Optical Imaging in Human Lymph Node Specimens for Detecting Breast Cancer Metastases: A Review.

Assessment of regional lymph node status in breast cancer is of important staging and prognostic value. Even though formal histological examination is the currently accepted standard of care, optical imaging techniques have shown promising results in disease diagnosis. In the present article, we review six spectroscopic techniques and focus on their use as alternative tools for breast cancer lymph node assessment. Elastic scattering spectroscopy (ESS) seems to offer a simple, cost-effective, and reproducible method for intraoperative diagnosis of breast cancer lymph node metastasis. Optical coherence tomography (OCT) provides high-resolution tissue scanning, along with a short data acquisition time. However, it is relatively costly and experimentally complex. Raman spectroscopy proves to be a highly accurate method for the identification of malignant axillary lymph nodes, and it has been further validated in the setting of head and neck cancers. Still, it remains time-consuming. Near-infrared fluorescence imaging (NIRF) and diffuse reflectance spectroscopy (DFS) are related to significant advantages, such as deep tissue penetration and efficiency. Fourier-transform infrared spectroscopy (FTIR) is a promising method but has significant drawbacks. Nonetheless, only anecdotal reports exist on their clinical use for cancerous lymph node detection. Our results indicate that optical imaging methods can create informative and rapid tools to effectively guide surgical decision-making.

The TNFR Wengen regulates the FGF pathway by an unconventional mechanism.

Unveiling the molecular mechanisms of receptor activation has led to much understanding of development as well as the identification of important drug targets. We use the Drosophila tracheal system to study the activity of two families of widely used and conserved receptors, the TNFRs and the RTK-FGFRs. Breathless, an FGFR, controls the program of differentiation of the tracheal terminal cells in response to ligand activation. Here we identify a role for Wengen, a TNFR, in repressing the terminal cell program by regulating the MAPK pathway downstream of Breathless. We find that Wengen acts independently of both its canonical ligand and downstream pathway genes. Wengen does not stably localise at the membrane and is instead internalised-a trafficking that seems essential for activity. We show that Breathless and Wengen colocalise in intracellular vesicles and form a complex. Furthermore, Wengen regulates Breathless accumulation, possibly regulating Breathless trafficking and degradation. We propose that, in the tracheal context, Wengen interacts with Breathless to regulate its activity, and suggest that such unconventional mechanism, involving binding by TNFRs to unrelated proteins, may be a general strategy of TNFRs.

Polyploidy-associated autophagy promotes larval tracheal histolysis at Drosophila metamorphosis.

Polyploidy is an extended phenomenon in biology. However, its physiological significance and whether it defines specific cell behaviors is not well understood. Here we study its connection to macroautophagy/autophagy, using the larval respiratory system of Drosophila as a model. This system comprises cells with the same function yet with notably different ploidy status, namely diploid progenitors and their polyploid larval counterparts, the latter destined to die during metamorphosis. We identified an association between polyploidy and autophagy and found that higher endoreplication status correlates with elevated autophagy. Finally, we report that tissue histolysis in the trachea during Drosophila metamorphosis is mediated by autophagy, which triggers the apoptosis of polyploid cells.Abbreviations: APF: after pupa formation; Atg: autophagy related; btl: breathless; CycE: Cyclin E; DT: dorsal trunk; fzr: fizzy-related; L3: larval stage 3; PBS: phosphate-buffered saline; RI: RNAi; Tr: tracheal metamere; yki: yorkie.

Antagonistic role of the BTB-zinc finger transcription factors Chinmo and Broad-Complex in the juvenile/pupal transition and in growth control.

During development, the growing organism transits through a series of temporally regulated morphological stages to generate the adult form. In humans, for example, development progresses from childhood through to puberty and then to adulthood, when sexual maturity is attained. Similarly, in holometabolous insects, immature juveniles transit to the adult form through an intermediate pupal stage when larval tissues are eliminated and the imaginal progenitor cells form the adult structures. The identity of the larval, pupal, and adult stages depends on the sequential expression of the transcription factors chinmo, Br-C, and E93. However, how these transcription factors determine temporal identity in developing tissues is poorly understood. Here, we report on the role of the larval specifier chinmo in larval and adult progenitor cells during fly development. Interestingly, chinmo promotes growth in larval and imaginal tissues in a Br-C-independent and -dependent manner, respectively. In addition, we found that the absence of chinmo during metamorphosis is critical for proper adult differentiation. Importantly, we also provide evidence that, in contrast to the well-known role of chinmo as a pro-oncogene, Br-C and E93 act as tumour suppressors. Finally, we reveal that the function of chinmo as a juvenile specifier is conserved in hemimetabolous insects as its homolog has a similar role in Blatella germanica. Taken together, our results suggest that the sequential expression of the transcription factors Chinmo, Br-C and E93 during larva, pupa an adult respectively, coordinate the formation of the different organs that constitute the adult organism.

