Tissue Elasticity as a Diagnostic Marker of Molecular Mutations in Morphologically Heterogeneous Colorectal Cancer.

The presence of molecular mutations in colorectal cancer (CRC) is a decisive factor in selecting the most effective first-line therapy. However, molecular analysis is routinely performed only in a limited number of patients with remote metastases. We propose to use tissue stiffness as a marker of the presence of molecular mutations in CRC samples. For this purpose, we applied compression optical coherence elastography (C-OCE) to calculate stiffness values in regions corresponding to specific CRC morphological patterns (n = 54). In parallel to estimating stiffness, molecular analysis from the same zones was performed to establish their relationships. As a result, a high correlation between the presence of KRAS/NRAS/BRAF driver mutations and high stiffness values was revealed regardless of CRC morphological pattern type. Further, we proposed threshold stiffness values for label-free targeted detection of molecular alterations in CRC tissues: for KRAS, NRAS, or BRAF driver mutation-above 803 kPa (sensitivity-91%; specificity-80%; diagnostic accuracy-85%), and only for KRAS driver mutation-above 850 kPa (sensitivity-90%; specificity-88%; diagnostic accuracy-89%). To conclude, C-OCE estimation of tissue stiffness can be used as a clinical diagnostic tool for preliminary screening of genetic burden in CRC tissues.

The Effect of Diabetes Mellitus Type 1 on the Energy Metabolism of Hepatocytes: Multiphoton Microscopy and Fluorescence Lifetime Imaging.

A decrease in the regenerative potential of the liver during the development of non-alcoholic fatty liver disease (NAFLD), which is observed in the vast majority of patients with diabetes mellitus type 1, significantly increases the risk of postoperative liver failure. In this regard, it is necessary to develop new approaches for the rapid intraoperative assessment of the condition of liver tissue in the presence of concomitant liver pathology. A modern label-free approach based on multiphoton microscopy, second harmonic generation (SHG), and fluorescence lifetime imaging microscopy (FLIM) allow for the evaluation of the structure of liver tissue as well as the assessment of the metabolic state of hepatocytes, even at the cellular level. We obtained optical criteria and identified specific changes in the metabolic state of hepatocytes for a reduced liver regenerative potential in the presence of induced diabetes mellitus type 1. The obtained criteria will expand the possibilities for the express assessment of the structural and functional state of liver tissue in clinical practice.

Effect of Hepatic Pathology on Liver Regeneration: The Main Metabolic Mechanisms Causing Impaired Hepatic Regeneration.

Liver regeneration has been studied for many decades, and the mechanisms underlying regeneration of normal liver following resection are well described. However, no less relevant is the study of mechanisms that disrupt the process of liver regeneration. First of all, a violation of liver regeneration can occur in the presence of concomitant hepatic pathology, which is a key factor reducing the liver's regenerative potential. Understanding these mechanisms could enable the rational targeting of specific therapies to either reduce the factors inhibiting regeneration or to directly stimulate liver regeneration. This review describes the known mechanisms of normal liver regeneration and factors that reduce its regenerative potential, primarily at the level of hepatocyte metabolism, in the presence of concomitant hepatic pathology. We also briefly discuss promising strategies for stimulating liver regeneration and those concerning methods for assessing the regenerative potential of the liver, especially intraoperatively.

Label-Free Imaging Techniques to Evaluate Metabolic Changes Caused by Toxic Liver Injury in PCLS.

Abuse with hepatotoxic agents is a major cause of acute liver failure. The search for new criteria indicating the acute or chronic pathological processes is still a challenging issue that requires the selection of effective tools and research models. Multiphoton microscopy with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are modern label-free methods of optical biomedical imaging for assessing the metabolic state of hepatocytes, therefore reflecting the functional state of the liver tissue. The aim of this work was to identify characteristic changes in the metabolic state of hepatocytes in precision-cut liver slices (PCLSs) under toxic damage by some of the most common toxins: ethanol, carbon tetrachloride (CCl4) and acetaminophen (APAP), commonly known as paracetamol. We have determined characteristic optical criteria for toxic liver damage, and these turn out to be specific for each toxic agent, reflecting the underlying pathological mechanisms of toxicity. The results obtained are consistent with standard methods of molecular and morphological analysis. Thus, our approach, based on optical biomedical imaging, is effective for intravital monitoring of the state of liver tissue in the case of toxic damage or even in cases of acute liver injury.

FLIM imaging revealed spontaneous osteogenic differentiation of stem cells on gradient pore size tissue-engineered constructs.

