Germline gain-of-function MMP11 variant results in an aggressive form of colorectal cancer

Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and nonmatrix proteins. Although most MMPs are secreted as inactive proenzymes and are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11's protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.     

Assessment of proadrenomedullin as diagnostic or prognostic biomarker of acute appendicitis in children with acute abdominal pain

BackgroundAcute appendicitis (AA) is one of the most frequent surgical pathologies in pediatrics.ObjectivesTo investigate the utility of proadrenomedullin (pro-ADM) for the diagnosis of AA.MethodsProspective, analytical, observational, and multicenter study conducted in 6 pediatric emergency departments. Children up to 18 years of age with suspected AA were included. Clinical, epidemiological, and analytical data were collected.ResultsWe studied 285 children with an average age of 9.5 years (95% confidence interval [CI], 9.1–9.9). AA was diagnosed in 103 children (36.1%), with complications in 10 of them (9.7%). The mean concentration of pro-ADM (nmol/L) was higher in children with AA (0.51 nmol/L, SD 0.16) than in children with acute abdominal pain (AAP) of another etiology (0.44 nmol/L, SD 0.14; p < 0.001). This difference was greater in complicated cases compared with uncomplicated AA (0.64 nmol/L, SD 0.17 and 0.50 nmol/L, SD 0.15, respectively; p = 0.005). The areas under the receiver-operating characteristic curves were 0.66 (95% CI, 0.59–0.72) for pro-ADM, 0.70 (95% CI, 0.63–0.76) for C-reactive protein (CRP), 0.84 (95% CI, 0.79–0.89) for neutrophils, and 0.84 (95% CI, 0.79–0.89) for total leukocytes. The most reliable combination to rule out AA was CRP ≤1.25 mg/dL and pro-ADM ≤0.35 nmol/L with a sensitivity of 96% and a negative predictive value of 93%.ConclusionChildren with AA presented higher pro-ADM values than children with AAP of other etiologies, especially in cases of complicated AA. The combination of low values of pro-ADM and CRP can help to select children with low risk of AA.     

Transcirculation Silk Vista Baby-assisted coiling in half-T configuration for the treatment of posterior communicating artery aneurysms associated with a fetal posterior circulation: An alternative flow diversion strategy

Flow diverter devices have become a routine first-line option for treatment of an increasing population of intracranial aneurysms at many neurovascular centers. Despite the promising results of flow diverter stents on anterior circulation, incomplete occlusion on the presence of fetal posterior circulation has been described on several reports. Here we describe a novel technical alternative to conventional flow diversion approach for this specific subgroup of aneurysms using the low-profile flow diverter, Silk Vista Baby. The device was selectively placed into the fetal type posterior cerebral artery in half-T configuration for the treatment of a posterior communicating aneurysm using a transcirculation approach through the anterior communicating artery. This represents a useful and effective technique and should be considered when encountering the above-described situation.     

Another (Internal) Epineurium: Beyond the Anatomical Barriers of Nerves

The epineurium has been accepted as the outer anatomical barrier of the peripheral nerves. Our objective was to characterize the microanatomy of the layers surrounding nerves using different tissue-specific staining methods. Two hundred forty-two cross sections of human sciatic and median nerves, and brachial plexuses of eight fresh unembalmed cadavers, were examined. The samples were fixed in formaldehyde solution and stained with hematoxylin–eosin, Masson's trichrome, or epithelial membrane antigen under standard conditions. Because epithelial membrane antigen only stains the perineurium, we demonstrated using hematoxylin–eosin and Masson's trichrome that there were different collagen layers inside and outside the nerves. All fascicles had a collagen layer that surrounded the perineurium and were in close contact with it, with no adipose tissue between them. Unlike the perineurium, this layer, an “internal epineurium,” contained no cells, and it surrounded one or a small group of fascicles. Bundling these fascicles or small groups of fascicles together was the true epineurium, and between the true and internal epineurium, we consistently found an adipose-containing compartment. More proximal to this, the tibial and common peroneal nerves were bundled together by another collagen layer, the circumneurium, which also had a fat-cell-containing compartment deep to it. There were scattered collagen fibers among the adipocytes. Using tissue-specific staining, we were able to demonstrate a collagen layer, the “internal epineurium.” Outside the nerves, we identified several fat-containing concentric compartments. Those compartments were limited by collagen fiber layers that were also similar to the epineurium. Clin. Anat. 33:199–206, 2020. © 2019 Wiley Periodicals, Inc.     

