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Lung cancer diagnosis is a challenge since it is also one of the most frequently diagnosed cancers. Diagnostic challenges are deeply related to the development of personalized therapy and molecular and precise histological characterizations of lung cancer. When addressing these features, it is very important to acknowledge the issue of tumour heterogeneity, as it imposes several questions. First of all, lung cancer is a very heterogeneous disease, at a cellular and histological level. Cellular and histological heterogeneity are addressed with emphasis on the diagnosis, pre-neoplastic lesions, and cell origin, trying to contribute to a better knowledge of carcinogenesis. Molecular intra-tumour and inter-tumour heterogeneity are also addressed as temporal heterogeneity. Lung cancer heterogeneity has implications in pathogenesis understanding, diagnosis, selection of tissue for molecular diagnosis, as well as therapeutic decision. The understanding of tumour heterogeneity is crucial and we must be aware of the implications and future developments regarding this field.

Neuroendocrine neoplasms (NENs) of the lung encompass neuroendocrine tumors (NETs) composed of typical (TC) and atypical (AC) carcinoids and full-fledged carcinomas (NECs) inclusive of large cell neuroendocrine carcinoma (LCNEC) and small cell carcinoma (SCLC). NETs and NECs are thought to represent distinct and separate lesions with neither molecular overlap nor common developmental continuum. Two perspectives were addressed regarding the morphologic and molecular classification of lung NENs: (i) a supervised approach by browsing the traditional classification, the relevant gene alterations, and their clinical implications; and (ii) an unsupervised approach, by reappraising neoplasms according to risk factors and natural history of disease to construct an interpretation model relied on biological data. We herein emphasize lights and shadows of the current classification of lung NENs and provide an alternative outlook on these tumors focused on what we currently know about the biological determinants and the natural history of disease.

At the end of the twentieth century, a new technology was developed that allowed an entire tissue section to be scanned on an objective slide. Originally called virtual microscopy, this technology is now known as Whole Slide Imaging (WSI). WSI presents new challenges for reading, visualization, storage, and analysis. For this reason, several technologies have been developed to facilitate the handling of these images. In this paper, we analyze the most widely used technologies in the field of digital pathology, ranging from specialized libraries for the reading of these images to complete platforms that allow reading, visualization, and analysis. Our aim is to provide the reader, whether a pathologist or a computational scientist, with the knowledge to choose the technologies to use for new studies, development, or research.

Bone tissue, with its complex structure, often necessitates decalcification of the hard tissue for ex vivo morphological studies. The choice of a suitable decalcification method plays a crucial role in preserving desired features and ensuring compatibility with diverse imaging techniques. The search for a universal decalcification method that is suitable for a range of biophotonic analyses remains an ongoing challenge. In this study, we systematically assessed five standard bone decalcification protocols, encompassing strong mineralic acids (3% and 5% nitric acid), a commercially available formulation of hydrochloric and formic acid), as well as weak organic acids (5% trichloroacetic acid and 8% formic acid), and a chelating agent (25% ethylenediamine-tetraacetic acid) with varying decalcification durations, using mouse long bones as our experimental model. Our imaging analysis panel included classical histological staining (Hematoxylin and Eosin, H&E), immunofluorescence staining, and label-free Raman microspectroscopic imaging. We used cryosections instead of paraffin sections since paraffin interferes with tissue Raman signals. This approach is not as commonly used as it is more prone to handling artifacts, but is the preferred method for subsequent Raman analysis. Decalcification efficacy was evaluated based on various qualitative and some quantitative imaging parameters by 2–3 independent observers. Our systematic approach revealed that the chelating agent, when used for 24 h, optimally preserved bone features and, thus, would be the ideal decalcifying agent for comprehensive subsequent analysis. However, the choice of decalcifier and the ideal decalcification duration may vary depending on the type and thickness of bone, necessitating tailored adjustments to meet specific experimental requirements.

