Liquid biopsy in triple-negative breast cancer: unlocking the potential of precision oncology.

In the era of precision oncology, the management of triple-negative breast cancer (TNBC) is rapidly changing and becoming more complicated with a variety of chemotherapy, immunotherapy, and targeted treatment options. Currently, TNBC treatment is based on prognostic and predictive factors including immunohistochemical biomarkers [e.g. programmed death-ligand 1 (PD-L1)] and germline BRCA mutations. Given the current limitation of existing biomarkers, liquid biopsies may serve as clinically useful tools to determine treatment efficacy and response in both the (neo)adjuvant and metastatic settings, for detecting early relapse, and for monitoring clonal evolution during treatment. In this review, we comprehensively summarize current and future liquid biopsy applications. Specifically, we highlight the role of circulating tumor cell characterization, circulating tumor DNA, and other preclinical liquid biopsy technologies including circulating exosomes, RNA liquid biopsy, and circulating immune-based biomarkers. In the near future, these biomarkers may serve to identify early disease relapse, therapeutic targets, and disease clonality for patients with TNBC in the clinical setting.

Development of innovative embedding procedures for the analyses of paint cross sections in ATR FITR microscopy.

We report the development of innovative embedding procedures for the analysis of paint cross sections by attenuated total reflection (ATR) Fourier transform IR microscopy. This technique was chosen because it is widely employed for the characterization and spatial location of organic and inorganic components in artistic samples. Moreover, the performance of the technique may be critically affected by sample preparation in terms of surface morphology and the presence of contamination. First, we evaluated the use of KBr as a barrier to contamination by the embedding synthetic medium. In this way, the sample cross section can be polished by means of a sample holder, which allows a controlled pressure to be applied to the sample, thus improving the reproducibility and quality of the surface cross section. In addition, argon ion milling was used for the polishing of samples embedded in KBr, and provided very promising results in terms of surface planarity and reduction of superficial contamination by KBr. Finally, the use of NaCl as an alternative to KBr was proposed thanks to its advantages in terms of hygroscopicity, cost, and toxicity. In addition, cross sections embedded in NaCl were characterized by greater hardness, a feature that allowed us to obtain improved contact with the ATR crystal.

Single and multiplexed immunoassays for the chemiluminescent imaging detection of animal glues in historical paint cross-sections.

The characterization of the organic components in a complex, multilayered paint structure is fundamental for studying painting techniques and for authentication and restoration purposes. Proteinaceous materials, such as animal glue, are of particular importance since they are widely used as binders, adhesives and for gilding. Even though proteins are usually detected by chromatographic and proteomic techniques, immunological methods represent an alternative powerful approach to protein analysis thanks to the high specificity of antigen-antibody reactions. Our previous studies demonstrated that ovalbumin and casein could be localized in paint cross-sections with high sensitivity and good spatial resolution (i.e. within the single painting layers) by using chemiluminescent (CL) immunochemical microscope imaging. In the present research work, we describe for the first time the immunolocalization of collagen (the main protein of animal glue) in paint cross-sections by CL imaging microscopy. Two different analytical protocols have been developed, allowing either the detection of collagen or the simultaneous detection of collagen and ovalbumin in the same paint sample. The assays were used to detect collagen and ovalbumin in cross-sections from model samples and historical paintings (a wall painting dated to 1773-1774 and a painted wood panel of the Renaissance period) in order to achieve information on paint techniques and past restoration interventions.

Digital restoration of colour cinematic films using imaging spectroscopy and machine learning.

Digital restoration is a rapidly growing methodology within the field of heritage conservation, especially for early cinematic films which have intrinsically unstable dye colourants that suffer from irreversible colour fading. Although numerous techniques to restore film digitally have emerged recently, complex degradation remains a challenging problem. This paper proposes a novel vector quantization (VQ) algorithm for restoring movie frames based on the acquisition of spectroscopic data with a custom-made push-broom VNIR hyperspectral camera (380-780 nm). The VQ algorithm utilizes what we call a multi-codebook that correlates degraded areas with corresponding non-degraded ones selected from reference frames. The spectral-codebook was compared with a professional commercially available film restoration software (DaVinci Resolve 17) tested both on RGB and on hyperspectral providing better results in terms of colour reconstruction.

