Modeling Absorption Dynamics of Differently Shaped Gold Glioblastoma and Colon Cells Based on Refractive Index Distribution in Holotomographic Imaging.

Herein, it is demonstrated that the toxic effect of gold nanoparticles (Au NPs) on three different cancer cell lines (U-118 and LN-299 glioblastoma and HCT-116 colon) depends on their absorption dynamics by cells, related to the shapes of the NPs. This hypothesis is confirmed by showing that i) based on refractive index (RI) values, typical for cell components and gold nanoparticles, it is possible to show the absorption dynamics and accumulation locations of the latter ones inside and outside of the cells. Moreover, ii) the saturation of the accumulated Au NPs volume in the cells depends on the nanoparticle shape and is reached in the shortest time for star-shaped Au NPs (AuS NPs) and in the longest time for spherical Au NPs (AuSph NPs) and on the cancer cells, where the longest and the shortest saturation are noticed for HCT-116 and LN-229 cells, respectively. A physical model of Au NPs absorption dynamics is proposed, where the diameter and shape of the Au NPs are used as parameters. The obtained theoretical data are consistent with experimental data in 85-98%.

Detection of primary myelofibrosis in blood serum via Raman spectroscopy assisted by machine learning approaches; correlation with clinical diagnosis.

Primary myelofibrosis (PM) is one of the myeloproliferative neoplasm, where stem cell-derived clonal neoplasms was noticed. Diagnosis of this disease is based on: physical examination, peripheral blood findings, bone marrow morphology, cytogenetics, and molecular markers. However, the molecular marker of PM, which is a mutation in the JAK2V617F gene, was observed also in other myeloproliferative neoplasms such as polycythemia vera and essential thrombocythemia. Therefore, there is a need to find methods that provide a marker unique to PM and allow for higher accuracy of PM diagnosis and consequently the treatment of the disease. Continuing, in this study, we used Raman spectroscopy, Principal Components Analysis (PCA), and Partial Least Squares (PLS) analysis as helpful diagnostic tools for PM. Consequently, we used serum collected from PM patients, which were classified using clinical parameters of PM such as the dynamic international prognostic scoring system (DIPSS) for primary myelofibrosis plus score, the JAK2V617F mutation, spleen size, bone marrow reticulin fibrosis degree and use of hydroxyurea drug features. Raman spectra showed higher amounts of C-H, C-C and C-C/C-N and amide II and lower amounts of amide I and vibrations of CH3 groups in PM patients than in healthy ones. Furthermore, shifts of amides II and I vibrations in PM patients were noticed. Machine learning methods were used to analyze Raman regions: (i) 800 cm-1 and 1800 cm-1, (ii) 1600 cm-1-1700 cm-1, and (iii) 2700 cm-1-3000 cm-1 showed 100 % accuracy, sensitivity, and specificity. Differences in the spectral dynamic showed that differences in the amide II and amide I regions were the most significant in distinguishing between PM and healthy subjects. Importantly, until now, the efficacy of Raman spectroscopy has not been established in clinical diagnostics of PM disease using the correlation between Raman spectra and PM clinical prognostic scoring. Continuing, our results showed the correlation between Raman signals and bone marrow fibrosis, as well as JAKV617F. Consequently, the results revealed that Raman spectroscopy has a high potential for use in medical laboratory diagnostics to quantify multiple biomarkers simultaneously, especially in the selected Raman regions.

Biosynthesis of Rubellins in Ramularia collo-cygni-Genetic Basis and Pathway Proposition.

The important disease Ramularia leaf spot of barley is caused by the fungus Ramularia collo-cygni. The disease causes yield and quality losses as a result of a decrease in photosynthesis efficiency due to the appearance of necrotic spots on the leaf surface. The development of these typical Ramularia leaf spot symptoms is thought to be linked with the release of phytotoxic secondary metabolites called rubellins in the host. However, to date, neither the biosynthetic pathways leading to the production of these metabolites nor their exact role in disease development are known. Using a combined in silico genetic and biochemistry approach, we interrogated the genome of R. collo-cygni to identify a putative rubellin biosynthetic gene cluster. Here we report the identification of a gene cluster containing homologues of genes involved in the biosynthesis of related anthraquinone metabolites in closely related fungi. A putative pathway to rubellin biosynthesis involving the genes located on the candidate cluster is also proposed.

