Ethanol and Higher Alcohols' Production in Fungal and Bacterial Laboratory Cultures and Significance for Forensic Samples.

Ethanol can be produced by many microorganisms that colonize a dead body. Ethanol's concentration depends on the congener higher alcohols, 1-propanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 1-butanol, as previous research has shown. This correlation is expressed by mathematical models which estimate the concentration of microbial ethanol. The aim of this contribution was to study the ethanol and higher alcohols' production in various laboratory bacterial and fungal cultures and the applicability of the bacterial and fungal models (which concern the bacteria E. coli, S. aureus, K. pneumoniae, and E. faecalis, and the fungus C. albicans) in these samples, as well as in blood samples from autopsy cases, with the overall objective of investigating the models' applicability in routine casework. The bacteria and fungus were cultured in conventional culture media and in denatured human blood cultures under various conditions. The alcohols' concentrations were determined using a head space-gas chromatography-flame ionization detector (HS-GC-FID). The previously reported bacterial and yeast models were applied in the cultured samples and in blood from 122 autopsy cases. Our results showed that 1-propanol was not produced by C. albicans and E. faecalis under certain conditions. Also, 1-butanol was not produced by C. albicans, E. faecalis, and K. pneumonia under certain conditions. Furthermore, the bacterial models were applicable in postmortem samples irrespective of the microbes that were possibly activated in the sample, while the EC models showed the best applicability among all the bacterial and yeast models. The best applicability of the bacterial models was observed in autopsy blood with 0.10 g/L < BAC < 1.0 g/L in cases of violent and undetermined causes of death and in cases with putrefaction. Finally, the yeast models were applicable in limited, possibly special, autopsy cases. In conclusion, it could be inferred that the source of ethanol in any given postmortem blood sample is likely microbial if either most bacterial models or at least one model from each distinct bacterial species is successfully applicable.

Intraoperative Flow Cytometry for the Rapid Diagnosis and Validation of Surgical Clearance of Non-Melanoma Skin Cancer: A Prospective Clinical Feasibility Study.

Non-melanoma skin cancer (NMSC) is the most prevalent cancer in humans, with a high global incidence. We present a prospective clinical feasibility study on the use of intraoperative flow cytometry (iFC) for the instant diagnosis of NMSC and its complete surgical clearance. Flow cytometry, a laser-based technique, quantifies cell features, which has applications in cancer research. This study aim is to explore the potential applicability of iFC in detecting and characterizing NMSC and its surgical margins. In total, 30 patients who underwent diagnosis for NMSC were recruited. The method demonstrated high sensitivity (95.2%) and specificity (87.1%), with an accuracy of 91.1%, as confirmed with a receiver operating characteristic curve analysis. The results also indicated that most tumors were diploid, with two cases being hypoploid. The average G0/G1 fractions for normal and tumor tissue samples were 96.03 ± 0.30% and 88.03 ± 1.29%, respectively, with the tumor index escalating from 3.89 ± 0.30% to 11.95 ± 1.29% in cancerous cells. These findings underscore iFC's capability for precise intraoperative NMSC characterization and margin evaluation, promising enhanced complete tumor excision rates. Given the technique's successful application in various other malignancies, its implementation in NMSC diagnosis and treatment holds significant promise and warrants further research in clinical trials.

BCC and Immunocryosurgery scar differentiation through computational resolution-enhanced OCT images and skin optical attenuation: A proof-of-concept study.

Monitoring medical therapy remains a challenging task across all non-surgical skin cancer treatment modalities. In addition, confirmation of residual tumours after treatment is essential for the early detection of potential relapses. Optical coherence tomography (OCT), a non-invasive method for real-time cross-sectional imaging of living tissue, is a promising imaging approach for assessing relatively flat, near-surface skin lesions, such as those that occur in most basal cell carcinomas (BCCs), at the time of diagnosis. However, the skin's inherent property of strong light scattering impedes the implementation of OCT in these cases due to the poor image quality. Furthermore, translating OCT's optical parameters into practical use in routine clinical settings is complicated due to substantial observer subjectivity. In this retrospective pilot study, we developed a workflow based on the upscale of the OCT images resolution using a deep generative adversarial network and the estimation of the skin optical attenuation coefficient. At the site of immunocryosurgery-treated BCC, the proposed methodology can extract optical parameters and discriminate objectively between tumour foci and scar tissue.

Towards non-invasive monitoring of non-melanoma skin cancer using spatially offset Raman spectroscopy.