Combining elemental and immunochemical analyses to characterize diagenetic alteration patterns in ancient skeletal remains.

Bones and teeth are biological archives, but their structure and composition are subjected to alteration overtime due to biological and chemical degradation postmortem, influenced by burial environment and conditions. Nevertheless, organic fraction preservation is mandatory for several archeometric analyses and applications. The mutual protection between biomineral and organic fractions in bones and teeth may lead to a limited diagenetic alteration, promoting a better conservation of the organic fraction. However, the correlation between elemental variations and the presence of organic materials (e.g., collagen) in the same specimen is still unclear. To fill this gap, chemiluminescent (CL) immunochemical imaging analysis has been applied for the first time for collagen localization. Then, Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS) and CL imaging were combined to investigate the correlation between elemental (i.e., REE, U, Sr, Ba) and collagen distribution. Teeth and bones from various archeological contexts, chronological periods, and characterized by different collagen content were analyzed. Immunochemical analysis revealed a heterogeneous distribution of collagen, especially in highly degraded samples. Subsequently, LA-ICP-MS showed a correlation between the presence of uranium and rare earth elements and areas with low amount of collagen. The innovative integration between the two methods permitted to clarify the mutual relation between elemental variation and collagen preservation overtime, thus contributing to unravel the effects of diagenetic alteration in bones and teeth.

New advances in the application of FTIR microscopy and spectroscopy for the characterization of artistic materials.

Fourier transform infrared (FTIR) spectroscopy is one of the most widely applied techniques for the investigation of cultural heritage materials. FTIR microscopy is well established as an essential tool in the microdestructive analysis of small samples, and the recent introduction of mapping and imaging equipment allows the collection of a large number of FTIR spectra on a surface, providing a distribution map of identified compounds. In this Account, we report recent advances in FTIR spectroscopy and microscopy in our research group. Our laboratory develops, tests, and refines new and less-studied IR spectroscopy and microscopy methods, with the goal of their adoption as routine analytical techniques in conservation laboratories. We discuss (i) the analysis of inorganic materials inactive in the mid-IR region by means of far-IR spectroscopy, (ii) the development of new methods for preparing cross sections, (iii) the characterization and spatial location of thin layers and small particles, and (iv) the evaluation of protective treatments. FTIR spectroscopy and microscopy have been mostly used in the mid-IR region of 4000-600 cm(-1). Some inorganic pigments, however, are inactive in this region, so other spectroscopic techniques have been applied, such as Raman spectroscopy. We suggest an alternative: harnessing the far-IR (600-50 cm(-1)). Our initial results show that far-IR spectroscopy is exceptionally useful with mural paintings or with corrosion products from which larger sample quantities can generally be collected. Moreover, the inorganic composition of a sample can be characterized by the presence of several compounds that are inactive in the mid-IR range (such as sulfides, oxides, and so forth). Stratigraphical analyses by FTIR microscopy can be hindered by the process of cross section preparation, which often involves an embedding organic polymer penetrating the sample's porous structure. Here, the polymer bands may completely cover the bands of organic compounds in the sample. However, a correct methodological approach can prevent such limitations. For example, it is always advisable to analyze the sample surface before preparing the cross section in order to characterize the preparation layers and the varnish layers, which are generally applied to the surface of a painting both to protect it and improve the color saturation. Furthermore, the innovative use of IR-transparent salts as embedding material for cross sections can prevent contamination of the embedding resin and improve detection of organic substances. Another key point in the use of FTIR microscopy in artwork analysis is spatial resolution. The high-energy output of a new integrated FTIR microscope enhances the ability to characterize and spatially locate small particles and thin layers. Moreover, the new configuration proves extremely useful in the evaluation of protective treatments, because larger areas may be analyzed in less time in comparison to traditional systems, allowing the collection of more statistical data.

Near-infrared hyperspectral imaging (NIR-HSI) and normalized difference image (NDI) data processing: An advanced method to map collagen in archaeological bones.