In recent years, gold nanoparticles (AuNPs) have aroused the interest of many researchers due to their unique physicochemical and optical properties. AuNPs are being explored in a variety of biomedical fields, either in diagnostics or therapy, particularly for localized thermal ablation of cancer cells after light irradiation. Besides the promising therapeutic potential of AuNPs, their safety constitutes a highly important issue for any medicine or medical device. For this reason, in the present work, the production and characterization of physicochemical properties and morphology of AuNPs coated with two different materials (hyaluronic and oleic acids (HAOA) and bovine serum albumin (BSA)) were firstly performed. Based on the above importantly referred issue, the in vitro safety of developed AuNPs was evaluated in healthy keratinocytes, human melanoma, breast, pancreatic and glioblastoma cancer cells, as well as in a three-dimensional human skin model. Ex vivo and in vivo biosafety assays using, respectively, human red blood cells and Artemia salina were also carried out. HAOA-AuNPs were selected for in vivo acute toxicity and biodistribution studies in healthy Balb/c mice. Histopathological analysis showed no significant signs of toxicity for the tested formulations. Overall, several techniques were developed in order to characterize the AuNPs and evaluate their safety. All these results support their use for biomedical applications.

Cancer stem cells (CSCs) are a small population of resistant cells inhabiting the tumors. Although comprising only nearly 3% of the tumor mass, these cells were demonstrated to orchestrate tumorigenesis and differentiation, underlie tumors’ heterogeneity and mediate therapy resistance and tumor relapse. Here we show that CSCs may be formed by dedifferentiation of terminally differentiated tumor cells under stress conditions. Using a elegant co-culture cellular system, we were able to prove that nutrients and oxygen deprivation activated non-malignant stromal fibroblasts, which in turn established with tumor cells a paracrine loop mediated by Interleukine-6 (IL-6), Activin-A and Granulocyte colony-stimulating factor (G-CSF), that drove subsequent tumor formation and cellular dedifferentiation. However, by scavenging these cytokines from the media and/or blocking exosomes’ mediated communication it was possible to abrogate dedifferentiation thus turning these mechanisms into potential therapeutic targets against cancer progression.

Breast cancer is a high-burden malignancy for society, whose impact boosts a continuous search for novel diagnostic and therapeutic tools. Among the recent therapeutic approaches, photothermal therapy (PTT), which causes tumor cell death by hyperthermia after being irradiated with a light source, represents a high-potential strategy. Furthermore, the effectiveness of PTT can be improved by combining near infrared (NIR) irradiation with gold nanoparticles (AuNPs) as photothermal enhancers. Herein, an alternative synthetic method using rosmarinic acid (RA) for synthesizing AuNPs is reported. The RA concentration was varied and its impact on the AuNPs physicochemical and optical features was assessed. Results showed that RA concentration plays an active role on AuNPs features, allowing the optimization of mean size and maximum absorbance peak. Moreover, the synthetic method explored here allowed us to obtain negatively charged AuNPs with sizes favoring the local particle accumulation at tumor site and maximum absorbance peaks within the NIR region. In addition, AuNPs were safe both in vitro and in vivo. In conclusion, the synthesized AuNPs present favorable properties to be applied as part of a PTT system combining AuNPs with a NIR laser for the treatment of breast cancer.

Bisphosphonate-associated osteonecrosis of the jaw (BRONJ), a post-surgical non-healing wound condition, is one of the most common side effects in patients treated with nitrogen-containing bisphosphonates. Its physiopathology has been related with suppression of bone turnover, of soft tissue healing and infection. Biphasic calcium phosphates (BCP) are used as a drug delivery vehicle and as a bone substitute in surgical wounds. Due to their capacity to adsorb zoledronate, it was hypothesized these compounds might have a protective effect on the soft tissues in BRONJ wounds. To address this hypothesis, a reproducible in vivo model of BRONJ in Wistar rats was used. This model directly relates chronic bisphosphonate administration with the development of osteonecrosis of the jaw after tooth extraction. BCP granules were placed in the alveolus immediately after tooth extraction in the test group. The animals were evaluated through nuclear medicine, radiology, macroscopic observation, and histologic analysis. Encouragingly, calcium phosphate ceramics were able to limit zoledronate toxicity in vivo and to favor healing, which was evidenced by medical imaging (nuclear medicine and radiology), macroscopically, and through histology. The studied therapeutic option presented itself as a potential solution to prevent the development of maxillary osteonecrosis.

