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Abstract: We show that treatment with non-toxic doses of zinc in association to the ionophore compound pyrrolidine dithiocarbamate (PDTC) inhibits p53−/− pancreatic cancer cell growth much more efficiently than gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Both the metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine and the radical scavenger N-acetyl-l-cysteine are able to recover cell growth inhibition by Zn/PDTC, demonstrating that this effect depends on the increased levels of intracellular zinc and of reactive oxygen species (ROS). Zn/PDTC treatment induces a strong apoptotic cell death that is associated to ROS-dependent nuclear translocation of the mitochondrial factor AIF, but not to the regulation of apoptotic genes and caspase activation. Primary fibroblasts are more resistant than pancreatic cancer cells to Zn/PDTC treatment and exhibit a lower basal and Zn/PDTC-induced enhancement of intracellular zinc. We show that Zn/PDTC induces p53 proteasomal degradation and that the proteasome inhibitor MG132 further increases fibroblast growth inhibition by Zn/PDTC, suggesting that p53 degradation plays an important role in fibroblast resistance to Zn/PDTC. [Copyright &y& Elsevier]

Introduction: Above elbow transplants represent 19% of the upper extremity transplants. Previous large-animal models have been too distal or heterotopic, did not use immunosuppression and had short survival. We hypothesize that an orthotopic forelimb transplant model, under standard immunosuppression, is feasible and can be used to address questions on peri-transplant ischemia reperfusion injury, and post-transplantation vascular, immunologic, infectious, and functional outcomes., Materials and Methods: Four forelimbs were used for anatomical studies. Four mock transplants were performed to establish technique/level of muscle/tendon repairs. Four donor and four recipient female Yucatan minipigs were utilized for in-vivo transplants (endpoint 90-days). Forelimbs were amputated at the midarm and preserved through ex vivo normothermic perfusion (EVNP) utilizing an RBC-based perfusate. Hourly perfusate fluid-dynamics, gases, electrolytes were recorded. Contractility during EVNLP was graded hourly using the Medical Research Council scale. EVNP termination criteria included systolic arterial pressure ≥115 mmHg, compartment pressure ≥30 mmHg (at EVNP endpoint), oxygen saturation reduction of 20%, and weight change ≥2%. Indocyanine green (ICG) angiography was performed after revascularization. Limb rejection was evaluated clinically (rash, edema, temperature), and histologically (BANFF classification) collecting per cause and protocol biopsies (POD 1, 7, 30, 60 and endpoint). Systemic infections were assessed by blood culture and tissue histology. CT scan was used to confirm bone bridging at endpoint., Results: Animals 2, 4 reached endpoint with grade 0-I rejection. Limbs 1, 3 presented grade III rejection on days 6, 61. CsA troughs averaged 461 ± 189 ng/mL. EVNLP averaged 4.3 ± 0.52 h. Perfusate lactate, PO 2 , and pH were 5.6 ± 0.9 mmol/L, 557 ± 72 mmHg and 7.5 ± 0.1, respectively. Muscle contractions were 4 [1] during EVNLP. Transplants 2, 3, 4 showed bone bridging on CT., Conclusion: We present preliminary evidence supporting the feasibility of an orthotopic, mid-humeral forelimb allotransplantation model under standard immunosuppression regimen. Further research should validate the immunological, infectious, and functional outcomes of this model., (© 2024 The Authors. Microsurgery published by Wiley Periodicals LLC.)

The mild oxidative stress induced by low doses of gaseous ozone (O 3 ) activates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing beneficial effects without cell damage. Mitochondria are sensitive to mild oxidative stress and represent a susceptible O 3 target. In this in vitro study, we investigated the mitochondrial response to low O 3 doses in the immortalized, non-tumoral muscle C2C12 cells; a multimodal approach including fluorescence microscopy, transmission electron microscopy and biochemistry was used. Results demonstrated that mitochondrial features are finely tuned by low O 3 doses. The O 3 concentration of 10 μg maintained normal levels of mitochondria-associated Nrf2, promoted the mitochondrial increase of size and cristae extension, reduced cellular reactive oxygen species (ROS) and prevented cell death. Conversely, in 20 μg O 3 -treated cells, where the association of Nrf2 with the mitochondria drastically dropped, mitochondria underwent more significant swelling, and ROS and cell death increased. This study, therefore, adds original evidence for the involvement of Nrf2 in the dose-dependent response to low O 3 concentrations not only as an Antioxidant Response Elements (ARE) gene activator but also as a regulatory/protective factor of mitochondrial function.