In the present study, an innovative and highly efficient near-infrared hyperspectral imaging (NIR-HSI) method is proposed to provide spectral maps able to reveal collagen distribution in large-size bones, also offering semi-quantitative estimations. A recently introduced method for the construction of chemical maps, based on Normalized Difference Images (NDI), is declined in an innovative approach, through the exploitation of the NDI values computed for each pixel of the hyperspectral image to localize collagen and to extract information on its content by a direct comparison with known reference samples. The developed approach addresses an urgent issue of the analytical chemistry applied to bioarcheology researches, which rely on well-preserved collagen in bones to obtain key information on chronology, paleoecology and taxonomy. Indeed, the high demand for large-sample datasets and the consequent application of a wide variety of destructive analytical methods led to the considerable destruction of precious bone samples. NIR-HSI pre-screening allows researchers to properly select the sampling points for subsequent specific analyses, to minimize costs and time and to preserve integrity of archaeological bones (which are available in a very limited amount), providing further opportunities to understand our past.

Mutation of a bHLH transcription factor allowed almond domestication.

Wild almond species accumulate the bitter and toxic cyanogenic diglucoside amygdalin. Almond domestication was enabled by the selection of genotypes harboring sweet kernels. We report the completion of the almond reference genome. Map-based cloning using an F1 population segregating for kernel taste led to the identification of a 46-kilobase gene cluster encoding five basic helix-loop-helix transcription factors, bHLH1 to bHLH5. Functional characterization demonstrated that bHLH2 controls transcription of the P450 monooxygenase-encoding genes PdCYP79D16 and PdCYP71AN24, which are involved in the amygdalin biosynthetic pathway. A nonsynonymous point mutation (Leu to Phe) in the dimerization domain of bHLH2 prevents transcription of the two cytochrome P450 genes, resulting in the sweet kernel trait.

Attenuated total reflection micro FTIR characterisation of pigment-binder interaction in reconstructed paint films.

The interaction of pigments and binding media may result in the production of metal soaps on the surface of paintings which modifies their visible appearance and state of conservation. To characterise more fully the metal soaps found on paintings, several historically accurate oil and egg yolk tempera paint reconstructions made with different pigments and naturally aged for 10 years were submitted to attenuated total reflectance Fourier transform infrared (ATR FTIR) microspectroscopic analyses. Standard metal palmitates were synthesised and their ATR spectra recorded in order to help the identification of metal soaps. Among the different lead-based pigments, red lead and litharge seemed to produce a larger amount of carboxylates compared with lead white, Naples yellow and lead tin yellow paints. Oil and egg tempera litharge and red lead paints appeared to be degraded into lead carbonate, a phenomenon which has been observed for the first time. The formation of metal soaps was confirmed on both oil and egg tempera paints based on zinc, manganese and copper and in particular on azurite paints. ATR mapping analyses showed how the areas where copper carboxylates were present coincided with those in which azurite was converted into malachite. Furthermore, the key role played by manganese in the production of metals soaps on burnt and raw sienna and burnt and raw umber paints has been observed for the first time. The formation of copper, lead, manganese, cadmium and zinc metal soaps was also identified on egg tempera paint reconstructions even though, in this case, the overlapping of the spectral region of the amide II band with that of metal carboxylates made their identification difficult.

New Frontiers in Application of FTIR Microscopy for Characterization of Cultural Heritage Materials.

We present an overview of recent advances in the application of Fourier Transform Infrared (FTIR) microscopy for analysis of complex, multicomponent, and multilayer samples such as those typically encountered in the field of heritage materials. This technique is particularly useful since it allows identification and localization of both organic and inorganic (if IR active) compounds. New improvements have been possible thanks to the introduction of ad hoc sample preparation methods to obtain either thin or cross sections that allow both avoidance of contamination from organic embedding resin and improvement of the quality of the acquired spectra. Moreover, integrated use of spectra registered in the near-infrared (NIR) and mid-infrared (MIR) regions allows better comprehension of cross section composition. Data interpretation has been improved thanks to the development of chemometric methods for elaboration of hyperspectral data. A new and very promising field is the development of enhanced FTIR methods for detection of trace components in microextracts. These systems, allowing detection of extractable organic compounds from about 0.1 mg of sample, will be extremely useful in the future for analysis of natural and synthetic colorants, varnishes extracted, for instance, from cotton swabs used during cleaning of paintings, and organic residues on archeological remains.

Analyses of trace amounts of dyes with a new enhanced sensitivity FTIR spectroscopic technique: MU-ATR (metal underlayer ATR spectroscopy).