BCC (basal cell carcinoma) and SCC (squamous cell carcinoma) account for the vast majority of cases of non-melanoma skin cancer (NMSC). The gold standard for the diagnosis remains biopsy, which, however, is an invasive and time-consuming procedure. In this study, we employed spatially offset Raman spectroscopy (SORS), a non-invasive approach, allowing the assessment of deeper skin tissue levels and collection of Raman photons with a bias towards the different layers of epidermis, where the non-melanoma cancers are initially formed and expand. Ex vivo Raman measurements were acquired from 22 skin biopsies using conventional back-scattering and a defocused modality (with and without a spatial offset). The spectral data were assessed against corresponding histopathological data to determine potential prognostic factors for lesion detection. The results revealed a positive correlation of protein and lipid content with the SCC and BCC types, respectively. By further correlating with patient data, multiple factor analysis (MFA) demonstrated a strong clustering of variables based on sex and age in all modalities. Specifically for the defocused modality (zero and 2 mm offset), further clustering occurred based on pathology. This study demonstrates the utility of the SORS technology in NMSC diagnosis prior to histopathological examination on the same tissue.

Treatment of nail clippings with ethyl alcohol improves the efficacy of Raman spectroscopy in the diagnosis of Trichophyton rubrum onychomycosis.

The purpose of this work was to enhance the diagnostic accuracy of nail Raman spectroscopy for fungal nail infections, specifically onychomycosis caused by Trichophyton rubrum. The study assessed the different ethyl alcohol retention rates between control and infected nails after soaking nail clippings in ethanolic solutions and drying. Results revealed that ethyl alcohol completely evaporated from infected nail samples, while significant amounts were still present in control samples. Principal component analysis (PCA) was applied to discriminate control from infected nails and showed superior group separation when nails were treated with ethyl alcohol. PCA loadings plot attributed the efficient classification to the νs (CCO) Raman vibrational mode of ethyl alcohol. As Raman spectroscopy can detect minute concentration changes of ethyl alcohol in nails and the deterioration caused by onychomycosis accelerates its evaporation, a simple and rapid method for detecting T. rubrum onychomycosis is proposed.

A Fiber Optic Sensor for Monitoring the Spectral Alterations and Depth in Ex Vivo and In Vivo Cryosurgery.

This article discusses how to monitor the freezing depth during cryotherapy using a fiber optic array sensor. The sensor was used to measure the backscattered and transmitted light from frozen and unfrozen ex vivo porcine tissue and in vivo human skin tissue (finger). The technique exploited the variations in optical diffusion properties of the frozen and unfrozen tissues to determine the extent of freezing. Ex vivo and in vivo measurements yielded comparable results, despite spectral variations attributable to the hemoglobin absorption peak in the human frozen and unfrozen tissues. However, because the spectral fingerprints of the freeze-thaw process in the ex vivo and in vivo experiments were similar, we could extrapolate the maximum depth of freezing. Therefore, this sensor has the potential to be utilized for monitoring cryosurgery in real time.

Substrate Specificity of the Highly Thermostable Esterase EstDZ3.

Esterases are among the most studied enzymes, and their applications expand into several branches of industrial biotechnology. Yet, despite the fact that information on their substrate specificity is crucial for selecting or designing the best fitted biocatalyst for the desired application, it cannot be predicted from their amino acid sequence. In this work, we studied the substrate scope of the newly discovered hydrolytic extremozyme, EstDZ3, against a library of esters with variable carbon chain lengths in an effort to understand the crucial amino acids for the substrate selectivity of this enzyme. EstDZ3 appears to be active against a wide range of esters with high selectivity towards medium- to long-carbon chain vinyl esters. In-silico studies of its 3D structure revealed that the selectivity might arise from the mainly hydrophobic nature of the active site's environment.

Molecular Dynamics Simulation of 2-Benzimidazolyl-Urea with DPPC Lipid Membrane and Comparison with a Copper(II) Complex Derivative.

Benzimidazole derivatives have gained attention recently due to their wide pharmacological activity acting as anti-inflammatory, hypotensive, analgesic, and anti-aggregatory agents. They are also common ligands in transition metal coordination chemistry, forming complex compounds with enhanced biological properties, especially in targeted cancer therapy. A key issue to understand anti-tumour effects is drug permeability through cellular membranes, as poor permeability outcomes can avert further futile drug development. In this work, we conducted atomistic molecular dynamics (MD) simulations and biased MD simulations to explore the interactions of 2-benzimidazolyl-urea with a phospholipid bilayer (dipalmitoylphosphatidylcholine, DPPC) together with a previously synthesized copper(II) complex compound. The aim was to study the permeability of these compounds by assessing their free energy profile along the bilayer normal. The simulations indicated that both the ligand (2-benzimidazolyl-urea, BZIMU) and the complex show a similar behaviour, yielding high energy barriers for the permeation process. However, with increasing concentration of BZIMU, the molecules tend to aggregate and form a cluster, leading to the formation of a pore. Clustering and pore formation can possibly explain the previously observed cytotoxicity of the BZIMU molecule via membrane damage.