Selected Fungal Natural Products with Antimicrobial Properties.

Fungal natural products and their effects have been known to humankind for hundreds of years. For example, toxic ergot alkaloids produced by filamentous fungi growing on rye poisoned thousands of people and livestock throughout the Middle Ages. However, their later medicinal applications, followed by the discovery of the first class of antibiotics, penicillins and other drugs of fungal origin, such as peptidic natural products, terpenoids or polyketides, have altered the historically negative reputation of fungal "toxins". The development of new antimicrobial drugs is currently a major global challenge, mainly due to antimicrobial resistance phenomena. Therefore, the structures, biosynthesis and antimicrobial activity of selected fungal natural products are described here.

Discovery and reconstitution of the cycloclavine biosynthetic pathway--enzymatic formation of a cyclopropyl group.

The ergot alkaloids, a class of fungal-derived natural products with important biological activities, are derived from a common intermediate, chanoclavine-I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast-based expression platform along with in vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine-I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500 mg L(-1) , thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.

Biosynthesis of the ergot alkaloids.

The ergots are a structurally diverse group of alkaloids derived from tryptophan and dimethylallyl pyrophosphate (DMAPP) . The potent bioactivity of ergot alkaloids have resulted in their use in many applications throughout human history. In this highlight, we recap some of the history of the ergot alkaloids, along with a brief description of the classifications of the different ergot structures and producing organisms. Finally we describe what the advancements that have been made in understanding the biosynthetic pathways, both at the genomic and the biochemical levels. We note that several excellent review on the ergot alkaloids, including one by Wallwey and Li in Nat. Prod. Rep., have been published recently. We provide a brief overview of the ergot alkaloids, and highlight the advances in biosynthetic pathway elucidation that have been made since 2011 in Section 4.

Chemical changes in childhood obesity blood as a marker of the disease. A Raman-based machine learning study.

Obesity in children is a global problem, leading to different medical conditions that may contribute to metabolic syndrome and increase the risk of diabetes, dyslipidemia, hypertension, and cardiovascular diseases in future health. Metabolic disorders are the results of the body's chemical process. The changes in the chemical compositions could be determined by Raman spectroscopy. Therefore, in this study, we measured blood collected from children with obesity to show chemical changes caused by obesity disease. Moreover, we will also show characteristic Raman peaks/regions, which could be used as a marker of obesity, not other metabolic syndromes. Children with obesity had higher glucose levels, proteins, and lipids than the control ones. Furthermore, it was noticed that the ratio between CO and C-H is 0.23 in control patients and 0.31 in children with obesity, as well as the ratio between amide II and amide I was 0.72 in control and 1.15 in obesity, which suggests an imbalance in these two fractions in childhood obesity. PCA with discrimination analyses showed that the accuracy, selectivity, and specificity of Raman spectroscopy in differentiation between childhood obesity and healthy children was between 93% and 100%. There is an increased risk of metabolic changes in childhood obesity with higher glucose levels, lipids, and proteins in children with obesity. Also, there were differences in the ratio between proteins and lipids functional groups and glucose, amide II, and amide I vibrations as a marker of obesity. The results of the study offer valuable insights into potential alterations in protein structure and lipid composition in children with obesity, emphasizing the importance of considering metabolic changes beyond traditional anthropometric, measurements.

Application of Fourier Transform InfraRed spectroscopy of machine learning with Support Vector Machine and principal components analysis to detect biochemical changes in dried serum of patients with primary myelofibrosis.