The identification of organic dyes is a challenging task in all the fields such as the forensic and conservation sciences, especially in cases where the amount of sample is extremely small. In this paper we propose a new enhanced FTIR method (MU-ATR metal underlayer ATR spectroscopy), which we believe is the first of its kind, for the analysis of a few ng of dyes. With this method, dyed fiber micro-extracts can be analyzed using a commercial FTIR microscope with a fixed incident angle, obtaining the same separation between the different classes of dyes investigated as we obtained analyzing pure dyes in transmission mode. Moreover, the new enhancement method has been validated on a real sample dated back to the 1893, showing how it can be promising for the analysis of trace amounts of organic substances in artistic samples such as dyes in paintings or textiles, varnishes and organic residues on archaeological objects.

Exercise does not modify spatial memory, brain autoimmunity, or antibody response in aged F-344 rats.

Old F-344 rats were given endurance training over a 10-week period on a motorized treadmill. This treatment resulted in substantial heart-to-body weight ratio increases, indicative of effective training. To determine whether endurance training might alter some of the known immune system and cognitive changes observed during aging, exercised old rats were compared to nonexercised old and young controls on three variables: in vivo antigen-specific immune activity, brain-reactive antibody formation, and spatial memory. The exercise training did not influence any of these measures in the old rats. Both groups of old rats showed poorer antibody response to a specific antigen, more brain-reactive antibody formation, and poorer spatial memory than the young controls. There was, however, a significant relationship between brain-reactive antibody formation and spatial memory performance, regardless of training condition.

Spaceflight downregulates antioxidant defense systems in rat liver.

Liver antioxidant enzyme activities, mRNA abundance, and glutathione (GSH) status were investigated in male Sprague-Dawley rats placed in an enclosure module aboard Space Shuttle STS-63 for 8 d (F, n = 6). F animals were compared to rats housed in an enclosure module on the ground (G, n = 9), which simulated the vibration and temperature conditions associated with launch and flight, and rats kept under conventional ground vivarium conditions in individual cages (V, n = 6). Spaceflight significantly decreased catalase, GSH reductase, and GSH sulfur-transferase activities in the liver (p < .05). Neither enzyme activity nor enzyme protein content of Cu-Zn and Mn superoxide dismutase (SOD) was affected by flight. The relative abundance of mRNA for Cu-Zn SOD and catalase was significantly decreased comparing F with G rats (p < .05). Spaceflight resulted in a dramatic decrease of liver GSH, glutathione disulfide, and total GSH contents (p < .01), which were accompanied by a lower gamma-glutamyl transpeptidase activity (p < .05). F rats showed a 47% (p < .05) increase in liver malondialdehyde concentration compared to G and V rats. Liver protein content was not affected by flight. These results indicate that spaceflight can downregulate antioxidant defense capacity and elicit an oxidative stress in the liver.

Effects of training on weight, food intake, and body composition in aging rats.

This study investigated the effect of endurance training on body weight, food intake, and body composition in female rats age 6, 15, and 27 mo. Animals underwent 12 wk of treadmill running. There was a significant increase in percent body fat associated with age. Training induced significant reductions in body fat which were of the same relative magnitude in all age groups. The 6-mo and 15-mo exercising rats significantly increased their food intake in response to the training stimulus; the 27-mo trained animals were unable to increase their food consumption relative to controls. It was concluded that 27-mo old animals are capable of changes in body composition similar to those of their younger counterparts, however, physiological mechanisms responsible for these shifts are age dependent. Older animals do not compensate their dietary intake for the increased energy expenditure of exercise as do the younger animals.

Effects of age and endurance training on beta-adrenergic receptor characteristics in Fischer 344 rats.

