Detection of Sialic Acid to Differentiate Cervical Cancer Cell Lines Using a Sambucus nigra Lectin Biosensor.

Pap smear screening is a widespread technique used to detect premalignant lesions of cervical cancer (CC); however, it lacks sensitivity, leading to identifying biomarkers that improve early diagnosis sensitivity. A characteristic of cancer is the aberrant sialylation that involves the abnormal expression of α2,6 sialic acid, a specific carbohydrate linked to glycoproteins and glycolipids on the cell surface, which has been reported in premalignant CC lesions. This work aimed to develop a method to differentiate CC cell lines and primary fibroblasts using a novel lectin-based biosensor to detect α2,6 sialic acid based on attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and chemometric. The biosensor was developed by conjugating gold nanoparticles (AuNPs) with 5 µg of Sambucus nigra (SNA) lectin as the biorecognition element. Sialic acid detection was associated with the signal amplification in the 1500-1350 cm-1 region observed by the surface-enhanced infrared absorption spectroscopy (SEIRA) effect from ATR-FTIR results. This region was further analyzed for the clustering of samples by applying principal component analysis (PCA) and confidence ellipses at a 95% interval. This work demonstrates the feasibility of employing SNA biosensors to discriminate between tumoral and non-tumoral cells, that have the potential for the early detection of premalignant lesions of CC.

Polyphenolic Compounds Nanostructurated with Gold Nanoparticles Enhance Wound Repair.

Gold nanoparticles (AuNPs) have been used in a wide range of applications, conferring to bio-molecules diverse properties such as delivery, stabilization, and reduction of the adverse effects of drugs or plant extracts. Polyphenolic compounds from Bacopa procumbens (B. procumbens) (BP) can modulate proliferation, adhesion, migration, and cell differentiation, reducing the artificial scratch area in fibroblast cultures and promoting wound healing in an in vivo model. Here, chemically synthesized AuNPs conjugated with BP (AuNP-BP) were characterized using UV-Vis, ATR-FTIR, DLS, zeta-potential, and TEM analysis. The results showed an overlap of the FTIR spectra of the polyphenolic compounds from B. procumbens adhered to the surface of the AuNPs. UV-vis analysis indicated that the average size of the AuNP-BP was 28 nm, while DLS analysis showed a size of 44.58 nm and, by TEM, a size of 16.5 nm with an icosahedral morphology was observed. These measurements suggest an increase in the size of the nanoparticles after conjugation with BP, compared to the sizes of 9 nm, 44.51 nm, and 14.17 nm for the unconjugated AuNPs, respectively. Furthermore, the zeta potential of the AuNPs, which was originally -36.3 ± 12.3 mV shifted to -18.2 ± 7.02 mV after conjugation with BP, indicating improved stability of the nanoparticles. Enhancement of the wound healing effect was evaluated by morphometric, histochemical, and FTIR changes in a rat wound excision model. Results showed that the nanoconjugation process reduced the BP concentrations by 100-fold to have the same wound healing effect as BP alone. Besides, histological and FTIR spectroscopy analyses demonstrated that AuNP-BP treatment exhibited better macroscopical performance, showing a reduction in inflammatory cells and an increased synthesis and improved organization of collagen fibers.

Dynamic response antibodies SARS-CoV-2 human saliva studied using two-dimensional correlation (2DCOS) infrared spectral analysis coupled with receiver operation characteristics analysis.

