistent documentation of results. The aim of this study was to retrospectively review the completeness of the operative reports for ileocolic Crohn's resections (ICR) and to propose a synoptic report. METHODS: A draft synoptic operative report for ICR for Crohn's disease was presented in the IBD multidisciplinary meeting and a Delphi process used to gain consensus for inclusion in the synoptic report. Retrospective analysis of consecutive ICR from January 2010 to April 2023 was undertaken to determine the presence of the standardized criteria. RESULTS: A total of 66 ICR were performed in 63 patients during the study period. No operation reports were excluded. The examination of bowel for macroscopic disease was partially documented in 88% cases. The extent of mesenteric resection and any difficulty encountered during dissection were poorly documented. The remaining length of small and large intestines was not documented in most operative reports. The clinical sections that were compulsory entrance in the electronic operative report achieved 100% compliance. CONCLUSION: This study has demonstrated that key information was often deficient in the operative report. This may have a significant impact on the future management of Crohn's patients and affects the interpretation of research outcomes. A proposed clinical synoptic operative report is easy to use and ensures compliance.

A single centre experience on the formation of double barrelled uro-colostomy in pelvic exenteration surgery: a cohort study.

BACKGROUND: Double barrelled uro-colostomy (DBUC) is an alternative to traditional ileal conduit (IC) and separate colostomy in patients requiring simultaneous urinary and faecal diversion for reconstruction in pelvic exenteration surgery (PES). METHODS: This cohort study evaluated short- and long-term morbidity and mortality associated with DBUC formation in 20 consecutive adult patients undergoing PES in an Australian Complex Pelvic Surgical Unit. Data were obtained from a prospective database. RESULTS: Mean age 59 years (range 27-76 years). PES was performed for malignant disease in 18 patients (curative intent in 17). Mean operative duration 11.8 h (range 7-17 h). Mean follow-up duration 29.1 months (range 2.6-90.1 months). Early DBUC-related complications occurred in four patients (20.0%): urinary tract infection (UTI)/urosepsis (n = 4) and early ureteric stenosis requiring intervention (n = 1). Late DBUC-related complications occurred in five patients (25.0%): recurrent UTI/urosepsis (n = 4), chronic kidney disease (n = 4), ureteric stenosis (n = 2) and parastomal hernia (n = 4). No mortality occurred secondary to a DBUC complication. CONCLUSION: DBUC is a safe reconstructive option with acceptable morbidity profile in patients requiring simultaneous urinary and faecal diversion.

Australian experience of side-to-side isoperistaltic stricturoplasty in Crohn's disease.

BACKGROUND: The Michelassi stricturoplasty has demonstrated efficacy for Crohn's disease in European and American series but has not had uptake in Australia. We report the short-term results of side-to-side isoperistaltic stricturoplasty (SSIS) in an Australian Practice. METHODS: Between March 2015 and October 2021 SSIS procedures were performed on Crohn's patients with long segment Crohn's strictures associated with obstructive symptoms, despite best medical therapy. Surgical demographics and results were recorded via inpatient and outpatient follow-up in a prospective database. RESULTS: Twenty-one SSIS performed in 16 patients, nine female, mean age 40 years. Single incision laparoscopic surgery (SILS) was used in 10 patients. The standard Michelassi SSIS used for 11 strictures and a Poggioli variant used for 10. Mean stricture length 32 cm (range 5-100); mean SSIS length 24 cm (range 6-55). Associated bowel resection in seven cases with a mean length of 47 mm. Ten patients had an average of three additional stricturoplasties. Complications included central line sepsis in one, deep surgical site infection in one and superficial wound infection in four patients. Mean duration of operation; 346 min and length of stay 10 days. CONCLUSION: SSIS techniques are safe for the management of long segment stricturing Crohn's disease. Although not widely used in Australia, surgeons should consider the Michelassi stricturoplasty, and its variants, for long Crohn's strictures as they are isoperistaltic whilst avoiding bowel resection and blind pouches.

Colonic Epstein-Barr Virus-Associated Mucocutaneous Ulcer Associated With Ulcerative Colitis.

Epstein-Barr virus-associated mucocutaneous ulcer is a rare lymphoproliferative disorder that occurs in immunosuppressed states that can develop in the gastrointestinal tract and mimic inflammatory bowel disease or other malignancies. We present the case of a 61-year-old man who presented with concurrent acute severe ulcerative colitis and colonic Epstein-Barr virus-associated mucocutaneous ulcer requiring rituximab therapy and a subtotal colectomy.

