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.

Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization.

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

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.

Bax exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming.

The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol. Using live-cell spinning-disk confocal imaging with a combination of FLIP, FRAP, and photoactivatable GFP-Bax, we demonstrate that disrupting adhesion-dependent survival signals slows the rate of Bax's dissociation from mitochondria, leading to its accumulation on the outer mitochondrial membrane. The overall accumulation of mitochondrial Bax following loss of survival signaling sensitizes cells to proapoptotic BH3 proteins. Our findings show that Bax is normally in a dynamic equilibrium between cytosol and mitochondria, enabling fluctuations in survival signals to finely adjust apoptotic sensitivity.

E2F1 interacts with BCL-xL and regulates its subcellular localization dynamics to trigger cell death.

E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes. This prevents efficient control of BCL-xL over its binding partners, in particular over BAK resulting in the induction of cell death. We thus identify a new, non-BH3-binding regulator of BCL-xL localization dynamics that influences its anti-apoptotic activity.

Concomitant Abdominoplasty and Laparoscopic Umbilical Hernia Repair.

BACKGROUND: Umbilical hernia is a common finding in patients undergoing abdominoplasty, especially those who are postpartum with rectus divarication. Concurrent surgical treatment of the umbilical hernia at abdominoplasty presents a "vascular challenge" due to the disruption of dermal blood supply to the umbilicus, leaving the stalk as the sole axis of perfusion. To date, there have been no surgical techniques described to adequately address large umbilical herniae during abdominoplasty. OBJECTIVES: To present an effective and safe technique that can address large umbilical herniae during abdominoplasty. METHODS: A prospective series of 10 consecutive patients, undergoing concurrent abdominoplasty and laparoscopic umbilical hernia repair between 2014 and 2017 were included in the study. All procedures were performed by the same general surgeon and plastic surgeon at the Macquarie University Hospital in North Ryde, NSW, Australia. Data were collected with approval of our ethics committee. RESULTS: At 12-month follow up there were no instances of umbilical necrosis, wound complications, seroma, or recurrent hernia. The mean body mass index was 23.8 kg/m2 (range, 16.1-30.1 kg/m2). Rectus divarication ranged from 35 to 80 mm (mean, 53.5 mm). Umbilical hernia repair took a mean of 25.9 minutes to complete (range, 18-35 minutes). CONCLUSIONS: We present a technique that avoids incision of the rectus fascia minimizes dissection of the umbilical stalk and is able to provide a gold standard hernia repair with mesh. This procedure is particularly suited to postpartum patients with large herniae (>3-4 cm diameter) and wide rectus divarication, where mesh repair with adequate overlap is the recommended treatment.

The Integrin-Mediated ILK-Parvin-αPix Signaling Axis Controls Differentiation in Mammary Epithelial Cells.

Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via ß1-integrin (ß1-itg) to establish apico-basal polarity and to differentiate in response to prolactin. Downstream of ß1-itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico-basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue-specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA-mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin-binding guanine nucleotide exchange factor αPix prevented prolactin-induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation-specific bifurcation point in ß1-itg-ILK adhesive signaling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin-ILK signaling axis for MEC differentiation. J. Cell. Physiol. 231: 2408-2417, 2016. © 2016 Wiley Periodicals, Inc.

Mitosis and mitochondrial priming for apoptosis.

Cell division is a period of danger for cells, as inaccurate segregation of chromosomes can lead to loss of cell viability or aneuploidy. In order to protect against these dangers, cells ultimately initiate mitochondrial apoptosis if they are unable to correctly exit mitosis. A number of important chemotherapeutics exploit this response to delayed mitotic exit, but despite this, the molecular mechanism of the apoptotic timer in mitosis has proved elusive. Some recent studies have now shed light on this, showing how passage through the cell cycle fine-tunes a cell's apoptotic sensitivity such that it can respond appropriately when errors arise.

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.

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.

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.

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.

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.

Bcl-2 proteins and mitochondria--specificity in membrane targeting for death.

The localization and control of Bcl-2 proteins on mitochondria is essential for the intrinsic pathway of apoptosis. Anti-apoptotic Bcl-2 proteins reside on the outer mitochondrial membrane (OMM) and prevent apoptosis by inhibiting the activation of the pro-apoptotic family members Bax and Bak. The Bcl-2 subfamily of BH3-only proteins can either inhibit the anti-apoptotic proteins or directly activate Bax or Bak. How these proteins interact with each other, the mitochondrial surface and within the OMM are complex processes we are only beginning to understand. However, these interactions are fundamental for the transduction of apoptotic signals to mitochondria and the subsequent release of caspase activating factors into the cytosol. In this review we will discuss our knowledge of how Bcl-2 proteins are directed to mitochondria in the first place, a crucial but poorly understood aspect of their regulation. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

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.

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.