Worldwide the incidence of andrological diseases is rising every year and, together with it, also the interest in them is increasing due to their strict association with disorders of the reproductive system, including impairment of male fertility, alterations of male hormones production, and/or sexual function. Prevention and early diagnosis of andrological dysfunctions have long been neglected, with the consequent increase in the incidence and prevalence of diseases otherwise easy to prevent and treat if diagnosed early. In this review, we report the latest evidence of the effect of andrological alterations on fertility potential in both young and adult patients, with a focus on the link between gonadotropins' mechanism of action and mitochondria. Indeed, mitochondria are highly dynamic cellular organelles that undergo rapid morphological adaptations, conditioning a multitude of aspects, including their size, shape, number, transport, cellular distribution, and, consequently, their function. Since the first step of steroidogenesis takes place in these organelles, we consider that mitochondria dynamics might have a possible role in a plethora of signaling cascades, including testosterone production. In addition, we also hypothesize a central role of mitochondria fission boost on the decreased response to the commonly administrated hormonal therapy used to treat urological disease in pediatric and adolescent patients as well as infertile adults.

In this study, the transdermal fate of vesicular nanosystems was investigated. Particularly, ethosomes based on phosphatidylcholine 0.9% w / w and transethosomes based on phosphatidylcholine 0.9 or 2.7% w / w plus polysorbate 80 0.3% w / w as an edge activator were prepared and characterized. The vesicle mean size, morphology and deformability were influenced by both phosphatidylcholine and polysorbate 80. Indeed, the mean diameters of ethosome were around 200 nm, while transethosome's mean diameters were 146 or 350 nm in the case of phosphatidylcholine 0.9 or 2.7%, w / w , respectively. The highest deformability was achieved by transethosomes based on phosphatidylcholine 0.9%, w / w . The three types of vesicular nanosystems were applied on explanted human skin maintained in a bioreactor. Transmission electron microscopy demonstrated that all vesicles were able to enter the skin, keeping their structural integrity. Notably, the vesicle penetration capability was influenced by their physical-chemical features. Indeed, ethosomes reached keratinocytes and even the dermis, phosphatidylcholine 0.9% transethosomes were found in keratinocytes and phosphatidylcholine 2.7% transethosomes were found only in corneocytes of the outer layer. These findings open interesting perspectives for a differentiated application of these vesicles for transdermal drug delivery as a function of the cutaneous pathology to be addressed.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an extremely variable clinical course. We have recently shown that high catalase (CAT) expression identifies patients with an aggressive clinical course. Elucidating mechanisms regulating CAT expression in CLL is preeminent to understand disease mechanisms and develop strategies for improving its clinical management. In this study, we investigated the role of the CAT promoter rs1001179 single nucleotide polymorphism (SNP) and of the CpG Island II methylation encompassing this SNP in the regulation of CAT expression in CLL. Leukemic cells harboring the rs1001179 SNP T allele exhibited a significantly higher CAT expression compared with cells bearing the CC genotype. CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β transcription factors. Moreover, CLL cells exhibited lower methylation levels than normal B cells, in line with the higher CAT mRNA and protein expressed by CLL in comparison with normal B cells. Methylation levels at specific CpG sites negatively correlated with CAT levels in CLL cells. Inhibition of methyltransferase activity induced a significant increase in CAT levels, thus functionally validating the role of CpG methylation in regulating CAT expression in CLL. Finally, the CT/TT genotypes were associated with lower methylation and higher CAT levels, suggesting that the rs1001179 T allele and CpG methylation may interact in regulating CAT expression in CLL. This study identifies genetic and epigenetic mechanisms underlying differential expression of CAT, which could be of crucial relevance for the development of therapies targeting redox regulatory pathways in CLL., (© 2022. The Author(s).)