Modeling microbial ethanol production by S. aureus, K. pneumoniae, and E. faecalis under aerobic/anaerobic conditions - applicability to laboratory cultures and real postmortem cases.

A quite intriguing subject being intensively researched in the forensic toxicology field is the source of postmortem determined blood ethanol concentration: antemortem ingestion or postmortem microbial production. Our previous research on microbial ethanol production has reported a quantitative relationship between the ethanol and the higher alcohols and 1-butanol produced by Escherichia coli, Clostridium perfrigens, and Clostridium sporogenes. In this contribution, we continue our research reporting on the following: (i) the patterns of ethanol, higher alcohols, and 1-butanol production by the microbes Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis (all being aerobic/facultative anaerobic species, common corpse's colonizers, and ethanol producers), under controlled laboratory conditions, (ii) the mathematical modeling, with simple mathematical equations, of the correlation between ethanol concentration and the other studied alcohols' concentrations, by performing multiple linear regression analysis of the results, and (iii) the applicability of the constructed models in microbial cultures developed under different temperature than that used to build the models, in denatured blood cultures and in real postmortem cases. The aforementioned alcohols were proved to be all indicators of ethanol production, both in qualitative and quantitative terms. 1-Propanol was the most significant alcohol in modeling microbial ethanol production, followed by methyl-butanol. The K. pneumoniae's models achieved the best scoring in applicability (E < 40%) compared to the S. aureus and E. faecalis models, both at laboratory microbial cultures at 37 °C and real postmortem cases. Overall, a noteworthy accuracy in estimating the microbial ethanol in cultures and autopsy blood is achieved by the employed simple linear models.

New bioelectrical impedance analysis equations for children and adolescents based on the deuterium dilution technique.

BACKGROUND AND AIMS: Body composition in childhood is not only a marker of the prevalence of obesity, but it can also be used to assess associated metabolic complications. Bioelectrical impedance analysis (BIA) shows promise as an easy to use, rapid, and non-invasive tool to evaluate body composition. The objectives of this study were to: (a) develop BIA prediction equations to estimate total body water (TBW) and fat-free mass (FFM) in European children and early adolescents and to validate the analysis with the deuterium dilution as the reference technique and (b) compare our results with previously published paediatric BIA equations. METHODS: The cohort included 266 healthy children and adolescents between 7 and 14 years of age, 46% girls, in five European countries: Bosnia and Herzegovina, Latvia, Montenegro, North Macedonia, and Portugal. TBW and FFM were the target variables in the developed regression models. For model development, the dataset was randomly split into training and test sets, in 70:30 ratio, respectively. Model tuning was performed with 10-fold cross-validation that confirmed the unbiased estimate of its performance. The final regression models were retrained on the whole dataset. RESULTS: Cross-validated regression models were developed using resistance index, weight, and sex as the optimal predictors. The new prediction equations explained 87% variability in both TBW and FFM. Limits of agreement between BIA and reference values, were within ±17% of the mean, (-3.4, 3.7) and (-4.5, 4.8) kg for TBW and FFM, respectively. BIA FFM and TBW estimates were within one standard deviation for approximately 83% of the children. BIA prediction equations underestimated TBW and FFM by 0.2 kg and 0.1 kg respectively with no proportional bias and comparable accuracy among different BMI-for-age subgroups. Comparison with predictive equations from published studies revealed varying discrepancy rates with the deuterium dilution measurements, with only two being equivalent to the equations developed in this study. CONCLUSIONS: The small difference between deuterium dilution and BIA measurements validated by Bland-Altman analysis, supports the application of BIA for epidemiological studies in European children using the developed equations.

Quantification and Classification of Diclofenac Sodium Content in Dispersed Commercially Available Tablets by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis.