The purpose of this investigation was to examine changes in beta-adrenergic receptor characteristics in various tissues with age and endurance training. Forty-eight young (6 months), middle-aged (15 months), and old (25 months) male Fischer 344 rats were assigned to either a trained or sedentary running group. Animals were endurance trained by 10 weeks of treadmill running at 75% maximal capacity, 1 h/day, 5 days/week. Animals were sacrificed at rest and the heart, liver, and soleus were removed for analysis. Percent of high and low affinity binding sites were determined by competitive binding experiments. Competition curves were generated using 12 concentrations of ICI-89406 (beta 1 antagonist) and ICI-118551 (beta 2 antagonist) to inhibit the total binding of (-) [125I] pindolol (IPIN). Maximal binding site number (Bmax) and affinity (KD) were determined by Scatchard analysis. Heart Bmax did not differ with age or training. An aging effect was observed in liver such that middle-aged and old animals had greater Bmax compared to young animals. In soleus, Bmax was not altered with training but decreased with age. While training had no affect on affinity in the liver and soleus, heart affinity increased with training in both the middle-aged (21%) and old (27%) animals. In soleus, affinity increased but remained unaltered in heart and liver with age. The ratio of beta 1:beta 2 receptors in the heart and liver did not differ with age or training. The influence of age and training on beta-adrenergic receptor characteristics appear to be tissue specific.

A comparison of skeletal muscle morphology with training between young and old Fischer 344 rats.

Muscle mass, fiber area, total fiber number, fiber population and capillarity were assessed in 6- and 25-month-old animals trained by treadmill running at 75% mean maximal capacity for 10 weeks. Serial cross-sections were stained for ATPase activity to differentiate fiber types and expose capillaries. Aging and training effects were demonstrated in maximal running speed and endurance running time. Soleus muscle mass increased in the aged, however, soleus and EDL total fiber number were declining. When adjusted for muscle weight, a significant reduction existed in fiber number for the aged soleus. Fiber area increased in the old soleus compared to young. All changes in the EDL were specific to the deep region. While there was a tendency for the capillaries/fiber and fiber area to be higher with training in the Type I fibers of the soleus, it was only significant for the young animals. Thus, while the majority of variables that showed a training effect did so across both age groups, this was not the case for vascularity. Age-associated changes in muscle morphology appear to be both muscle and fiber specific.

Influence of aging and endurance training on lactate dehydrogenase in liver and skeletal muscle.

The purpose of this investigation was to determine the effects of aging and endurance training on lactate dehydrogenase (LDH) activity and isozyme pattern in liver and skeletal muscle. Male Fischer 344 rats (n = 30) of three different age groups (young, 4 months; middle-aged, 12 months and old, 22 months) were trained on a treadmill at 75% running capacity for 1 h/day, five times per week for 10 weeks. Age-matched sedentary controls (n = 36) were used for comparison. Total LDH enzyme activity was measured spectrophotometrically; LDH isozymes were separated by native 5.5% polyacrylamide gel electrophoresis and quantified densitometrically. With increasing age, hepatic LDH activity decreased 28%. Old sedentary animals displayed significantly less (22%) hepatic LDH 5 than young and middle-aged animals, and significantly more (40%) hepatic LDH 4 than middle-aged animals. Training resulted in a significant decrease (38%) in total hepatic LDH activity in young rats only. Young animals displayed a significant increase in hepatic LDH 3 (28%), whereas middle-aged animals exhibited a significant decrease in hepatic LDH 3 (40%) with training. No change in total hepatic LDH activity was exhibited in middle-aged or old rats with training. Neither aging or training had a significant effect on LDH activity or isozyme pattern in extensor digitorum longus (EDL). Similarly, LDH activity was maintained in soleus with age, and isozyme pattern was only negligibly affected. We conclude that with age there is a decline in hepatic LDH activity and a decrease in the LDH 5 isozyme. Endurance training induced significant decreases in hepatic LDH activity of young animals. However, these decreases were not a result of shifts in isozymal pattern. Further, LDH activity was maintained in EDL and soleus muscle with age. Finally, endurance training did not have a significant effect on LDH activity or isozymal pattern of EDL or soleus.

Influence of endurance training on the age-related decline in hepatic glyconeogenesis.