Skin to origin of mesenteric artery study (STOMA): prospective study of distances between stoma location and proximal superior mesenteric artery.

BACKGROUND: Traditional siting of stomas, in the lower abdomen, has been guided by surgical dogma lacking evidence. In the lower abdomen, the combination of a thick and pendulous abdominal apron, can create a challenging and suboptimal site for a stoma. The anatomical determinant limiting delivery of a stoma to the abdominal skin is the distance of the SMA from the lower border of the pancreas. The aim of this cross-sectional study was to compare the distance between the traditional stoma site, and upper abdominal stoma sites, to both the superior mesenteric artery (SMA) origin and SMA at the inferior border of the pancreas on abdominal computed tomography (CT). METHODS: A cross-sectional study at a single academic university hospital of adult patients who underwent abdominal CT in Australia. RESULTS: Two hundred and thirteen patients were included. Stoma sites in the upper abdomen were 57-76 mm shorter to the origin of the SMA and inferior border of the pancreas than those positioned at the traditional stoma site (P < 0.001). The mean panniculus thickness in the upper abdomen was 10 mm thinner than in the lower abdomen and increased with increasing BMI (P < 0.001). The ratio between the distance from the xiphisternum to umbilicus, and the umbilicus to pubic symphysis, was 1.10; this ratio increased with increasing BMI. CONCLUSION: The distance of the SMA to the skin is always shorter in the upper abdomen compared to the traditional stoma site. Consideration should be given to placing stomas in the upper abdomen, particularly in overweight or obese patients.

Evaluation of a COVID-19 convalescent plasma program at a U.S. academic medical center.

Amidst the therapeutic void at the onset of the COVID-19 pandemic, a critical mass of scientific and clinical interest coalesced around COVID-19 convalescent plasma (CCP). To date, the CCP literature has focused largely on safety and efficacy outcomes, but little on implementation outcomes or experience. Expert opinion suggests that if CCP has a role in COVID-19 treatment, it is early in the disease course, and it must deliver a sufficiently high titer of neutralizing antibodies (nAb). Missing in the literature are comprehensive evaluations of how local CCP programs were implemented as part of pandemic preparedness and response, including considerations of the core components and personnel required to meet demand with adequately qualified CCP in a timely and sustained manner. To address this gap, we conducted an evaluation of a local CCP program at a large U.S. academic medical center, the University of North Carolina Medical Center (UNCMC), and patterned our evaluation around the dimensions of the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework to systematically describe key implementation-relevant metrics. We aligned our evaluation with program goals of reaching the target population with severe or critical COVID-19, integrating into the structure of the hospital-wide pandemic response, adapting to shifting landscapes, and sustaining the program over time during a compassionate use expanded access program (EAP) era and a randomized controlled trial (RCT) era. During the EAP era, the UNCMC CCP program was associated with faster CCP infusion after admission compared with contemporaneous affiliate hospitals without a local program: median 29.6 hours (interquartile range, IQR: 21.2-48.1) for the UNCMC CCP program versus 47.6 hours (IQR 32.6-71.6) for affiliate hospitals; (P<0.0001). Sixty-eight of 87 CCP recipients in the EAP (78.2%) received CCP containing the FDA recommended minimum nAb titer of ≥1:160. CCP delivery to hospitalized patients operated with equal efficiency regardless of receiving treatment via a RCT or a compassionate-use mechanism. It was found that in a highly resourced academic medical center, rapid implementation of a local CCP collection, treatment, and clinical trial program could be achieved through re-deployment of highly trained laboratory and clinical personnel. These data provide important pragmatic considerations critical for health systems considering the use of CCP as part of an integrated pandemic response.

Apoptotic priming is defined by the dynamic exchange of Bcl-2 proteins between mitochondria and cytosol.

Apoptosis is regulated by interactions between the BH3-only and multi-domain Bcl-2 family proteins. These interactions are integrated on the outer mitochondrial membrane (OMM) where they set the threshold for apoptosis, known as mitochondrial priming. However, how mitochondrial priming is controlled at the level of single cells remains unclear. Retrotranslocation of Bcl-XL has been proposed as one mechanism, removing pro-apoptotic Bcl-2 proteins from the OMM, thus reducing priming. Contrary to this view, we now show that Bcl-XL retrotranslocation is inhibited by binding to its BH3-only partners, resulting in accumulation of these protein complexes on mitochondria. We find that Bcl-XL retrotranslocation dynamics are tightly coupled to mitochondrial priming. Quantifying these dynamics indicates the heterogeneity in priming between cells within a population and predicts how they subsequently respond to a pro-apoptotic signal.