Primary myelofibrosis (PM) is a myeloproliferative neoplasm characterized by stem cell-derived clonal neoplasms. Several factors are involved in diagnosing PM, including physical examination, peripheral blood findings, bone marrow morphology, cytogenetics, and molecular markers. Commonly gene mutations are used. Also, these gene mutations exist in other diseases, such as polycythemia vera and essential thrombocythemia. Hence, understanding the molecular mechanism and finding disease-related biomarker characteristics only for PM is crucial for the treatment and survival rate. For this purpose, blood samples of PM (n = 85) vs. healthy controls (n = 45) were collected for biochemical analysis, and, for the first time, Fourier Transform InfraRed (FTIR) spectroscopy measurement of dried PM and healthy patients' blood serum was analyzed. A Support Vector Machine (SVM) model with optimized hyperparameters was constructed using the grid search (GS) method. Then, the FTIR spectra of the biomolecular components of blood serum from PM patients were compared to those from healthy individuals using Principal Components Analysis (PCA). Also, an analysis of the rate of change of FTIR spectra absorption was studied. The results showed that PM patients have higher amounts of phospholipids and proteins and a lower amount of H-O=H vibrations which was visible. The PCA results indicated that it is possible to differentiate between dried blood serum samples collected from PM patients and healthy individuals. The Grid Search Support Vector Machine (GS-SVM) model showed that the prediction accuracy ranged from 0.923 to 1.00 depending on the FTIR range analyzed. Furthermore, it was shown that the ratio between α-helix and ß-sheet structures in proteins is 1.5 times higher in PM than in control people. The vibrations associated with the CO bond and the amide III region of proteins showed the highest probability value, indicating that these spectral features were significantly altered in PM patients compared to healthy ones' spectra. The results indicate that the FTIR spectroscope may be used as a technique helpful in PM diagnostics. The study also presents preliminary results from the first prospective clinical validation study.

Optical monitoring of hemodialysis using noninvasive measurement of uric acid in the dialysate.

The aim of this study was to present a methodology for predicting changes in uric acid concentrations in the blood of chronically hemodialyzed patients based on an optical measurement of the intensity of selected wavelengths in the dialysate. Blood samples were taken from the arterial line every 30 min throughout the hemodialysis period, to measure uric acid levels. Simultaneously, optical measurements were made on dialysate flowing from the dialyzer. Uric acid concentration can be measured either directly from the blood or from dialyzer outflow with acceptable error. In addition, both methods reveal any increased dynamics in uric acid concentration in the initial phase of hemodialysis. The wavelength of the light was adjusted for optimal uric acid particle detection. Comparing the uric acid concentration measured in the blood of patients with the intensity of wave absorption in the dialysate, the functional relationship between the uric acid concentration levels was determined. Using the optical method for measuring uric acid concentration in the dialysate, the concentration of uric acid in the blood during hemodialysis can be non-invasively and accurately estimated. This method can be used to assess the adequacy of hemodialysis by computer acquisition of uric acid concentrations determined in on-line dialysate.

FTIR- based serum structure analysis in molecular diagnostics of essential thrombocythemia disease.

Essential thrombocythemia (ET) reflects the transformation of a multipotent hematopoietic stem cell, but its molecular pathogenesis remains obscure. Nevertheless, tyrosine kinase, especially Janus kinase 2 (JAK2), has been implicated in myeloproliferative disorders other than chronic myeloid leukaemia. FTIR analysis was performed on the blood serum of 86 patients and 45 healthy volunteers as control with FTIR spectra-based machine learning methods and chemometrics. Thus, the study aimed to determine biomolecular changes and separation of ET and healthy control groups illustration by applying chemometrics and ML techniques to spectral data. The FTIR-based results showed that in ET disease with JAK2 mutation, there are alterations in functional groups associated with lipids, proteins and nucleic acids significantly. Moreover, in ET patients the lower amount of proteins with simultaneously higher amount of lipids was noted in comparison with the control one. Furthermore, the SVM-DA model showed 100% accuracy in calibration sets in both spectral regions and 100.0% and 96.43% accuracy in prediction sets for the 800-1800 cm-1 and 2700-3000 cm-1 spectral regions, respectively. While changes in the dynamic spectra showed that CH2 bending, amide II and CO vibrations could be used as a spectroscopy marker of ET. Finally, it was found a positive correlation between FTIR peaks and first bone marrow fibrosis degree, as well as the absence of JAK2 V617F mutation. The findings of this study contribute to a better understanding of the molecular pathogenesis of ET and identifying biomolecular changes and may have implications for early diagnosis and treatment of this disease.

Raman spectroscopy-based biomarker screening by studying the fingerprint and lipid characteristics of Polycythem..a Vera cases blood serum.