A new methodology, based on Fourier transform infrared spectroscopy equipped with an attenuated total reflectance accessory (ATR FT-IR), was developed for the determination of diclofenac sodium (DS) in dispersed commercially available tablets using chemometric tools such as partial least squares (PLS) coupled with discriminant analysis (PLS-DA). The results of PLS-DA depicted a perfect classification of the tablets into three different groups based on their DS concentrations, while the developed model with PLS had a sufficiently low root mean square error (RMSE) for the prediction of the samples' concentration (~5%) and therefore can be practically used for any tablet with an unknown concentration of DS. Comparison with ultraviolet/visible (UV/Vis) spectrophotometry as the reference method revealed no significant difference between the two methods. The proposed methodology exhibited satisfactory results in terms of both accuracy and precision while being rapid, simple and of low cost.

Modeling postmortem ethanol production by C. albicans: Experimental study and multivariate evaluation.

In previous research, we modeled the ethanol production by certain bacteria under controlled experimental conditions in an attempt to quantify the production of microbial postmortem ethanol in cases where other alcohols were co-detected. This contribution on the modeling of postmortem ethanol production by Candida albicans is complementary to these previous studies. Τhis work aimed to study ethanol, higher alcohols (1-propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol), and 1-butanol production by Candida albicans: (i) in different culture media (Brain Heart Infusion, BHI and, Sabouraud Dextrose Broth, SDB), (ii) under mixed aerobic/anaerobic or strict anaerobic conditions, and (iii) at different temperatures (37 °C, 25 °C and, 4 °C), and develop simple mathematical models, resulted from fungal cultures at 25 °C, to predict the microbially produced ethanol in correlation with the other alcohols. The applicability of the models was tested in the C. albicans cultures in BHI and SDB media at 37 °C, in denatured human blood at 25 °C, acidic and neutral with different concentrations of additional glucose, in acidic denatured blood diluted with dextrose solution and in blood from autopsy cases. The received results indicated that the C. albicans models could apply in cases where yeasts have been activated in blood with elevated glucose levels. Overall, the in vitro ethanol production by C. albicans in blood depended on temperature, time, glucose (or carbohydrate) content, pH of the medium and endogenous changes in the medium composition through time. Our results showed that methyl-butanol is the most significant indicator of fungal ethanol production, followed by the equally important isobutanol and 1-propanol in qualitative and quantitative terms.

Silver Nanoparticles from Oregano Leaves' Extracts as Antimicrobial Components for Non-Infected Hydrogel Contact Lenses.

The oregano leaves' extract (ORLE) was used for the formation of silver nanoparticles (AgNPs(ORLE)). ORLE and AgNPs(ORLE) (2 mg/mL) were dispersed in polymer hydrogels to give the pHEMA@ORLE_2 and pHEMA@AgNPs(ORLE)_2 using hydroxyethyl-methacrylate (HEMA). The materials were characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), thermogravimetric differential thermal analysis (TG-DTA), derivative thermogravimetry/differential scanning calorimetry (DTG/DSC), ultraviolet (UV-Vis), and attenuated total reflection mode (ATR-FTIR) spectroscopies in solid state and UV-Vis in solution. The crystallite size value, analyzed with XRPD, was determined at 20 nm. The antimicrobial activity of the materials was investigated against Gram-negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The Gram-positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are known to be involved in microbial keratitis by the means of inhibitory zone (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The IZs, which developed upon incubation of P. aeruginosa, E. coli, S. epidermidis, and S. aureus with paper discs soaked in 2 mg/mL of AgNPs(ORLE), were 11.7 ± 0.7, 13.5 ± 1.9, 12.7 ± 1.7, and 14.3 ± 1.7 mm. When the same dose of ORLE was administrated, the IZs were 10.2 ± 0.7, 9.2 ± 0.5, 9.0 ± 0.0, and 9.0 ± 0.0 mm. The percent of bacterial viability when they were incubated over the polymeric hydrogel discs of pHEMA@AgNPs(ORLE)_2 was interestingly low (66.5, 88.3, 77.7, and 59.6%, respectively, against of P. aeruginosa, E. coli, S. epidermidis, and S. aureus) and those of pHEMA@ORLE_2 were 89.3, 88.1, 92.8, and 84.6%, respectively. Consequently, pHEMA@AgNPs(ORLE)_2 could be an efficient candidate toward the development of non-infectious contact lenses.

Self-Assembly Magnetic Micro- and Nanospheres and the Effect of Applied Magnetic Fields.