COVID-19 has affected the entire world due to the rapid spread of SARS-CoV-2, mainly through airborne particles from saliva, which, being easily obtained, help monitor the progression of the disease. Fourier transform infrared (FTIR) spectra combined with chemometric analysis could increase the diagnostic efficiency of the disease. However, two-dimensional correlation spectroscopy (2DCOS) is superior to conventional spectra as it helps to resolve the minute overlapped peaks. In this work, we aimed to use 2DCOS and receiver operating characteristic (ROC) analyses to compare the immune response in saliva associated with COVID-19, which could be important in biomedical diagnosis. FTIR spectra of human saliva samples from male (575) and female (366) patients ranging from 20 to 85 ± 2 years of age were used for the study. Age groups were segregated as G1 (20-40 ± 2 years), G2 (45-60 ± 2 years), and G3 (65-85 ± 2 years). The results of the 2DCOS analysis showed biomolecular changes in response to SARS-CoV-2. 2DCOS analyses of the male G1 + (1579,1644) and -(1531,1598) cross peaks evidenced changes such as amide I > IgG. Female G1 cross peaks -(1504,1645), (1504,1545) and -(1391,1645) resulted in amide I > IgG > IgM. The asynchronous spectra in 1300-900 cm-1 of the G2 male group showed that IgM is more important in diagnosing infections than IgA. Female G2 asynchronous spectra -(1027,1242) and + (1068,1176) showed that IgA > IgM is produced against SARS-CoV-2. The G3 male group evidenced antibody changes in IgG > IgM. The absence of IgM in the female G3 population diagnoses a specifically targeted immunoglobulin associated with sex. Moreover, ROC analysis showed sensitivity (85-89 % men; 81-88 % women) and specificity (90-93 % men; 78-92 % women) for the samples studied. The general classification performance (F1 score) of the studied samples is high for the male (88-91 %) and female (80-90 %) populations. This high PPV (positive predictive value) and NPV (negative predictive value) verify our segregation of COVID-19 positive and negative sample groups. Therefore, 2DCOS with ROC analysis using FTIR spectra have the potential for a non-invasive approach to monitoring COVID-19.

Comparison of the Immune Response in Vaccinated People Positive and Negative to SARS-CoV-2 Employing FTIR Spectroscopy.

Various immunopathological events characterize the systemic acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Moreover, it has been reported that coronavirus disease 2019 (COVID-19) vaccination and infection by SARS-CoV-2 induce humoral immunity mediated by B-cell-derived antibodies and cellular immunity mediated by T cells and memory B cells. Immunoglobulins, cytokines, and chemokines play an important role in shaping immunity in response to infection and vaccination. Furthermore, different vaccines have been developed to prevent COVID-19. Therefore, this research aimed to analyze and compare Fourier-transform infrared (FTIR) spectra of vaccinated people with a positive (V-COVID-19 group) or negative (V-Healthy group) real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) test, evaluating the immunoglobulin and cytokine content as an immunological response through FTIR spectroscopy. Most individuals that integrated the V-Healthy group (88.1%) were asymptomatic; on the contrary, only 28% of the V-COVID-19 group was asymptomatic. Likewise, 68% of the V-COVID-19 group had at least one coexisting illness. Regarding the immunological response analyzed through FTIR spectroscopy, the V-COVID-19 group showed a greater immunoglobulins G, A, and M (IgG, IgA, and IgM) content, as well as the analyzed cytokines interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-ɑ), and interleukins 1ß, 6, and 10 (IL-1ß, IL-6, and IL-10). Therefore, we can state that it was possible to detect biochemical changes through FTIR spectroscopy associated with COVID-19 immune response in vaccinated people.

Two-methods approach to follow up biomass by impedance spectroscopy: Bacillus thuringiensis fermentations as a study model.