This study aimed to develop a novel approach for diagnosing Polycythemia Vera (PV), a stem cell-derived neoplasm of the myeloid lineage. The approach utilized Raman spectroscopy coupled with multivariate analysis to analyze blood serum samples collected from PV patients. The results showed that PV serum exhibited lower protein and lipid levels and structural changes in the functional groups that comprise proteins and lipids. The study also demonstrated differences in lipid biosynthesis and protein levels in PV serum. Using the Partial Least Square Discriminant Analysis (PLS-DA) model, Raman-based multivariate analysis achieved high accuracy rates of 96.49 and 93.04% in the training sets and 93.10% and 89.66% in the test sets for the 800-1800 cm-1 and 2700-3000 cm-1 ranges, respectively. The area under the curve (AUC) values of the test datasets were calculated as 0.92 and 0.89 in the 800-1800 cm-1 and 2700-3000 cm-1 spectral regions, respectively, demonstrating the effectiveness of the PLS-DA models for the diagnosis of PV. This study highlights the potential of Raman spectroscopy-based analysis in the early and accurate diagnosis of PV, enabling the application of effective treatment strategies.

Interkingdom signaling: integration, conformation, and orientation of N-acyl-L-homoserine lactones in supported lipid bilayers.

N-Acyl-L-homoserine lactones (AHLs) are small cell-to-cell signaling molecules involved in the regulation of population density and local gene expression in microbial communities. Recent evidence shows that contact of this signaling system, usually referred to as quorum sensing, to living eukaryotes results in interactions of AHL with host cells in a process termed "interkingdom signaling". So far details of this process and the binding site of the AHLs remain unknown; both an intracellular and a membrane-bound receptor seem possible, the first of which requires passage through the cell membrane. Here, we used sum-frequency-generation (SFG) spectroscopy to investigate the integration, conformation, orientation, and translocation of deuterated N-acyl-L-homoserine lactones (AHL-d(n)) with varying chain length (8, 12, and 14 C atoms) in lipid bilayers consisting of a 1:1 mixture of POPC:POPG supported on SiO(2) substrates (prepared by vesicle fusion). We found that all AHL-d(n) derivatives are well-ordered within the supported lipid bilayer (SLB) in a preferentially all-trans conformation of the deuterated alkyl chain and integrated into the upper leaflet of the SLB with the methyl terminal groups pointing downward. For the bilayer system described above, no flip-flop of AHL-d(n) from the upper leaflet to the lower one could be observed. Spectral assignments and interpretations were further supported by Fourier transform infrared and Raman spectroscopy.

Deuterium-labelled N-acyl-L-homoserine lactones (AHLs)--inter-kingdom signalling molecules--synthesis, structural studies, and interactions with model lipid membranes.

N-Acyl-L-homoserine lactones (AHLs) are synthesized by Gram-negative bacteria. These quorum-sensing molecules play an important role in the context of bacterial infection and biofilm formation. They also allow communication between microorganisms and eukaryotic cells (inter-kingdom signalling). However, very little is known about the entire mechanism of those interactions. Precise structural studies are required to analyse the different AHL isomers as only one form is biologically most active. Theoretical studies combined with experimental infrared and Raman spectroscopic data are therefore undertaken to characterise the obtained compounds. To mimic interactions between AHL and cell membranes, we studied the insertion of AHL in supported lipid bilayers, using vibrational sum-frequency-generation spectroscopy. Deuterium-labelled AHLs were thus synthesized. Starting from readily available deuterated fatty acids, a two-step procedure towards deuterated N-acyl-L-homoserine lactones with varying chain lengths is described. This included the acylation of Meldrum's acid followed by amidation. Additionally, the detailed analytical evaluation of the products is presented herein.

Structural characterization of EasH (Aspergillus japonicus) - an oxidase involved in cycloclavine biosynthesis.

Aj_EasH is a non-heme iron- and α-keto-glutarate-dependent oxidase that is responsible for an unusual cyclopropyl ring formation in the biosynthesis of the fungal ergot alkaloid cycloclavine. The three dimensional structure of Aj_EasH (2.2 Å resolution) reported here provides insight into the mechanism of this unusual and complex reaction.

Discovery and Reconstitution of the Cycloclavine Biosynthetic Pathway-Enzymatic Formation of a Cyclopropyl Group.

The ergot alkaloids, a class of fungal-derived natural products with important biological activities, are derived from a common intermediate, chanoclavine-I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast-based expression platform along with in vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine-I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500 mg L-1, thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.