Anuradha Ramoji

Liver fibrosis is a major complication of chronic liver injury, driven in part by Signal Transducer and Activator of Transcription 1 (Stat1). This study evaluated passively liver-targeted polymeric nanocarriers delivering Stat1 siRNA (siSTAT1) to treat steatohepatitis and fibrosis in a thioacetamide (TAA) mouse model. Two polyplex nanocarriers with different core−corona ratios, EN15 and EN76, were tested in TAA-treated mice and activated human and murine hepatocytes. In vitro, both formulations enabled efficient cellular uptake and Stat1 silencing, reducing TAA-induced lipid accumulation. Through an In vivo approach, mice received TAA for 16 weeks followed by four weeks of siSTAT1 treatment. Both formulations reduced collagen deposition, hepatic inflammation, and fibrosis scores. Notably, EN76, which preferentially targets non-parenchymal cells, showed superior antifibrotic efficacy compared with EN15. These findings highlight Stat1 as a key driver of steatohepatitis and fibrosis and support passive siRNA delivery, particularly via EN76, as a promising therapeutic strategy.

Complex-valued chemometrics offers a promising extension of classical regression methods by exploiting both real and imaginary spectral components. Here, we show that conventional absorbance (χ⁽¹⁾) and Raman (χ⁽³⁾) spectra can be transformed into complex-valued forms by combining the measured intensities as imaginary parts with their Kramers-Kronig-derived real parts. We benchmark four regression methods─classical least squares (CLS), inverse least squares (ILS), principal component regression (PCR), and partial least-squares regression (PLSR)─across four representative systems: the quasi-ideal benzene-toluene and benzene-cyclohexane mixtures, the nonideal acetone-chloroform mixture, and blood plasma spiked with glucose and urea. Compared to conventional chemometrics, complex-valued approaches consistently reduce prediction errors (MAE, RMSE, and R²). Implementation is computationally inexpensive, since the Kramers-Kronig transform of absorbance or Raman spectra can be obtained within seconds using FFT-based routines, even for large data sets. Software implementation is straightforward, and programs can be adapted within minutes using standard environments such as Mathematica. Surprisingly, complex-valued ILS matches or surpasses complex-valued PLSR, echoing earlier results in infrared spectroscopy, using the complex refractive index function, and suggesting a re-evaluation of regression hierarchies when complex spectra are available. These findings demonstrate that complex-valued chemometrics is broadly applicable, physically grounded, and capable of enhancing both classical and modern regression strategies in analytical spectroscopy.

Heme is involved in many critical processes in pathogenic bacteria as iron acquisition by these microorganisms is achieved by either direct uptake of heme or use of heme‐binding proteins called hemophores. Exploring the underlying mechanisms on a molecular level can open new avenues in understanding the host‐pathogen interactions. Any imbalance of the heme concentration has a direct impact on the bacterial growth and survival. Thus, heme‐regulated proteins that are involved in heme homeostasis poise to be promising targets for research. Similarly, naturally occurring compounds, including cysteine‐rich peptides from either plant secondary metabolites or venom toxins from vertebrates and invertebrates, have been studied for their therapeutic potential. NCR247 is such a cysteine‐rich peptide, known to be crucial for nitrogenase activity in M. truncatula and its symbiotic relation with S. meliloti . NCR247‐derived peptides were suggested to serve as high‐affinity heme‐binding molecules with remarkable heme‐capturing properties. A comprehensive biochemical and computational analysis of NCR247‐derived peptides, however, redefines their heme‐binding capacity and consequently their potential therapeutic role.

Sepsis is a life-threatening condition that results from an overwhelming and disproportionate host response to an infection. Currently, the quality and extent of the immune response are evaluated based on clinical symptoms and the concentration of inflammatory biomarkers released or expressed by the immune cells. However, the host response toward sepsis is heterogeneous, and the roles of the individual immune cell types have not been fully conceptualized. During sepsis, the spleen plays a vital role in pathogen clearance, such as bacteria by an antibody response, macrophage bactericidal capacity, and bacterial endotoxin detoxification. This study uses Raman spectroscopy to understand the splenic T-lymphocyte compartment profile changes during bona fide bacterial sepsis versus hyperinflammatory endotoxemia. The Raman spectral analysis showed marked changes in splenocytes of mice subjected to septic peritonitis principally in the DNA region, with minor changes in the amino acids and lipoprotein areas, indicating significant transcriptomic activity during sepsis. Furthermore, splenocytes from mice exposed to endotoxic shock by injection of a high dose of lipopolysaccharide showed significant changes in the protein and lipid profiles, albeit with interindividual variations in inflammation severity. In summary, this study provided experimental evidence for the applicability and informative value of Raman spectroscopy for profiling the immune response in a complex, systemic infection scenario. Importantly, changes within the acute phase of inflammation onset (24 h) were reliably detected, lending support to the concept of early treatment and severity control by extracorporeal Raman profiling of immunocyte signatures.

The intestinal epithelial barrier, among other compartments such as the mucosal immune system, contributes to the maintenance of intestinal homeostasis. Therefore, any disturbance within the epithelial layer could lead to intestinal permeability and promote mucosal inflammation. Considering that disintegration of the intestinal epithelial barrier is a key element in the etiology of ulcerative colitis, further assessment of barrier integrity could contribute to a better understanding of the role of epithelial barrier defects in ulcerative colitis (UC), one major form of chronic inflammatory bowel disease. Herein, we employ fast, non-destructive, and label-free non-linear methods, namely coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and two-photon fluorescence lifetime imaging (2P-FLIM), to assess the morpho-chemical contributions leading to the dysfunction of the epithelial barrier. For the first time, the formation of epithelial barrier gaps was directly visualized, without sophisticated data analysis procedures, by the 3D analysis of the colonic mucosa from severely inflamed UC patients. The results were compared with histopathological and immunofluorescence images and validated using transmission electron microscopy (TEM) to indicate structural alterations of the apical junction complex as the underlying cause for the formation of the epithelial barrier gaps. Our findings suggest the potential advantage of non-linear multimodal imaging is to give precise, detailed, and direct visualization of the epithelial barrier in the gastrointestinal tract, which can be combined with a fiber probe for future endomicroscopy measurements during real-time in vivo imaging.

Non-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner.

The intestinal epithelial barrier, among other compartments such as the mucosal immune system, contributes to the maintenance of intestinal homeostasis. Therefore, any disturbance within the epithelial layer could lead to intestinal permeability and promote mucosal inflammation. Considering that disintegration of the intestinal epithelial barrier is a key element in the etiology of ulcerative colitis, further assessment of barrier integrity could contribute to a better understanding of the role of epithelial barrier defects in ulcerative colitis (UC), one major form of chronic inflammatory bowel disease. Herein, we employ fast, non-destructive, and label-free non-linear methods, namely coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and two-photon fluorescence lifetime imaging (2P-FLIM) to assess the morpho-chemical contributions leading to the dysfunction of the epithelial barrier. For the first time, the formation of epithelial barrier gaps was directly visualized, without sophisticated data analysis procedures, by the 3D analysis of the colonic mucosa from severely inflamed UC patients. The results were compared with histopathological and immunofluorescence images and validated using transmission electron microscopy (TEM) to indicate structural alterations of the apical junction complex as the underlying cause for the formation of the epithelial barrier gaps. Our findings suggest the potential of non-linear multimodal imaging to give precise, detailed, and direct visualization of the epithelial barrier in the gastrointestinal tract, which can be combined with a fiber probe for future endomicroscopy measurements during real-time in-vivo imaging.

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

SARS-CoV-2 infection remains to spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman hallmark of molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.