BACKGROUND: There is an urgent clinical need for targeted strategies aimed at the treatment of bone defects resulting from fractures, infections or tumors. 3D scaffolds represent an alternative to allogeneic MSC transplantation, due to their mimicry of the cell niche and the preservation of tissue structure. The actual structure of the scaffold itself can affect both effective cell adhesion and its osteoinductive properties. Currently, the effects of the structural heterogeneity of scaffolds on the behavior of cells and tissues at the site of damage have not been extensively studied. METHODS: Both homogeneous and heterogeneous scaffolds were generated from poly(L-lactic acid) methacrylated in supercritical carbon dioxide medium and were fabricated by two-photon polymerization. The homogeneous scaffolds consist of three layers of cylinders of the same diameter, whereas the heterogeneous (gradient pore sizes) scaffolds contain the middle layer of cylinders of increased diameter, imitating the native structure of spongy bone. To evaluate the osteoinductive properties of both types of scaffold, we performed in vitro and in vivo experiments. Multiphoton microscopy with fluorescence lifetime imaging microscopy was used for determining the metabolic states of MSCs, as a sensitive marker of cell differentiation. The results obtained from this approach were verified using standard markers of osteogenic differentiation and based on data from morphological analysis. RESULTS: The heterogeneous scaffolds showed improved osteoinductive properties, accelerated the metabolic rearrangements associated with osteogenic differentiation, and enhanced the efficiency of bone tissue recovery, thereby providing for both the development of appropriate morphology and mineralization. CONCLUSIONS: The authors suggest that the heterogeneous tissue constructs are a promising tool for the restoration of bone defects. And, furthermore, that our results demonstrate that the use of label-free bioimaging methods can be considered as an effective approach for intravital assessment of the efficiency of differentiation of MSCs on scaffolds.

Optical Biomedical Imaging Reveals Criteria for Violated Liver Regenerative Potential.

To reduce the risk of post-hepatectomy liver failure in patients with hepatic pathologies, it is necessary to develop an approach to express the intraoperative assessment of the liver's regenerative potential. Traditional clinical methods do not enable the prediction of the function of the liver remnant. Modern label-free bioimaging, using multiphoton microscopy in combination with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), can both expand the possibilities for diagnosing liver pathologies and for assessing the regenerative potential of the liver. Using multiphoton and SHG microscopy, we assessed the structural state of liver tissue at different stages of induced steatosis and fibrosis before and after 70% partial hepatectomy in rats. Using FLIM, we also performed a detailed analysis of the metabolic state of the hepatocytes. We were able to determine criteria that can reveal a lack of regenerative potential in violated liver, such as the presence of zones with reduced NAD(P)H autofluorescence signals. Furthermore, for a liver with pathology, there was an absence of the jump in the fluorescence lifetime contributions of the bound form of NADH and NADPH the 3rd day after hepatectomy that is characteristic of normal liver regeneration. Such results are associated with decreased intensity of oxidative phosphorylation and of biosynthetic processes in pathological liver, which is the reason for the impaired liver recovery. This modern approach offers an effective tool that can be successfully translated into the clinic for express, intraoperative assessment of the regenerative potential of the pathological liver of a patient.

Effect of Collagen Matrix on Doxorubicin Distribution and Cancer Cells' Response to Treatment in 3D Tumor Model.

The extracellular matrix (ECM) plays an important role in regulation of many aspects of tumor growth and response to therapies. However, the specifics of the interaction of chemotherapeutic agents with cancer cells in the presence of collagen, the major component of ECM, is still poorly investigated. In this study, we explored distribution of doxorubicin (DOX) and its effects on cancer cells' metabolism in the presence of collagen with different structures in 3D models. For this, a combination of second harmonic generation imaging of collagen and multiphoton fluorescence microscopy of DOX, and metabolic cofactor NAD(P)H was used. It was found that collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen. As a result, the cells in collagen displayed better viability upon treatment with DOX. Taken together, our data illustrate that tumor collagen contributes to heterogeneous and sub-optimal response to DOX and highlight the challenges in improving drug delivery and efficacy.

SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative.

The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.

Direct transmembrane clustering and cytoplasmic dimerization of focal adhesion kinase initiates its tyrosine phosphorylation.

We investigated mechanisms for inducing focal adhesion kinase (FAK) tyrosine phosphorylation and their ability to trigger MAP kinase signaling using transmembrane chimeras that localize FAK and its mutants to the plasma membrane. We tested whether tyrosine phosphorylation was triggered by FAK transmembrane aggregation using antibodies against the chimeric extracellular domain. Experimental clustering of chimeras containing integrin beta cytoplasmic domains or FAK induced FAK tyrosine phosphorylation and trans-phosphorylation of endogenous FAK, as well as strong ERK activation. Next, we examined whether lower-order molecular proximity, namely dimerization, could regulate FAK tyrosine phosphorylation. We found that even relatively low-affinity FAK dimerization (K(d)=3.9 x 10(-5) M), in either of two different orientations, could induce FAK tyrosine phosphorylation. However, this cytoplasmic FAK dimerization could not induce MAP kinase activation or trans-phosphorylation of endogenous FAK. We conclude that dimerization of FAK is sufficient to induce its tyrosine phosphorylation, but that higher-order molecular proximity (clustering) at the cell membrane is apparently needed for additional biochemical events. This study identifies a proximity mechanism for regulating the initiation of FAK-mediated biochemical signaling.