Egg, larva, pupa, adult: the life of many insects is structured around these four well-defined stages of development. After hatching, the larva grows until it reaches a certain size; when the right conditions are met, it then becomes a pupa and metamorphoses into an adult. Most larval cells die during metamorphosis; only a group known as imaginal cells survives, dividing and maturing to create pupal and adult tissues. Each of these developmental steps are linked to a particular genetic program deployed in response to a single stage-specifying gene. For instance, the activation of the Br-C gene triggers the transition from larva to pupa, while E93 initiates the transformation of the pupa into an adult. However, which stage-specifying gene controls larval identity remains unclear. Recent studies suggest that in fruit flies, a gene known as chinmo could be playing this role. In response, Chafino et al. explored how chinmo shapes the development of fruit fly larvae. The experiments showed that chinmo is activated in the juvenile stage, and that it is required for the larvae to grow properly and for larval and imaginal tissues to form. Conversely, it must be switched off for the insect to become a pupa and then an adult. Further work suggested that the role of chinmo as a larval specifier could have emerged early in insect evolution. Moreover, Chafino et al. revealed that chinmo could repress Br-C, an important characteristic since stage-specifying genes usually switch on sequentially by regulating each other. A closer look suggested that, in imaginal cells, chinmo promotes development by inhibiting Br-C; in larval cells, however, chinmo not only has a Brc-repressing role but it is also necessary for larval cells to grow. Additional experiments exploring the role of the stage-specifying genes in tumor formation showed that chinmo promotes cells proliferation while Br-C and E93 had tumor-suppressing properties. Overall, the work by Chafino et al. sheds new light on the genetic control of insect development, while also potentially providing a new perspective on how genes related to chinmo and Br-C contribute to the emergence of human cancers.

A dynamic interplay between chitin synthase and the proteins Expansion/Rebuf reveals that chitin polymerisation and translocation are uncoupled in Drosophila.

Chitin is a highly abundant polymer in nature and a principal component of apical extracellular matrices in insects. In addition, chitin has proved to be an excellent biomaterial with multiple applications. In spite of its importance, the molecular mechanisms of chitin biosynthesis and chitin structural diversity are not fully elucidated yet. To investigate these issues, we use Drosophila as a model. We previously showed that chitin deposition in ectodermal tissues requires the concomitant activities of the chitin synthase enzyme Kkv and the functionally interchangeable proteins Exp and Reb. Exp/Reb are conserved proteins, but their mechanism of activity during chitin deposition has not been elucidated yet. Here, we carry out a cellular and molecular analysis of chitin deposition, and we show that chitin polymerisation and chitin translocation to the extracellular space are uncoupled. We find that Kkv activity in chitin translocation, but not in polymerisation, requires the activity of Exp/Reb, and in particular of its conserved Nα-MH2 domain. The activity of Kkv in chitin polymerisation and translocation correlate with Kkv subcellular localisation, and in absence of Kkv-mediated extracellular chitin deposition, chitin accumulates intracellularly as membrane-less punctae. Unexpectedly, we find that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane. We propose a model in which Exp/Reb regulate the organisation of Kkv complexes at the apical membrane, which, in turn, regulates the function of Kkv in extracellular chitin translocation.

Complex refractive index of freshly excised human breast tissue as a marker of disease.

We report differences in the refractive index of healthy and tumorous freshly excised human breast tissue as determined from reflectance profile measurements at five wavelengths (432 nm, 532 nm, 633 nm, 964 nm, 1551 nm) in the visible and near-infrared using a standard prism-coupling refractometer. These refractive index differences, particularly in the near-infrared, can be used to distinguish fibroadenomas and cancerous growths not only from normal breast tissue but also from each other.

Systemic and local effect of the Drosophila headcase gene and its role in stress protection of Adult Progenitor Cells.

During the development of a holometabolous insect such as Drosophila, specific group of cells in the larva survive during metamorphosis, unlike the other larval cells, and finally give rise to the differentiated adult structures. These cells, also known as Adult Progenitor Cells (APCs), maintain their multipotent capacity, differentially respond to hormonal and nutritional signals, survive the intrinsic and environmental stress and respond to the final differentiation cues. However, not much is known about the specific molecular mechanisms that account for their unique characteristics. Here we show that a specific Drosophila APC gene, headcase (hdc), has a dual role in the normal development of these cells. It acts at a systemic level by controlling the hormone ecdysone in the prothoracic gland and at the same time it acts locally as a tissue growth suppressor in the APC clusters, where it modulates the activity of the TOR pathway and promotes their survival by contributing in the regulation of the Unfolded Protein Response. We also show that hdc provides protection against stress in the APCs and that its ectopic expression in cells that do not usually express hdc can confer these cells with an additional stress protection. Hdc is the founding member of a group of homolog proteins identified from C. elegans to humans, where has been found associated with cancer progression. The finding that the Drosophila hdc is specifically expressed in progenitor cells and that it provides protection against stress opens up a new hypothesis to be explored regarding the role of the human Heca and its contribution to carcinogenesis.

Impact of Deserosalization on Small Bowel Anastomosis Healing in Swine: A Pilot Study.