Impedance spectroscopy is used for the characterization of electrochemical systems as well as for the monitoring of bioprocesses. However, the data obtained using this technique allow multiple interpretations, depending on the methodology implemented. Hence, it is necessary to establish a robust methodology to reliably follow-up biomass in fermentations. In the present work, two methodological approaches, mainly used for the characterization of electrochemical systems, were employed to characterize and determine a frequency that allows the monitoring of biomass in Bacillus thuringiensis fermentations by impedance spectroscopy. The first approach, based on a conventional analysis, revealed a single distribution with a characteristic frequency of around 2 kHz. In contrast, the second approach, based on the distribution of relaxation times, gave three distributions (A, B, and C). The C distribution, found near 9 kHz, was more related to the microbial biomass than the distribution at 2 kHz using the equivalent circuits. The time course of the B. thuringiensis fermentation was followed; bacilli, spores, glucose, and acid and base consumption for pH were determined out of line; and capacitance at 9 kHz was monitored. The correlation between the time course data and the capacitance profile indicated that the monitoring of B. thuringiensis at 9 kHz mainly corresponds to extracellular activity and, in a second instance, to the cellular concentration. These results show that it is necessary to establish a robust and reliable methodology to monitor fermentation processes by impedance spectroscopy, and the distribution of relaxation times was more appropriate. KEY POINTS: • Application of impedance spectroscopy for bioprocess monitoring • Low-frequency monitoring of biomass in fermentations • Analysis of impedance data by two methodological approaches.

Use of equivalent circuit analysis and Cole-Cole model in evaluation of bioreactor operating conditions for biomass monitoring by impedance spectroscopy.

The most important parameter in bioprocesses is biomass, where not only the quantity produced in a culture but also the behavior that is presented are important concerns. It is clear that conditions of operation in a bioreactor affect biomass production, but how operation conditions affect the measurement of biomass on-line is of special interest. We studied the effect of bioreactor operating condition variations on model parameters using impedance spectroscopy for biomass monitoring. The model parameters analyzed were capacitance, resistance, alpha (α), conductivity delta (∆σ) and critical frequency (fc). These model parameters were obtained by fitting data from impedance measurements to an equivalent circuit model and Cole-Cole conductivity model. The effect of operating conditions on the medium with no cells was estimated by the percentage of change in each model parameter. The operating conditions with the most significant percentage of change were determined, by comparing to the percentage of change of the same model parameters obtained, during a fermentation of Bacillus thuringiensis as a cellular model. Equivalent circuit parameters were mainly affected by variation in pH, temperature and aeration, whereas Cole-Cole parameters were affected by variation in agitation, aeration, temperature and pH. Therefore, any variation in these operating conditions (within the test interval) during a fermentation may generate changes in monitoring parameters, which will not be a direct consequence of any change in the properties of the biomass.

Morphological, molecular and FTIR spectroscopic analysis during the differentiation of kidney cells from pluripotent stem cells.

BACKGROUND: Kidney diseases are a global health problem. Currently, over 2 million people require dialysis or transplant which are associated with high morbidity and mortality; therefore, new researches focused on regenerative medicine have been developed, including the use of stem cells. RESULTS: In this research, we generate differentiated kidney cells (DKCs) from mouse pluripotent stem cells (mPSCs) analyzing their morphological, genetic, phenotypic, and spectroscopic characteristics along differentiation, highlighting that there are no reports of the use of Fourier transform infrared (FTIR) spectroscopy to characterize the directed differentiation of mPSCs to DKCs. The genetic and protein experiments proved the obtention of DKCs that passed through the chronological stages of embryonic kidney development. Regarding vibrational spectroscopy analysis by FTIR, bands related with biomolecules were shown on mPSCs and DKCs spectra, observing distinct differences between cell lineages and maturation stages. The second derivative of DKCs spectra showed changes in the protein bands compared to mPSCs. Finally, the principal components analysis obtained from FTIR spectra allowed to characterize chemical and structurally mPSCs and their differentiation process to DKCs in a rapid and non-invasive way. CONCLUSION: Our results indicated that we obtained DKCs from mPSCs, which passed through the chronological stages of embryonic kidney development. Moreover, FTIR spectroscopy resulted in a non-invasive, rapid and precise technic that together with principal component analysis allows to characterize chemical and structurally both kind of cells and also discriminate and determine different stages along the cell differentiation process.

Potential Therapeutic Strategies of Regenerative Medicine for Renal Failure.