Background: Ischemia-reperfusion injury remains a primary concern in upper extremity transplantation. Ex vivo normothermic perfusion (EVNP) enables near-physiological organ preservation, avoiding the deleterious effects of hypoxia and cooling. We investigated the effectiveness of human limb EVNP compared with static cold storage (SCS)., Methods: Twenty human upper extremities were procured. Ten were perfused at 38 °C with an oxygenated red blood cell-based solution, and contralateral limbs served as SCS control (4 °C). EVNP was terminated with systolic arterial pressure ≥115 mm Hg, compartment fullness, or a 20% decline in oxygen saturation. Weight, contractility, compartment pressure, tissue oxygen saturation, and uptake rates were assessed. Perfusate fluid dynamics, gases, electrolytes, and metabolites were measured. Myocyte injury scores and liquid chromatography-mass spectrometry analysis were performed., Results: EVNP duration was 41.6 ± 9.4 h. Vascular resistance averaged 173.0 ± 29.4 mm Hg × min/L. Weight change and compartment pressures were 0.4 ± 12.2% ( P  = 0.21) and 21.7 ± 15.58 mm Hg ( P  = 0.003), respectively. Arterial and venous carbon dioxide partial pressure, oxygen saturation, and pH were 509.5 ± 91.4 mm Hg, 15.7 ± 30.2 mm Hg, 87.4 ± 11.4%, and 7.3 ± 0.2, respectively. Oxygen uptake rates averaged 5.7 ± 2.8 mL/min/g. Lactate reached 20 mmol/L after 15 (interquartile range = 6) h. Limb contractility was preserved for 30.5 (interquartile range = 15.8) h ( P  < 0.001) and negatively correlated with perfusate potassium (ρ = -0.7, P  < 0.001). Endpoint myocyte injury scores were 28.9 ± 11.5% (EVNP) and 90.2 ± 11.8% (SCS) ( P  < 0.001). A significant increase in taurine ( P  = 0.002) and decrease in tryptophan ( P  = 0.002) were detected. Infrared thermography and indocyanine green angiography confirmed the presence of peripheral perfusion., Conclusions: EVNP can overcome the limitations of cold preservation by extending preservation times, enabling limb quality assessment, and allowing limb reconditioning before transplantation., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs.

A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of Plasmodium falciparum GAPDH ( Pf GAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH. They showed an antiproliferative effect on pancreatic ductal-adenocarcinoma cells (PDAC) and pancreatic-cancer stem cells (CSCs), and among them two promising compounds were selected to be tested in vivo. Interestingly, these compounds were not effective in fibroblasts. The AXP-3019 derivative was able to block PDAC-cell growth in mice xenograft without apparent toxicity. The overall results support the assumption that selective inhibition of the glycolytic pathway, by targeting GAPDH, is an effective therapy for pancreatic cancer and that 3-bromo-isoxazoline derivatives represent a new class of anti-cancer compounds targeting glycolysis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors; it is often diagnosed at an advanced stage and is hardly treatable. These issues are strictly linked to the absence of early diagnostic markers and the low efficacy of treatment approaches. Recently, the study of the metabolic alterations in cancer cells has opened the way to important findings that can be exploited to generate new potential therapies. Within this scenario, mitochondria represent important organelles within which many essential functions are necessary for cell survival, including some key reactions involved in energy metabolism. These organelles remodel their shape by dividing or fusing themselves in response to cellular needs or stimuli. Interestingly, many authors have shown that mitochondrial dynamic equilibrium is altered in many different tumor types. However, up to now, it is not clear whether PDAC cells preferentially take advantage of fusion or fission processes since some studies reported a wide range of different results. This review described the role of both mitochondria arrangement processes, i.e., fusion and fission events, in PDAC, showing that a preference for mitochondria fragmentation could sustain tumor needs. In addition, we also highlight the importance of considering the metabolic arrangement and mitochondria assessment of cancer stem cells, which represent the most aggressive tumor cell type that has been shown to have distinctive metabolic features to that of differentiated tumor cells.

Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC.