Cloning of hamster osteopontin and expression distribution in normal tissues and experimentally induced oral squamous-cell carcinoma

Osteopontin (OPN) is a non-collagenous extracellular matrix (ECM) protein expressed and secreted by several human cancers. This study investigated the expression pattern of OPN during development of oral squamous-cell carcinoma by using 7,12-dimethylbenz[a]anthracene (DMBA)-induced squamous-cell carcinomas in buccal pouch of syrian golden hamsters. We first identified the hamster OPN cDNA sequence by screening of a hamster calvariae cDNA library with a rat OPN cDNA probe. The resulting 1,449 bp of hamster OPN cDNA led to a deduced protein sequence of 305 amino acids containing several putative binding sites to integrins, CD44 receptors, calcium ions and hydroxyapatite, as well as multiple sites for phosphorylation, glycosylation and sulphation. Hamster OPN cDNA was then used as a probe to analyze the expression of OPN mRNA by Northern blot and in situ hybridization analyses of normal and malignant tissues. OPN mRNA was detected in several non-mineralized tissues as well as in mineralized tissues, but was not present in normal hamster buccal epithelium. DMBA-treated hamster buccal pouches expressed OPN mRNA as early as 4 weeks and displayed the highest level of expression at 15 weeks. The specimens treated with DMBA for 15 weeks exhibited histological features of squamous-cell carcinoma, presented microcrystalline deposits and showed OPN expression associated with malignant epithelium and tumor-associated macrophages. To summarize, our results suggest that buccal-pouch carcinogenesis of Syrian golden hamster may constitute an excellent experimental model to study the mechanisms by which OPN is associated with oral cancer pathogenesis, and to validate OPN-based therapeutic approaches to ameliorate oral cancer progression and metastasis.     

Experimental mandibular regeneration by distraction osteogenesis with submerged devices: preliminary results of a canine model

The authors describe a new technique for reconstruction of mandibular body defects. The feasibility of distraction osteogenesis with submerged (internal) devices for reconstruction of segmental mandibular defects is investigated in an experiment with five adult dogs. A segmental mandibulectomy was performed on the horizontal ramus. The bony defect was regenerated using distraction osteogenesis (bone transport) at a rate of 1 mm daily. The animals were killed after the consolidation period. Complete bone regeneration of the surgically created gap was successful in three of five dogs. Two animals failed to create new bone. In these two cases, the screws did not offer proper stability to the bony fragments, and this caused a lack of ossification. This experimental study demonstrates the possibility to use internal distraction devices to reconstruct segmental mandibular defects in a canine model. Internal devices show enormous advantages in comparison with the external ones. This method with no donor-site morbidity may become a very useful option in human mandibular reconstruction.     

Pediatric transplantation in Europe during the COVID-19 pandemic: Early impact on activity and healthcare

The current pandemic SARS-CoV-2 has required an unusual allocation of resources that can negatively impact chronically ill patients and high-complexity procedures. Across the European Reference Network on Pediatric Transplantation (ERN TransplantChild), we conducted a survey to investigate the impact of the COVID-19 outbreak on pediatric transplant activity and healthcare practices in both solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT). The replies of 30 professionals from 18 centers in Europe were collected. Twelve of 18 centers (67%) showed a reduction in their usual transplant activity. Additionally, outpatient visits have been modified and restricted to selected ones, and the use of telemedicine tools has increased. Additionally, a total of 14 COVID-19 pediatric transplanted patients were identified at the time of the survey, including eight transplant recipients and six candidates for transplantation. Only two moderate-severe cases were reported, both in HSCT setting. These survey results demonstrate the limitations in healthcare resources for pediatric transplantation patients during early stages of this pandemic. COVID-19 disease is a major worldwide challenge for the field of pediatric transplantation, where there will be a need for systematic data collection, encouraging regular discussions to address the long-term consequences for pediatric transplantation candidates, recipients, and their families.     

