Micro-Raman spectroscopy study of optically trapped erythrocytes in malaria, dengue and leptospirosis infections.

Malaria, dengue and leptospirosis are three tropical infectious diseases that present with severe hematological derangement causing significant morbidity and mortality, especially during the seasonal monsoons. During the course of these infectious diseases, circulating red blood cells are imperiled to the direct ill-effects of the infectious pathogen in the body as well as to the pro-inflammatory cytokines generated as a consequence of the infection. RBCs when exposed to such inflammatory and/or pathogenic milieu are susceptible to injuries such as RBC programmed eryptosis or RBC programmed necrosis. This research aimed to explore the Raman spectra of live red cells that were extracted from patients infected with malaria, dengue, and leptospirosis. Red cells were optically trapped and micro-Raman probed using a 785 nm Diode laser. RBCs from samples of all three diseases displayed Raman signatures that were significantly altered from the normal/healthy. Distinct spectral markers that were common across all the four groups were obtained from various standardized multivariate analytical methods. Following comprehensive examination of multiple studies, we propose these spectral wavenumbers as "Raman markers of RBC injury." Findings in our study display that anemia-triggering infections can inflict variations in the healthy status of red cells, easily identifiable by selectively analyzing specific Raman markers. Additionally, this study also highlights relevant statistical tools that can be utilized to study Raman spectral data from biological samples which could help identify the very significant Raman peaks from the spectral band. This approach of RBC analysis can foster a better understanding of red cell behavior and their alterations exhibited in health and disease.

Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold.

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1ß (IL-1ß). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1ß. Seeking to understand the evolutionary origin of this caspase, we utilized the large amount of data available in public databases to perform ancestral sequence reconstruction of an inflammatory caspase of a Carnivora ancestor. We expressed the catalytic domain of this putative ancestor in Escherichia coli, purified it, and compared its substrate specificity on synthetic and protein substrates to extant caspases. We demonstrated that it activates gasdermin D but has reduced ability to activate IL-1ß. Our reconstruction suggests that caspase-1 was lost in a Carnivora ancestor, perhaps upon a selective pressure for which the generation of biologically active IL-1ß by caspase-1 was detrimental. We speculate that later, a Carnivora encountered selective pressures that required the production of IL-1ß, and caspase-4 subsequently gained this activity. This hypothesis would explain why extant Carnivora possess an inflammatory caspase with caspase-1 catalytic function placed on a caspase-4 scaffold.

Red blood cells under varying extracellular tonicity conditions: an optical tweezers combined with micro-Raman study.

Extracellular tonicity has a significant influence on human red blood cell deformation capability. Advancements in the area of laser physics and optical trapping have opened up a plethora of applications for understanding cell structure and dynamics. Here, Raman Tweezers technique was employed to investigate the impact of extracellular tonicity by exposing human red blood cells to both hypertonic and hypotonic intravenous fluids. Heme aggregation was observed in hypertonic saline solution, accompanied with damage in membrane protein. Loss of intracellular hemoglobin in hypotonic solution was evident from the decrease in porphyrin breathing mode present at 752 cm-1. Oxygen binding to the central iron in the red blood cell heme was also affected under both hyper/hypo tonicity conditions. Morphological deviation of discocytes to echinocytes/spherocytes were also evident from quantitative phase imaging. Principal component analysis have showed clear differentiation of samples in order to classify the control erythrocytes and the tonicity stressed erythrocytes. Present study has also demonstrated the application of Raman Tweezers spectroscopy as a potential tool for probing red blood cell under different stress conditions.

Automatic segmentation of lung parenchyma in the presence of diseases based on curvature of ribs.

RATIONALE AND OBJECTIVES: Segmentation of lungs using high-resolution computer tomographic images in the setting of diffuse lung diseases is a major challenge in medical image analysis. Threshold-based techniques tend to leave out lung regions that have increased attenuation, such as in the presence of interstitial lung disease. In contrast, streak artifacts can cause the lung segmentation to "leak" into the chest wall. The purpose of this work was to perform segmentation of the lungs using a technique that selects an optimal threshold for a given patient by comparing the curvature of the lung boundary to that of the ribs. METHODS: Our automated technique goes beyond fixed threshold-based approaches to include lung boundary curvature features. One would expect the curvature of the ribs and the curvature of the lung boundary around the ribs to be very close. Initially, the ribs are segmented by applying a threshold algorithm followed by morphologic operations. The lung segmentation scheme uses a multithreshold iterative approach. The threshold value is verified until the curvature of the ribs and the curvature of the lung boundary are closely matched. The curve of the ribs is represented using polynomial interpolation, and the lung boundary is matched in such a way that there is minimal deviation from this representation. Performance of this technique was compared with conventional (fixed threshold) lung segmentation techniques on 25 subjects using a volumetric overlap fraction measure. RESULTS: The performance of the rib segmentation technique was significantly different from conventional techniques with an average higher mean volumetric overlap fraction of about 5%. CONCLUSIONS: The technique described here allows for accurate quantification of volumetric computed tomography and more advanced segmentation of abnormal areas.

Three-dimensional mapping of gallbladder wall thickness on computed tomography using Laplace's equation.

RATIONALE AND OBJECTIVES: Traditionally, maximum gallbladder wall thickness is measured at a single point on ultrasonography. The purpose of this work was to develop an automated technique to measure the thickness of the gallbladder wall over the entire gallbladder surface using computer tomography (CT). MATERIALS AND METHODS: Subjects who had (5-mm) thick and thin (2.5-mm) reconstruction through the abdomen were selected from a research database. Their volumetric computed tomographic images were acquired using a multidetector GE Medical Systems LightSpeed 16 scanner at 120 kVp, approximately 250 mAs, with standard filter reconstruction algorithm and segmented in three dimensions. Two segmentation boundaries were obtained, an inner and an outer boundary of the gallbladder wall. The thickness of the wall was quantified by computing the distance between the boundaries over the entire volume using Laplace's equation from mathematical physics. The distance between the surfaces is found by computing normalized gradients that form a vector field, representing tangent vectors along field lines connecting both boundaries. The Laplacian technique was compared with the well-known Euclidean distance transformation (EDT) technique that provides a three-dimensional Euclidean distance mapping between the two extracted surfaces. RESULTS: The technique was tested on 10 subjects who had thin- and thick-section computed tomographic datasets reconstructed from a single scan. The mean thickness for the thick- and thin-section CT using Laplace was 3.18 and 2.93 mm, respectively. The smooth transition between surfaces resulting from the Laplace technique resulted in a coefficient of variation that was less than 1% compared to EDT. CONCLUSIONS: EDT technique is very sensitive to imperfect segmentations, resulting in higher variation compared to the Laplacian technique. The smooth transition between surfaces makes the Laplacian technique more robust compared to EDT for the measurement of CT gallbladder thickness.