Introduction: With increasing popularity of minimally invasive approaches to abdominoperineal resection (APR), thigh-based flaps are becoming the preferred option for reconstruction. The gluteal-thigh flap provides sufficient bulk, albeit with a high complication rate. We reevaluated the vascularization and design of the gluteal-thigh flap. The purpose of this study is to highlight the importance of the vascularization of the posterior thigh skin by the descending branch of the inferior gluteal artery (IGA) and the profunda femoris artery (PFA) perforators to design a more reliable and versatile gluteal thigh flap. This flap is indicated in selected cases in which use of vertical rectus abdominis musculocutaneous flap is not feasible., Methods: Eleven fresh cadavers were used. The course, distribution, and diameter of IGA and PFA perforators were recorded. A wide posterior gluteal-thigh propeller flap (WPGTPF) was designed including the distance between the ischiatic tuberosity and greater trochanter; and extending it to within 8 cm of the popliteal fossa to improve flap reliability. Ten patients (mean age of 58.7 ± 10.6 years) underwent APR due to anal cancer (2) and rectal cancer (8); the approach was open in 3, laparoscopic in 6, and robotic in 1. All 10 patients received unilateral flap with a width of 12 ± 3.3 cm and surface of 405.5 ± 175.9 cm 2 ., Results: The descending branch of the IGA was dominant in 72.7% of the specimens. In 22.7% of the specimens, the pedicle of the flap derived from the first or second PFA perforators. In one case, there was a double vascularization. Descending branch of the IGA was mapped at 46 ± 7.96 mm on the X-axis (horizontal line from the ischial tuberosity [IT] to the greater trochanter) and -12.1 ± 17.9 mm on the Y-axis (vertical line from the IT to the Medial Femoral condyle). Its average caliber measured 2.18 ± 0.3 mm. The first and second PFA perforators were located at 101.6 ± 17.9 mm and 104.5 ± 15.5 mm on the X-axis; 35.9 ± 27.1 mm and 89.2 ± 37.6 mm on the Y-axis. Their average diameters were 1.84 ± 0.41 mm and 1.48 ± 0.3 mm. In two cases, the flap was based on the first PFA perforator, the rest were on the descending branch of the IGA. Neither complete nor partial flap necrosis was observed. One patient developed coccyx osteomyelitis treated and resolved with bone debridement and one patient developed a seroma of the lateral thigh that was treated conservatively. Three patients underwent a debulking procedure by a combination of liposuction and resection to improve the gluteal symmetry. All ten flaps survived completely., Conclusions: Harvest of a wide flap that includes the PFA perforators and implementation of the propeller design increase the survival and versatility of the flap., (© 2020 Wiley Periodicals LLC.)

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance., Methods: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs., Results: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters' expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions., Conclusion: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.

Background: Facial allotransplantation including the temporomandibular joints may improve the functional outcomes in face transplant candidates who have lost or damaged this joint., Methods: Linear and angular measurements were taken in 100 dry skulls and mandibles and in 100 three-dimensionally-reconstructed facial computed tomographic scans to determine the variability of the temporomandibular joint, glenoid fossa, and mandible. A vascular study was performed in six fresh cadaveric heads, followed by harvest of the face allograft in three heads. Next, four heads were used for mock transplantation (two donors and two recipients). The full facial allograft included four different segments: a Le Fort III, a mandibular tooth-bearing, and two condyle and temporomandibular joint-bearing segments. Statistical analysis was performed using SAS software., Results: In only one-third of the skulls, the condylar shape was symmetric between right and left sides. There was a wide variability in the condylar coronal (range, 14.3 to 23.62 mm) and sagittal dimensions (range, 5.64 to 10.96 mm), medial intercondylar distance (range, 66.55 to 89.91 mm), and intercondylar angles (range, 85.27 to 166.94 degrees). This high variability persisted after stratification by sex, ethnicity, and age. The temporomandibular joint was harvested based on the branches of the superficial temporal and maxillary arteries. The design of the allograft allowed fixation of the two condyle and temporomandibular joint-bearing segments to the recipient skull base, preserving the articular disk-condyle-fossa relationship, and differences were adjusted at the bilateral sagittal split osteotomy sites., Conclusion: Procurement and transplantation of a temporomandibular joint-containing total face allograft is technically feasible in a cadaveric model.