GAP,an aequorin-based fluorescent indicator for imaging Ca2+ in organelles

Genetically encoded calcium indicators allow monitoring subcellular Ca²⁺ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca²⁺ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg²⁺, tunable Ca²⁺ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca²⁺ indicators for organelles and becomes a valuable tool for in vivo Ca²⁺ imaging applications.Ca²⁺ is involved in the regulation of many intracellular processes that take place both in the cytosol and inside organelles (1–3). Therefore, accurate measurement of the calcium concentration ([Ca²⁺]) inside organelles is essential to discriminate discrete Ca²⁺ signals between the different compartments. Although synthetic Ca²⁺ indicators can be loaded into organelles, the signal has poor selectivity, as the dye is also present in the cytosol and must be carefully removed before measurements (4). The main advantage of Genetically Encoded Ca²⁺ Indicators (GECIs) is their ability to be targeted to specific intracellular locations. Both bioluminescent and fluorescent proteins have been successfully used to measure subcellular [Ca²⁺]. The photoprotein aequorin (5), purified from the jellyfish Aequorea victoria, was the first protein-based Ca²⁺ indicator, injected into cells in the early 1970s (6). After cloning of its cDNA (7), recombinant aequorin became the most frequently used probe to measure Ca²⁺ in organelles, including mitochondria (8), the endoplasmic reticulum (ER) (9), the nucleus (10), the Golgi apparatus (11), or secretory vesicles (12).Fluorescent GECIs achieve a better spatial resolution than bioluminescent sensors. They are generally composed of one or two fluorescent proteins, most of them variants of GFP, fused to a Ca²⁺-binding protein (13). Recently, a single EF-hand motif has been inserted in the GFP moiety to generate a Ca²⁺ fluorescent probe (14). Since the first cameleon based on FRET (15), the number of GECIs has exponentially increased, attempting optimization of critical features such as adequate expression, signal strength, or dynamic range. However, the in vivo use in mammals, one of the main applications of GECIs, has grown more slowly and has disclosed severe limitations (16, 17). Transgenic sensors usually showed a low expression, often resulting in its inactivation or reduced dynamic range. With the exception of troponin derivatives, most of the available GECIs, namely cameleons, camgaroos, pericams, or GCaMPs (circularly permutated EGFP-based Ca²⁺ sensors), are based on calmodulin, a highly regulated ubiquitous protein that binds a large number of targets (13). Although the interference with endogenous calmodulin has been reduced in the improved cameleons (18), the interaction with other cellular proteins cannot be ruled out. Thus, the loss of Ca²⁺ sensitivity observed in vivo may reflect the interaction of the probe with endogenous partners, which may disturb cellular functions.The jellyfish aequorin exhibits a number of advantages over mammalian EF-hand proteins. It is not toxic and appears not to interfere with other intracellular Ca²⁺-binding molecules, even when microinjected at high concentrations in mammalian cells. Moreover, the use of aequorin as a bioluminescence sensor has been extensively reported, ranging from subcellular Ca²⁺ measurements in many different cell types up to whole organisms, including transgenic animals (19–21).Here we describe a family of fluorescent Ca²⁺ sensors based on the fusion of two jellyfish proteins, GFP and apoaequorin. This Ca²⁺ probe shows a larger dynamic range compared with other GECIs and a robust photonic and thermal stability. It can be targeted to distinct compartments such as the nucleus, cytosol, or mitochondria, where it selectively and accurately monitors dynamic Ca²⁺ changes. In addition, we have generated a variant with a lower Ca²⁺ affinity suited for imaging Ca²⁺ changes in organelles with high resting [Ca²⁺] such as the ER or the Golgi apparatus. Finally, we demonstrate its in vivo applicability by generating transgenic mice where the Ca²⁺ biosensor maintained its in vitro features.