Mitochondrial dynamics regulate genome stability via control of caspase-dependent DNA damage.

Mitochondrial dysfunction is interconnected with cancer. Nevertheless, how defective mitochondria promote cancer is poorly understood. We find that mitochondrial dysfunction promotes DNA damage under conditions of increased apoptotic priming. Underlying this process, we reveal a key role for mitochondrial dynamics in the regulation of DNA damage and genome instability. The ability of mitochondrial dynamics to regulate oncogenic DNA damage centers upon the control of minority mitochondrial outer membrane permeabilization (MOMP), a process that enables non-lethal caspase activation leading to DNA damage. Mitochondrial fusion suppresses minority MOMP and its associated DNA damage by enabling homogeneous mitochondrial expression of anti-apoptotic BCL-2 proteins. Finally, we find that mitochondrial dysfunction inhibits pro-apoptotic BAX retrotranslocation, causing BAX mitochondrial localization and thereby promoting minority MOMP. Unexpectedly, these data reveal oncogenic effects of mitochondrial dysfunction that are mediated via mitochondrial dynamics and caspase-dependent DNA damage.

Matrikines as mediators of tissue remodelling.

Extracellular matrix (ECM) proteins confer biomechanical properties, maintain cell phenotype and mediate tissue repair (via release of sequestered cytokines and proteases). In contrast to intracellular proteomes, where proteins are monitored and replaced over short time periods, many ECM proteins function for years (decades in humans) without replacement. The longevity of abundant ECM proteins, such as collagen I and elastin, leaves them vulnerable to damage accumulation and their host organs prone to chronic, age-related diseases. However, ECM protein fragmentation can potentially produce peptide cytokines (matrikines) which may exacerbate and/or ameliorate age- and disease-related ECM remodelling. In this review, we discuss ECM composition, function and degradation and highlight examples of endogenous matrikines. We then critically and comprehensively analyse published studies of matrix-derived peptides used as topical skin treatments, before considering the potential for improvements in the discovery and delivery of novel matrix-derived peptides to skin and internal organs. From this, we conclude that while the translational impact of matrix-derived peptide therapeutics is evident, the mechanisms of action of these peptides are poorly defined. Further, well-designed, multimodal studies are required.

Elevated EDAR signalling promotes mammary gland tumourigenesis with squamous metaplasia.

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong ß-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes ß-catenin. Deletion of this exon yields unconstrained ß-catenin signalling activity. We also demonstrate that ß-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which ß-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways.

Laparoscopic Resection of an Acute Rectosigmoid Intussusception Due to a Giant Pedunculated Lipoma.

Intussusception is the invagination and telescopic migration of a proximal segment of the gastrointestinal tract distally and is classically described in the paediatric age group, accounting for 95% of reported cases. Intussusception in adults is highly uncommon; its aetiology involves a wide spectrum of pathologies, namely, concerning malignancy and differing management pathways. In the management of adult intussusception, consideration must be given to the potential malignant nature of the lesion, as reducing a malignant segment could render dissemination. Intussusception with non-malignant lesions like lipomas has been described, usually involving the right and transverse colons. In this case report, we review a rare case of adult colorectal intussusception secondary to a submucosal sigmoid lipoma.

Natural orifice specimen extraction for colorectal surgery: Early adoption in a Western population.