Ischemia and reperfusion injury remains a significant limiting factor for the successful revascularization of amputated extremities. Ex vivo normothermic perfusion is a novel approach to prolong the viability of the amputated limbs by maintaining physiologic cellular metabolism. This study aimed to evaluate the outcomes of extended ex vivo normothermic limb perfusion (EVNLP) in preserving the viability of amputated limbs for over 24 hours. A total of 10 porcine forelimbs underwent EVNLP. Limbs were perfused using an oxygenated colloid solution at 38°C containing washed RBCs. Five forelimbs (Group A) were perfused for 12 hours and the following 5 (Group B) until the vascular resistance increased. Contralateral forelimbs in each group were preserved at 4°C as a cold storage control group. Limb viability was compared between the 2 groups through assessment of muscle contractility, compartment pressure, tissue oxygen saturation, indocyanine green (ICG) angiography and thermography. EVNLP was performed for 12 hours in group A and up to 44 hours (24-44 hours) in group B. The final weight increase (-1.28 ± 8.59% vs. 7.28 ± 15.05%, P = .548) and compartment pressure (16.50 ± 8.60 vs. 24.00 ± 9.10) (P = .151) were not significantly different between the two groups. Final myoglobin and CK mean values in group A and B were: 875.0 ± 325.8 ng/mL (A) versus 1133.8 ± 537.7 ng/mL (B) (P = .056) and 53 344.0 ± 16 603.0 U/L versus 64 333.3 ± 32 481.8 U/L (P = .286). Tissue oxygen saturation was stable until the end in both groups. Infra-red thermography and ICG-angiography detected variations of peripheral limb perfusion. Our results suggest that extended normothermic preservation of amputated limbs is feasible and that the outcomes of prolonged EVNLP (>24 hours) are not significantly different from short EVNLP (12 hours)., (© 2020 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Pancreatic ductal adenocarcinoma (PDAC) is typically characterized by high chemoresistance and metastatic spread, features mainly attributable to cancer stem cells (CSCs). It is of central interest the characterization of CSCs and, in particular, the study of their metabolic features in order to selectively identify their peculiarities for an efficient therapeutic approach. In this study, CSCs have been obtained by culturing different PDAC cell lines with a specific growth medium. Cells were characterized for the typical stem/mesenchymal properties at short-, medium-, and long-term culture. Metabolomics, proteomics, analysis of oxygen consumption rate in live cells, and the effect of the inhibition of lactate transporter on cell proliferation have been performed to delineate the metabolism of CSCs. We show that gradually de-differentiated pancreatic cancer cells progressively increase the expression of both stem and epithelial-to-mesenchymal transition markers, shift their metabolism from a glycolytic to an oxidative one, and lastly gain a quiescent state. These quiescent stem cells are characterized by high chemo-resistance, clonogenic ability, and metastatic potential. Re-differentiation reverts these features, re-activating their proliferative capacity and glycolytic metabolism, which generally correlates with high aggressiveness. These observations add an important piece of knowledge to the comprehension of the biology of CSCs, whose metabolic plasticity could be exploited for the generation of promising and selective therapeutic approaches for PDAC patients., Competing Interests: The authors declare that they have no conflict of interests.

Succinate dehydrogenase (SDH) has been classically considered a mitochondrial enzyme with the unique property to participate in both the citric acid cycle and the electron transport chain. However, in recent years, several studies have highlighted the role of the SDH substrate, i.e. succinate, in biological processes other than metabolism, tumorigenesis being the most remarkable. For this reason, SDH has now been defined a tumor suppressor and succinate an oncometabolite. In this review, we discuss recent findings regarding alterations in SDH activity leading to succinate accumulation, which include SDH mutations, regulation of mRNA expression, post-translational modifications and endogenous SDH inhibitors. Further, we report an extensive examination of the role of succinate in cancer development through the induction of epigenetic and metabolic alterations and the effects on epithelial to mesenchymal transition, cell migration and invasion, and angiogenesis. Finally, we have focused on succinate and SDH as diagnostic markers for cancers having altered SDH expression/activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: A number of authors have proposed retrograde arterial embolism as the responsible mechanism for filler-induced blindness. However, no previous human study has substantiated this proposed mechanism., Objectives: The aim of this study was to investigate the pathophysiology of filler-induced blindness using a fresh cadaver perfusion technique., Methods: A fresh cadaver head perfusion model that simulates both physiologic blood pressure and flow rate of the carotid artery, ophthalmic artery, and supratrochlear artery was used. The common carotid artery was cannulated and the internal jugular vein exposed for open venous drainage. A plasma-based perfusate was circulated through the cadaver head, which was connected to a perfusion system consisting of a roller pump, preload reservoir, and pressure monitor. The hyaluronic acid filler mixed with methylene blue was injected into the cannulated superficial branch of the supratrochlear artery. Cadaver dissection, angiographic study, and histology were used to investigate filler-induced blindness., Results: Cannulation of the superficial branch of the supratrochlear artery was successful in all six cadavers. Emboli to the ophthalmic artery was successfully demonstrated in the three out of 6 fresh cadaver heads. The C-arm angiogram documented a cut-off sign in the ophthalmic artery due to hyaluronic acid filler emboli. An average intravascular volume of the intraorbital part of the supratrochlear artery was 50.0 µL. The average depth of location of the superficial branch of the supratrochlear artery from the epidermal surface was 1.5 mm., Conclusions: Our cadaveric study demonstrated that retrograde hyaluronic acid filler emboli to the ophthalmic artery could be produced by the cannulation of the supratrochlear artery. The superficial location of the supratrochlear artery, the rich vasculature surrounding it, and the variability in the anatomy make this possible.