Background: Gastrointestinal (GI) disturbances occur frequently in the early premotor stage of Parkinson’s disease (PD). These GI impairments are associated, at least in part, with dopaminergic dysfunction in the myenteric plexus. However, the enteric nervous system (ENS) pathophysiology underlying GI dysfunction in PD has been overlooked. Objectives: The aim of this study was to evaluate the premotor GI disturbances in rats submitted to intranasal (i.n.) MPTP, a valid experimental model of the premotor stage of PD. Methods: Ileum segments from male Wistar rats (21 weeks old) were collected 12 days following the i.n. MPTP administration for functional studies. Isometric contractile concentration–response (CR) curves (cumulative) for dopamine (DA) were performed in both the presence and absence of sulpiride, a selective dopamine D2-like receptor (D2R) antagonist. Results: Functional studies showed that DA induced a concentration-dependent contractile response in the ileum, which exhibited marked contraction at lower concentrations (0.01–0.9 µM) and relaxation at higher concentrations (3–90 µM). MPTP significantly attenuated both the contraction and the ensuing relaxation. Furthermore, sulpiride significantly reduced the contractile response to DA in the control group and blocked the relaxation in the MPTP group. The MPTP-induced dysmotility occurred with preserved DA homeostasis, as shown by normal DA, TH, and D2R ileal levels in the MPTP group. However, MPTP seemed to impose a decrease in S100β and GFAP (enteroglial markers) immunostaining in the ileal myenteric plexus. Conclusions: In summary, we provide pioneering functional, neurochemical, and morphological evidence showing that rats submitted to the i.n. MPTP model exhibited premotor DA-dependent ileum motile dysfunction accompanied by enteroglial disturbance within the myenteric plexus, but with preserved DA markers.

Inflammatory bowel disease is characterized by chronic relapsing idiopathic inflammation of the gastrointestinal tract and persistent inflammation. Studies focusing on the immune-regulatory function of reactive oxygen species (ROS) are still largely missing. In this study, we analyzed an ROS-deficient mouse model leading to colon adenocarcinoma. Colitis was induced with dextran sulfate sodium (DSS) supplied the drinking water in wild-type (WT) and Ncf1-mutant (Ncf1) B10.Q mice using two different protocols, one mimicking recovery after acute colitis and another simulating chronic colitis. Disease progression was monitored by evaluation of clinical parameters, histopathological analysis, and the blood serum metabolome using H nuclear magnetic resonance spectroscopy. At each experimental time point, colons and spleens from some mice were removed for histopathological analysis and internal clinical parameters. Clinical scores for weight variation, stool consistency, colorectal bleeding, colon length, and spleen weight were significantly worse for Ncf1 than for WT mice. Ncf1 mice with only a 7-day exposure to DSS followed by a 14-day resting period developed colonic distal high-grade dysplasia in contrast to the low-grade dysplasia found in the colon of WT mice. After a 21-day resting period, there was still β-catenin-rich inflammatory infiltration in the Ncf1 mice together with high-grade dysplasia and invasive well-differentiated adenocarcinoma, while in the WT mice, high-grade dysplasia was prominent without malignant invasion and only low inflammation. Although exposure to DSS generated less severe histopathological changes in the WT group, the blood serum metabolome revealed an increased fatty acid content with moderate-to-strong correlations to inflammation score, weight variation, colon length, and spleen weight. Ncf1 mice also displayed a similar pattern but with lower coefficients and showed consistently lower glucose and/or higher lactate levels which correlated with inflammation score, weight variation, and spleen weight. In our novel, DSS-induced colitis animal model, the lack of an oxidative burst ROS was sufficient to develop adenocarcinoma, and display altered blood plasma metabolic and lipid profiles. Thus, oxidative burst seems to be necessary to prevent evolution toward cancer and may confer a protective role in a ROS-mediated self-control mechanism.