AIM: Natural orifice specimen extraction (NOSE) challenges the limits of minimally invasive colorectal surgery by exploiting a natural opening for specimen delivery. Technically challenging, it is less painful, requires smaller wounds and abolishes the possibility of incisional hernia. These advantages of NOSE are seen in the obese (body mass index [BMI] >30 kg/m2 ). This audit aims to demonstrate the feasibility of NOSE colectomy in an Australian population. METHOD: Prospective data collected from 2007 to the present were retrospectively analysed. Only patients with mucosally benign colorectal conditions were included: complex diverticulosis, post-malignant polypectomy and volvulus. Left sided mucosal malignancies were excluded. Study end-points included postoperative length of stay, anastomotic leak rate and wound complications. RESULTS: In total, 159 patients underwent NOSE, mean age 59 years (19-88), mean BMI 28.2 kg/m2 (17-45). Ten (6.2%) patients developed retroperitoneal small bowel herniation; seven required further surgery. There were five (3.1%) anastomotic leaks, seven (4.4%) postoperative ileus and three (1.9%) anastomotic bleeds. One (0.6%) patient had a superficial wound infection. There were no port site hernias. Patients with BMI <30 kg/m2 (98 patients) and BMI >30 kg/m2 (59 patients) were compared; there was no difference in anastomotic leak rate (P = 0.60), complication rate (P = 0.71) and length of stay (P = 0.63). However, duration of operation increased with BMI (P = 0.000). CONCLUSION: This large series of NOSE colectomy from Australia suggests that NOSE is comparable to conventional laparoscopic colectomy in terms of postoperative outcome. Given that obesity has not featured in the NOSE literature, our study suggests that NOSE, for benign disease, is safe in obese patients, without added morbidity.

Laparoscopic right hemicolectomy with intracorporeal anastomosis and natural orifice surgery extraction/minimal extraction site surgery in the obese.

BACKGROUND: Despite advantages associated with laparoscopic colorectal surgery, there is significant morbidity associated with incisions required for specimen extraction and restoration of bowel continuity. In laparoscopic colorectal surgery, the length of the longest incision depends upon that required to facilitate extra-corporeal steps. The purpose of this study was to analyse obese patients (body mass index >30 kg/m2 ) who have undergone laparoscopic small bowel or right-sided colonic resection with intracorporeal anastomosis (ICA) and natural orifice surgery extraction (NOSE)/minimal extraction site (MES) surgery. METHODS: A retrospective review of 11 obese patients who have undergone laparoscopic small bowel and right-sided colonic resection with ICA and NOSE/MES was conducted. RESULTS: Mean body mass index was 40.4 kg/m2 (range 32.7-56 kg/m2 ) in 11 patients. Procedures performed were laparoscopic right hemicolectomy (7) - one with high anterior resection, pelvic peritonectomy, bilateral salpingo-oophorectomy and greater omentectomy, small bowel resection (2), transverse colotomy (1) and segmental transverse colectomy (1). All colonic specimens were extracted via NOSE (vaginal colpotomy or transcolonic), except two requiring a miniaturized extraction wound. Small bowel specimens were extracted via a 12-mm port hole, without extension. Mean operating time was 240 min (range 100-510 min). Mean time to discharge was 4 days (range 4-6 days). Complications included a superficial wound infection in a patient presenting with an obstructed tumour and a second patient developed a seroma following small bowel resection for an incarcerated hernia. CONCLUSION: Obese patients can undergo laparoscopic small bowel and right-sided colonic resection with ICA and NOSE/MES surgery and benefit from short length of stay and low morbidity.

Short-term outcomes of parastomal hernia prophylaxis with Stapled Mesh stomA Reinforcement Technique (SMART) in permanent stomas.

BACKGROUND: Parastomal hernias occur in 50-80% after stoma formation. Even with mesh repairs, recurrence can be as high as 33%. Stapled Mesh stomA Reinforcement Technique (SMART) places a prophylactic onlay mesh in the trephine during permanent stoma formation to prevent parastomal hernia. Our study aims to describe the short-term outcomes of SMART procedures. METHODS: A prospective study of patients receiving the SMART procedure from 2015 to 2020 was conducted. INCLUSION CRITERIA: non-Crohn's colorectal and urological surgery with permanent stoma formation. The SMART surgical technique incorporates a 70-mm circular piece of polypropylene mesh by stapling it to the muscular abdominal wall using a circular stapler, and attaching the edge of the mesh to the deep fascia. RESULTS: Fifty patients had a total of 53 SMART procedures. Median follow-up was 27 months. Procedures included: 35 end colostomies, five end ileostomies, eight ileal urinary conduits and five double-barrelled wet colostomies. Four patients had parastomal hernia during follow-up. One was acute, on day 1, due to very large size of trephine, one in a double-barrelled wet stoma that was repaired laparoscopically, one had a stomal prolapse requiring revision at 3 years and one patient had early small bowel obstruction due to very small size of trephine requiring another surgery. There were no wound infections or mesh-related sepsis. CONCLUSION: Symptomatic parastomal herniation occurred in 8% of the study population, and most complications were due to incorrect choice of stapled trephine diameter. Longer term follow-up is required to assess for problematic parastomal hernia.