BACKGROUND: Kidney diseases are a public health problem worldwide. Available therapies include function replacement by dialysis or transplant, which are associated with a high morbidity and mortality. Likewise, none of these treatments compensate all kidney functions. There is a great concern in developing more effective therapies with the ability to replace the wide range of renal functions, so that, new studies on developing therapeutic strategies have focused on regenerative medicine. OBJECTIVE: The aim of this paper is to review the new advances in regenerative medicine for renal failure treatment. RESULTS: Regenerative medicine comprises two therapeutic strategies: cell therapy and tissue engineering. Cell therapy techniques depend on cell and tissue cultures, with the aim to replace morphological structures, tissues, and functions. The main strategic strength of cell therapy in renal failure is the incorporation of additional cells in a damaged kidney, for which purpose different kind of Stem Cells (SCs) have been used such as Embryonic SCs, induced Pluripotent SCs, Multipotent SCs, Renal SCs, or drugs that increase survival and mobilization of SCs. Tissue engineering complements cell therapy combining techniques of biological sciences and engineering to create structures and devices as scaffolds, matrices or 3D biocompatible materials. CONCLUSION: Even though there is a significant advance in regenerative medicine strategies, we are far from using any of its techniques on health institutions, due to it is necessary to evaluate side effects, biodistribution, dosage, type of administration, vehicle of cell therapy, as well as the evaluation of response time and long-term studies, among other studies.

Morphological, molecular and FTIR spectroscopic analysis during the differentiation of kidney cells from pluripotent stem cells

BACKGROUND: Kidney diseases are a global health problem. Currently, over 2 million people require dialysis or transplant which are associated with high morbidity and mortality; therefore, new researches focused on regenerative medicine have been developed, including the use of stem cells. RESULTS: In this research, we generate differentiated kidney cells (DKCs) from mouse pluripotent stem cells (mPSCs) analyzing their morphological, genetic, phenotypic, and spectroscopic characteristics along differentiation, highlighting that there are no reports of the use of Fourier transform infrared (FTIR) spectroscopy to characterize the directed differentiation of mPSCs to DKCs. The genetic and protein experiments proved the obtention of DKCs that passed through the chronological stages of embryonic kidney development. Regarding vibrational spectroscopy analysis by FTIR, bands related with biomolecules were shown on mPSCs and DKCs spectra, observing distinct differences between cell lineages and maturation stages. The second derivative of DKCs spectra showed changes in the protein bands compared to mPSCs. Finally, the principal components analysis obtained from FTIR spectra allowed to characterize chemical and structurally mPSCs and their differentiation process to DKCs in a rapid and non-invasive way. CONCLUSION: Our results indicated that we obtained DKCs from mPSCs, which passed through the chronological stages of embryonic kidney development. Moreover, FTIR spectroscopy resulted in a non-invasive, rapid and precise technic that together with principal component analysis allows to characterize chemical and structurally both kind of cells and also discriminate and determine different stages along the cell differentiation process.

FTIR Spectroscopic and Molecular Analysis during Differentiation of Pluripotent Stem Cells to Pancreatic Cells.

Some of the greatest challenges in stem cells (SCs) biology and regenerative medicine are differentiation control of SCs and ensuring the purity of differentiated cells. In this work, we differentiated mouse pluripotent stem cells (mPSCs) toward pancreatic cells characterizing this differentiation process by molecular and spectroscopic technics. Both mPSCs and Differentiated Pancreatic Cells (DPCs) were subjected to a genetic, phenotypic, and biochemical analysis by real-time quantitative PCR (RT-qPCR), immunocytochemistry, and Fourier Transform Infrared (FTIR) spectroscopy. Cultured mPCSs expressed pluripotent genes and proteins (Nanog and SOX2). DPCs expressed endodermal genes (SOX17 and Pdx1) at day 11, an inductor gene of embryonic pancreas development (Pdx1) at day 17 and pancreas genes and proteins (Insulin and Glucagon) at day 21 of differentiation. Likewise, FTIR spectra of mPSCs and DPCs at different maturation stages (11, 17, and 21 days) were obtained and showed absorption bands related with different types of biomolecules. These FTIR spectra exhibited significant spectral changes agreeing with the differentiation process, particularly in proteins and nucleic acids bands. In conclusion, the obtained DPCs passed through the chronological stages of embryonic pancreas development and FTIR spectra provide a new biophysical parameter based on molecular markers indicating the differentiation process of mPSCs to specialized cells.