BACKGROUND: Healing is related to gastrointestinal anastomotic leak, which is a severe and common complication. This study aimed to investigate the feasibility and the impact of deserosalization on healing of jejuno-jejunal anastomoses in an animal model. MATERIALS AND METHODS: Seven swine underwent three types of side-to-side jejuno-jejunal anastomosis twice and survived seven days. Three different types of jejuno-jejunal side-to-side anastomoses were performed twice at 20-cm distance from each other in each animal: no serosa removal, one-sided, and two-sided serosa removal, respectively. Bursting pressure, tissue hydroxyproline concentration, and pathology scores were evaluated. RESULTS: Hydroxyproline tissue concentration was a mean±standard deviation of 0.37±0.09, 0.38±0.08, and 0.30±0.05 nmoI/ml respectively (p<0.05). Bursting pressure was a mean±standard deviation of 59.02±8.60, 73.20±11.09, and 100.01±7.49 mmHg, respectively (p<0.001). The histopathological assessment did not find any statistically significant differences. CONCLUSION: Deserosalization in jejuno-jejunal anastomosis was technically feasible and seemed to improve mechanical strength and collagen deposition in this experimental porcine model. Further investigation is warranted.

Targeting of copper-trafficking chaperones causes gene-specific systemic pathology in Drosophila melanogaster: prospective expansion of mutational landscapes that regulate tumor resistance to cisplatin.

Copper, a transition metal, is an essential component for normal growth and development. It acts as a critical co-factor of many enzymes that play key roles in diverse cellular processes. The present study attempts to investigate the regulatory functions decisively controlling copper trafficking during development and aging of the Drosophila model system. Hence, through engagement of the GAL4/UAS genetic platform and RNAi technology, we herein examined the in vivo significance of Atox1 and CCS genes, products of which pivotally govern cellular copper trafficking in fly tissue pathophysiology. Specifically, we analyzed the systemic effects of their targeted downregulation on the eye, wing, neuronal cell populations and whole-body tissues of the fly. Our results reveal that, in contrast to the eye, suppression of their expression in the wing leads to a notable increase in the percentage of malformed organs observed. Furthermore, we show that Atox1 or CCS gene silencing in either neuronal or whole-body tissues can critically affect the viability and climbing capacity of transgenic flies, while their double-genetic targeting suggests a rather synergistic mode of action of the cognate protein products. Interestingly, pharmacological intervention with the anti-cancer drug cisplatin indicates the major contribution of CCS copper chaperone to cisplatin's cellular trafficking, and presumably to tumor resistance often acquired during chemotherapy. Altogether, it seems that Atox1 and CCS proteins serve as tissue/organ-specific principal regulators of physiological Drosophila development and aging, while their tissue-dependent downregulation can provide important insights for Atox1 and CCS potential exploitation as predictive gene biomarkers of cancer-cell chemotherapy responses.

Complex refractive index of normal and malignant human colorectal tissue in the visible and near-infrared.

A multi-wavelength prism coupling refractometer is utilized to measure the angular reflectance of freshly excised human intestinal tissue specimens. Based on reflectance data, the real and imaginary part of the refractive index is calculated via Fresnel analysis for three visible (blue, green, red) and two near-infrared (963 nm and 1551 nm) wavelengths. Averaged values of the complex refractive index and corresponding Cauchy dispersion fits are given for the mucosa, submucosa and serosa layers of the colorectal wall at the normal state. The refractive constants of tumorous and normal mucosa are then cross-compared for the indicative cases of one patient diagnosed with a benign polyp and three patients diagnosed with adenocarcinomas of different phenotype. Significant index contrast exists between the normal and diseased states, indicating the potential use of refractive index as a marker of colorectal dysplasia.

Visible to near-infrared refractive properties of freshly-excised human-liver tissues: marking hepatic malignancies.

The refractive index is an optical constant that plays a significant role in the description of light-matter interactions. When it comes to biological media, refraction is understudied despite recent advances in the field of bio-optics. In the present article, we report on the measurement of the refractive properties of freshly excised healthy and cancerous human liver samples, by use of a prism-coupling technique covering the visible and near-infrared spectral range. Novel data on the wavelength-dependent complex refractive index of human liver tissues are presented. The magnitude of the real and imaginary part of the refractive index is correlated with hepatic pathology. Notably, the real index contrast is pointed out as a marker of discrimination between normal liver tissue and hepatic metastases. In view of the current progress in optical biosensor technologies, our findings may be exploited for the development of novel surgical and endoscopic tools.

The embryonic transcription factor Zelda of Drosophila melanogaster is also expressed in larvae and may regulate developmentally important genes.

The transcription factor Zelda plays a pivotal role in promoting the maternal to zygotic transition during embryogenesis in Drosophila melanogaster. However, little is known about its role later in development. Here we are showing that Zelda is essential for proper wing development through gain and loss of function experiments. Zelda's transcript variants RB, RC and RD are present in imaginal wing discs of third instar larvae and the production of 2 protein isoforms of ∼180 and ∼70kD was detected in the same tissue. In ChIP experiments using larval wing discs, Zelda was found to bind to a region of the optomotor-blind gene, suggesting an interaction with a Dpp target that promotes wing growth and patterning.