Background: Pancreatic cancer stem cells (CSCs) are responsible for resistance to standard therapy, metastatic potential, and disease relapse following treatments. The current therapy for pancreatic ductal adenocarcinoma (PDAC) preferentially targets the more differentiated cancer cell population, leaving CSCs as a cell source for tumor mass formation and recurrence. For this reason, there is an urgent need to improve current therapies and develop novel CSC-targeted therapeutic approaches., Methods: Hyaluronic acid (HA) decorated liposomes, containing diethyldithiocarbamate‑copper (Cu(DDC) 2 ), able to target the specific CSC marker CD44 receptor were prepared by ion gradient technique and fully characterized. Their antiproliferative effect was evaluated on pancreatic CSCs derived from PDAC cell lines or patients. To clarify the mechanism of action of Cu(DDC) 2 liposomes, ROS level neutralization assay in the presence of N-acetyl-L-cysteine was performed., Results: Liposomes showed high encapsulation efficiency and Cryo-TEM analysis revealed the presence of Cu(DDC) 2 crystals in the aqueous core of liposomes. In vitro test on pancreatic CSCs derived from PDAC cell lines or patients showed high ROS mediated anticancer activity of HA decorated liposomes. The sphere formation capability of CSCs obtained from patients was drastically reduced by liposomal formulations containing Cu(DDC) 2 ., Conclusions: The obtained results show that the encapsulation of Cu(DDC) 2 complex in HA decorated liposomes strongly increases its anti-proliferative activity on pancreatic CSCs., General Significance: This paper describes for the first time the use of HA decorated liposomes containing Cu(DDC) 2 against pancreatic CSCs and opens the way to the development of nanomedicine based CSC-targeted therapeutic approaches., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Negative pressure wound therapy, a tool widely applied to treat lower limb traumas, is useful in reconstructive procedures. However, obtaining an airtight vacuum seal when using a negative pressure dressing around an external fixation device can be complicated and time-consuming. The plastic drape seldom adheres to screws, pins, or wires and, as such, the vacuum seal is jeopardized. In surgical departments, colostomy paste is widely used, readily available, and applied where pins and wires make contact with the plastic drape. It is a fast, practical, and inexpensive method of preventing air leakage. In conclusion, to obtain an airtight seal between the skin and an external fixation device, colostomy paste may be used to ensure an optimal tight seal, even for complex and extended wounds, for 3-5 days without complications or the requirement for additional dressing changes. Furthermore, it is inexpensive, readily available, simple to use, and quick to apply.

Background: Hand transplantation in patients with severe upper extremity burns can be associated with an increased risk of exposure of vessels, tendons, and nerves because of extensive skin and soft-tissue deficit. This study evaluated how to reliably transfer additional extended skin flaps with a standard hand allograft., Methods: Twenty-five upper extremities were used. Sixteen were injected with latex to map the perforating branches of the brachial, superior ulnar collateral, radial, ulnar, and posterior interosseous arteries. Nine hand allografts were procured, injected with blue ink through the brachial artery to assess the perfusion of the skin flaps, and then mock transplanted., Results: Sizable perforators from the brachial, superior ulnar collateral, radial, ulnar, and posterior interosseous arteries were used to augment the vascularization of the skin flaps. The average stained area of the medial arm flap was between 85.7 and 93.9 percent. The stained area of the volar forearm flap was the smallest when based on perforators within 6 cm from the wrist crease (51.22 percent). The dorsal forearm flap showed the least amount of staining (34.7 to 46.1 percent). The average time to repair tendons, nerves, and vessels was longer when a single volar forearm-arm flap was harvested (171.6 minutes). Harvest of the allograft associated with a distally based forearm flap and islanded arm flap was the fastest (181.6 ± 17.55 minutes)., Conclusion: Extended skin flaps, based on perforators of the main axial vessels, can be reliably transplanted with a standard hand allograft based on the brachial or axillary vascular pedicle.