Hepatic gluconeogenic and glyconeogenic capabilities were investigated in Fischer 344 rat livers (ages 7, 15 and 25 months; n = 66) to determine if endurance training affected age related decrements in these hepatic functions. Animals were trained 1 h/day, 5 days/week for 10 weeks at treadmill speeds of 75% of age-specific maximal capacity. After training, rats were injected (300 mg/kg) with a known gluconeogenic inhibitor, 3-mercaptopicolinic acid (MPA). Two endurance tests were performed to help assess the contribution of gluconeogenesis to exercise performance, an initial test 4 days prior to injection and a final test immediately post-injection. MPA significantly (P < 0.05) reduced running times in all trained groups compared to their control test: 89%, 81%, and 51% in the young, middle-aged, and old, respectively. MPA reduced running times in the untrained animals 19%, 11%, and 8%, respectively. Three days after the last exercise bout, the animals were anesthetized and liver sections were sliced and incubated in [14C]lactic acid or [14C]fructose. An age-related decline was found in [14C]lactate incorporation (middle-aged decreases 66%, old decreases 54%) and in [14C]fructose incorporation (middle-aged decreases 51%, old decreases 48%) into glycogen. Differences existed in lactate incorporation in trained compared to untrained animals for the young, middle-aged, and old groups: 150.1 +/- 11.3 vs. 102.1 +/- 10.0; 75.3 +/- 6.2 vs. 34.9 +/- 6.4; and 69.3 +/- 14.9 vs. 47.0 +/- 4.7 nmol/g/h, respectively. No differences were found with training in any of the age groups for fructose. Phosphoenolpyruvate carboxykinase (PEPCK) activity and messenger RNA (mRNA) were significantly reduced in the old compared to the young rats (decreases 64% and decreases 58%, respectively). No training effects were found for either PEPCK activity or mRNA for any age group. These results suggest that hepatic gluconeogenic and glyconeogenic capabilities declined with age. Training had an effect in attenuating the glyconeogenic decline but had a minimal effect in offsetting the age-related decline in PEPCK.

Immune response to a single bout of exercise in young and elderly subjects.

The purpose of this investigation was to examine alterations in lymphocyte proliferation activity and T cell subsets following an acute bout of exercise in young and old subjects. Six young (26+/-3 years) and nine old (69+/-5 years) male subjects were tested at rest and immediately after 20 min of submaximal exercise at 50% peak work capacity. Arterial blood was sampled from an indwelling catheter for catecholamine and immunology assays. Peripheral blood lymphocytes were isolated for mitogen-induced phytohemagglutinin (PHA) proliferation capacity. Lymphocyte subsets were analyzed by dual-labeled flow cytometry. As has been shown in previous studies, baseline proliferative responsiveness was significantly lower in the old (down 22%) compared to the young subjects. In response to submaximal exercise, proliferative responsiveness to PHA increased significantly in the young subjects (up 55%), however, for the old subjects this response did not differ significantly from resting values (up 18%). The number of total lymphocytes, as well as CD4+ and CD8+ T cell subsets, at rest were lower for old subjects compared with young. Exercise-induced increases in T cell subset populations were similar across age groups. It was concluded that, while having lower initial T cell numbers and PHA responsiveness, immunoresponsiveness during a single bout of exercise is, in general, maintained in old when compared to young individuals.

Pulmonary function and hypoxic ventilatory response in subjects susceptible to high-altitude pulmonary edema.

To determine if spirometric changes reflect early high-altitude pulmonary edema (HAPE) formation, we measured the FVC, FEV1, and FEF25-75 serially during the short-term period following simulated altitude exposure (4,400 m) in eight male subjects, four with a history of HAPE and four control subjects who had never experienced HAPE. Three of the four HAPE-susceptible subjects developed acute mountain sickness (AMS), based on their positive Environmental Symptom Questionnaire (AMS-C) scores. Clinical signs and symptoms of mild pulmonary edema developed in two of the three subjects with AMS after 4 h of exposure, which prompted their removal from the chamber. Their spirometry showed small decreases in FVC and greater decreases in FEV1 and FEF25-75 after arrival at high altitude in the presence of rales or wheezing on clinical examination and normal chest radiographs. One of the two subjects had desaturation (59 percent) and tachycardia during mild exercise, and excessive fatigue and inability to complete the exercise protocol developed in the other at 4 h. The six other subjects had minimal changes in spirometry and did not develop signs of lung edema. Further, we measured each subject's ventilatory response to hypoxia (HVR) prior to decompression to determine whether the HVR would predict the development of altitude illness in susceptible subjects. In contrast to anticipated results, high ventilatory responses to acute hypoxia, supported by increased ventilation during exposure to high altitude, occurred in the two subjects in whom symptoms of HAPE developed. The results confirm that HAPE can occur in susceptible individuals despite the presence of a normal or high ventilatory response to hypoxia.