Reactance and resistance: main properties to follow the cell differentiation process in Bacillus thuringiensis by dielectric spectroscopy in real time.

During growth, Bacillus thuringiensis presents three phases: exponential phase (EP), transition state (TS), and sporulation phase (SP). In order to form a dormant spore and to synthesize delta-endotoxins during SP, bacteria must undergo a cellular differentiation process initiated during the TS. Dielectric spectroscopy is a technique that can be utilized for continuous and in situ monitoring of the cellular state. In order to study on-line cell behavior in B. thuringiensis cultures, we conducted a number of batch cultures under different conditions, by scanning 200 frequencies from 42 Hz to 5 MHz and applying fixed current and voltage of 20 mA and 5 V DC, respectively. The resulting signals included Impedance (Z), Angle phase (Deg), Voltage (V), Current (I), Conductance (G), Reactance (X), and Resistance (R). Individual raw data relating to observed dielectric property profiles were correlated with the different growth phases established using data from cellular growth, cry1Ac gene expression, and free spores obtained with conventional techniques and fermentation parameters. Based on these correlations, frequencies of 0.1, 0.5, and 1.225 MHz were selected for the purpose of measuring dielectric properties in independent batch cultures, at a fixed frequency. X and R manifest more propitious behavior in relation to EP, TS, SP, and spore release, due to particular changes in their signals. Interestingly, these profiles underwent pronounced changes during EP and TS that were not noticed when using conventional methods, but were indicative of the beginning of the B. thuringiensis cell differentiation process.

Histologic and spectroscopic study of pluripotent stem cells after implant in ocular traumatic injuries in a murine model.

INTRODUCTION: Ocular trauma is defined as a trauma caused by blunt or penetrating mechanisms on the eyeball and its peripheral structures, causing damage with different degrees of affection with temporary or permanent visual function compromise. Ocular trauma is a major cause of preventable blindness worldwide; it constitutes 7% of all corporal injury and 10% to 15% of all eye diseases. Regenerative medicine research has opened up the possibility to use stem cells as a source of cell replacement, so that experimental studies on embryonic stem cells and bone marrow stem cells have been carried out. In this study, we analyzed the histopathological and spectroscopic changes in ocular tissue with trauma, treated with mouse pluripotent stem cells. METHODS: Firstly, mouse embryonic stem cells were seeded. Subsequently, the obtained cells were implanted in a murine model of scleral and retinal damage at the first, second, and fourth weeks post-trauma. At week 12 post-trauma, the eyes were enucleated for histopathologic study (inflammatory response and histological integrity) and spectroscopic analysis by Fourier transform infrared spectroscopy in the attenuated total reflection configuration. Data were analyzed by one-way analysis of variance. RESULTS: Histopathological results showed that the experimental groups treated with stem cells presented a decrease in the inflammatory response, and the histological integrity was restored, which contrasted with the experimental group treated with saline solution. Moreover, in the spectroscopic analysis, characteristic bands of biological samples were observed in all tissues, highlighting in healthy tissues the presence of C = O bond at 1,745 cm⁻¹, which was not observed in the injured and treated tissues. Also, the absorption spectrum of the tissues treated with embryonic stem cells showed bands whose intensity was high at around 1,080 to 1,070 cm⁻¹. It has been reported that these bands are characteristic of pluripotent stem cells. CONCLUSIONS: The implant of embryonic stem cells could be a useful therapeutic treatment after traumatic eye injuries or many other eye diseases to reduce the inflammatory response and restore histological integrity. Furthermore, the spectroscopic technique could be used as a complementary technique for detecting stem cell incorporation into various tissues.