The epithelial-mesenchymal transition (EMT) is a biologic process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. EMT is involved in embryo development, wound healing, tissue regeneration, organ fibrosis and has also been proposed as the critical mechanism for the acquisition of malignant phenotypes by epithelial cancer cells. These cells have been shown to acquire a mesenchymal phenotype when localized at the invasive front of primary tumours increasing aggressiveness, invasiveness, metastatic potential and resistance to chemotherapy. There is now increasing evidence demonstrating that a crucial role in the development of this process is played by factors secreted by cells of the tumour microenvironment or by the tumour cells themselves. This review summarises the current knowledge of EMT induction in cancer by paracrine or autocrine mechanisms, by exosomes or free proteins and miRNAs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Its aggressiveness is driven by an intense fibrotic desmoplastic reaction in which the increasingly collagen I-rich extracellular matrix (ECM) and several cell types, including cancer stem cells (CSCs), create a tumor-supportive environment. However, how ECM composition regulates CSC dynamics and their relationship with the principle parenchymal tumor population to promote early invasive growth is not yet characterized. For this, we utilized a platform of 3D organotypic cultures composed of laminin-rich Matrigel, representative of an early tumor, plus increasing concentrations of collagen I to simulate malignant stroma progression. As ECM collagen I increases, CSCs progress from a rapidly growing, vascular phenotype to a slower growing, avascular phase, while maintaining their endothelial-like gene signatures. This transition is supported autocrinically by the CSCs and paracrinically by the parenchymal cells via their ECM-dependent secretomes. Indeed, when growing on an early tumor ECM, the CSCs are dedicated toward the preparation of a vascular niche by (a) activating their growth program, (b) secreting high levels of proangiogenic factors which stimulate both angiogenesis and vasculogenic mimicry, and (c) overexpressing VEGFR-2, which is activated by VEGF secreted by both the CSC and parenchymal cells. On Matrigel, the more differentiated parenchymal tumor cell population had reduced growth but a high invasive capacity. This concerted high local invasion of parenchymal cells into the CSC-derived vascular network suggests that a symbiotic relationship between the parenchymal cells and the CSCs underlies the initiation and maintenance of early PDAC infiltration and metastasis., (© 2018 Federation of European Biochemical Societies.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal of all human cancers with a high mortality rate. Resistance to conventional treatments and chemotherapeutics is a typical feature of PDAC. To investigate the causes of drug resistance it is essential to deeply investigate the mechanism of action of chemotherapeutics. In this study, we performed an in depth shotgun proteomic approach using the label-free proteomic SWATH-MS analysis to investigate novel insights of the mechanism of action of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in PDAC cells. This proteomic analysis in PaCa44 cells and data elaboration of TSA-regulated proteins by bioinformatics showed an overall up-regulation of cytokeratins and other proteins related to the cytoskeleton organization, keratinization, and apoptotic cell death. On the contrary, a large amount of the down-regulated proteins by TSA treatment belongs to the cellular energetic metabolism and to the machinery of protein synthesis, such as ribosomal proteins, determining synergistic cell growth inhibition by the combined treatment of TSA and the glycolytic inhibitor 2-deoxy-d-glucose in a panel of PDAC cell lines. Data are available via ProteomeXchange with identifier PXD007801., (© 2017 Wiley Periodicals, Inc.)

Several evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose. Through a bidimensional electrophoresis analysis, we identify 19 protein species differentially expressed after treatment with the UCP2 inhibitor genipin and, by bioinformatic analyses, we show that these proteins are mainly involved in metabolic processes. In particular, we demonstrate that the antioxidant UCP2 induces the expression of hnRNPA2/B1, which is involved in the regulation of both GLUT1 and PKM2 mRNAs, and of lactate dehydrogenase (LDH) increasing the secretion of L-lactic acid. We further demonstrate that the radical scavenger N-acetyl-L-cysteine reverts hnRNPA2/B1 and PKM2 inhibition by genipin indicating a role for reactive oxygen species in the metabolic reprogramming of cancer cells mediated by UCP2. We also observe an UCP2-dependent decrease in mtOXPHOS complex I (NADH dehydrogenase), complex IV (cytochrome c oxidase), complex V (ATPase) and in mitochondrial oxygen consumption, suggesting a role for UCP2 in the counteraction of pancreatic cancer cellular respiration. All these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation with the concomitant metabolic shift from mtOXPHOS to the glycolytic pathway., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Unlabelled: Skin-reduction mastectomy with prepectoral implant reconstruction is a novel technique for immediate breast reconstruction, with subcutaneous implant placement in patients eligible for skin-reducing mastectomy. Implants were placed above the pectoralis muscles in a compound pocket made by a dermal flap and acellular dermal matrix. The procedure was performed on 33 breasts in 27 selected patients. In three cases, there was skin ischemia; in one case, it healed spontaneously; and in two patients, a surgical necrosectomy and primary closure were needed. No implant loss occurred. This new technique proved to be a useful alternative, with good cosmetic results, in selected patients requiring mastectomy. These preliminary results need to be confirmed by long-term and comparative studies., Clinical Question/level of Evidence: Therapeutic, IV.

Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9. Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified., Biological Significance: The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-1 CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first proteomic study of pancreatic CSC secretome. We performed an iTRAQ-based analysis to compare the secretomes of Panc1 CSCs and Panc1 parental cell line and identified a total of 43 proteins secreted at higher level by pancreatic cancer stem cells. We found modulation of different vital physiological pathways (such as glycolysis and gluconeogenesis, pentose phosphate pathway) and the involvement of CSC secreted proteins (for example 72kDa type IV collagenase, galectin-3, alpha-actinin-4, and MARCKS) in pathological conditions including cancer differentiation, invasion and metastasis. By ELISA verification we found that MARCKS and ceruloplasmin discriminate between controls and PDAC patients; in addition ROC curve analyses indicate that MARCKS does not have diagnostic accuracy, while ceruloplasmin could be a promising marker only for patients negative for CA19-9. We think that the findings reported in our manuscript advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Many genetic alterations, including predisposing or somatic mutations, may contribute toward the development of melanoma. Although CDKN2A and CDK4 are high-penetrance genes for melanoma, MC1R is a low-penetrance gene that has been associated most consistently with the disease. Moreover, BRAF is the most frequently somatically altered oncogene and is a validated therapeutic target in melanoma. This paper reports a case of multiple primary melanoma with germline CDK4 mutation, MC1R variant, and somatic BRAF mutation in nine out of 10 melanomas, indicating that a common pathogenesis, because of a predisposing genetic background, may be shared among distinct subsequent melanomas of probable clonal origin. After 3 months of targeted therapy with BRAF inhibitor, our patient developed resistance with rapid progression of the disease leading to death. This is the first case in which early resistance to BRAF inhibitor has been reported in a patient with CDK4 germline mutation.

Cancer stem cells (CSCs) are a sub-population of quiescent cells endowed with self-renewal properties that can sustain the malignant behavior of the tumor mass giving rise to more differentiated cancer cells. For this reason, the specific killing of CSCs represents one of the most important challenges of the modern molecular oncology. However, their particular resistance to traditional chemotherapy and radiotherapy imposes a thorough understanding of their biological and biochemical features. The metabolic peculiarities of CSCs may be a therapeutic and diagnostic opportunity in cancer research. In this review, we summarize the most significant discoveries on the metabolism of CSCs describing and critically analyzing the studies supporting either glycolysis or mitochondrial oxidative phosphorylation as a primary source of energy for CSCs., (© 2015 International Union of Biochemistry and Molecular Biology.)

Background: The acellular dermal matrix (ADM)-assisted breast reconstruction technique is widely known, but discouraging results due to early postoperative complications have been reported. As the literature identifies seroma as the most common issue after breast surgery without identifying its pathogenesis, we aimed to report the trend of postoperative daily serum collection after ADM-assisted breast reconstruction and compare it with data in the literature in order to discover more about this little-known topic., Methods: A retrospective study on 28 consecutive patients who received ADM-assisted breast reconstruction between February 2013 and February 2014 was performed. In order to reduce the number of variables that could affect serum production, only one brand of ADM was used and all tissues were handled gently and precisely. The daily drainage volume was recorded per patient during the first four days of hospitalization. Likewise, postoperative complications were noted during routine follow-up., Results: In total, five (17.9%) bilateral and 23 (82.1%) unilateral ADM-assisted breast reconstructions (33 implants) were performed. The mean age, body mass index, and length of hospital stay were 53.6 years, 21.3 kg/m(2), and 4.5 days, respectively. One major complication led to implant loss (3.0%), and nine minor complications were successfully treated with ambulatory surgery (27.3%). Serum collection linearly decreased after 24 hours postoperatively., Conclusions: Daily drainage decreased following the theoretical decline of acute inflammation. In concordance with the literature, daily serum production may not be related to the use of ADM.