The impact of in-plane and perpendicular magnetic fields on the spatial arrangement of superparamagnetic nanospheres is explored. We utilize nanosphere self-organization methods like Spin Coating and Drop-Casting in the presence of magnetic fields. In this way, the additional parameter of the long range magnetic dipolar interactions is introduced to the competing nanosphere-surface and nanosphere-nanosphere interactions, which control order and agglomeration. We present a comparative analysis of the self-assembly characteristics with respect to the different methods and the effect of the applied field in different directions. Under zero field perfect hexagonal arrays can be obtained by spin coating. Parallel applied fields tend to create directional patterns, while perpendicular favor 3D-accumulation.

A Social Media-Promoted Educational Community of Joint Replacement Patients Using the WeChat App: Survey Study.

BACKGROUND: Much effort has been made to optimize the results of total hip arthroplasty and total knee arthroplasty. With the rapid growth of social media use, mobile apps, such as WeChat, have been considered for improving outcomes and patient satisfaction after total hip arthroplasty and total knee arthroplasty. OBJECTIVE: We aimed to evaluate the effectiveness of a WeChat-based community as an intervention for overall patient satisfaction. METHODS: The study was conducted among discharged in-hospital patients who received hip or knee procedures in the First Affiliated Hospital of the University of Science and Technology of China from April 2019 to January 2020. An educational online social community was constructed with the WeChat app. Participants willing to join the community were enrolled in a WeChat group and received 3 months of intervention and follow-up. Those who were not willing to use the account were included in a control group and received routine publicity via telephone, mail, and brochures. The Danish Health and Medicine Authority patient satisfaction questionnaire was used to score perioperative patient education and overall satisfaction. The contents in the group chat were analyzed using natural language processing tools. RESULTS: A total of 3428 patients were enrolled in the study, including 2292 in the WeChat group and 1236 in the control group. Participants in the WeChat group had higher overall satisfaction scores than those in the control group (mean 8.48, SD 1.12 vs mean 6.66, SD 1.80, P<.001). The difference between the two groups was significant for primary surgery based on subgroup stratification. To control confounding factors and explore the effects of WeChat participation as a mediating variable between perioperative patient education and overall satisfaction, hierarchical regression was utilized. An interpatient interaction model was found in the community group chat, and it contributed to overall satisfaction. Patients in the group with more interpatient interactions were more likely to have better overall satisfaction. CONCLUSIONS: The social media-promoted educational community using WeChat was effective among joint replacement patients. Provision of more perioperative education is associated with more active patient participation in the community and therefore more patient satisfaction in terms of the overall joint procedure. Community group chat could facilitate interactions among patients and contribute to overall satisfaction.

Biothiol modulated growth and aggregation of gold nanoparticles and their determination in biological fluids using digital photometry.

This work describes a novel and easy to use method for the determination of biologically important thiols that relies on their ability to inhibit the catalytic enlargement of AuNP seeds in the presence of ACl4- ions and trigger their aggregation. UV-vis spectroscopic monitoring of the plasmon resonance bands of the formed AuNPs showed that the spectral and color transitions depend both on the concentration and the structure of biothiols. The colorimetric changes induced by biothiols were quantified in the concentration range from 5 to 300 µM in the RGB color system with digital photometry using a commercially available flatbed scanner as detector. On the basis of these results, the applicability of the method was tested to the determination of glutathione in red blood cells and cysteine in blood plasma with satisfactory recoveries (88.7-96.5%), low detection limits (1.0 µM), good selectivity against major biomolecules under physiologically relevant conditions and satisfactory reproducibility (<8%). The method requires minimum technical expertise, is easy to use and is performed without scientific equipment, holding promise as a simple assay of biothiol testing even by non-experts.

Integrative Analysis of Genomics and Transcriptome Data to Identify Regulation Networks in Female Osteoporosis.

Background: Osteoporosis is a highly heritable skeletal muscle disease. However, the genetic mechanisms mediating the pathogenesis of osteoporosis remain unclear. Accordingly, in this study, we aimed to clarify the transcriptional regulation and heritability underlying the onset of osteoporosis. Methods: Transcriptome gene expression data were obtained from the Gene Expression Omnibus database. Microarray data from peripheral blood monocytes of 73 Caucasian women with high and low bone mineral density (BMD) were analyzed. Differentially expressed messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) were identified. Differences in BMD were then attributed to several gene modules using weighted gene co-expression network analysis (WGCNA). LncRNA/mRNA regulatory networks were constructed based on the WGCNA and subjected to functional enrichment analysis. Results: In total, 3,355 mRNAs and 999 lncRNAs were identified as differentially expressed genes between patients with high and low BMD. The WGCNA yielded three gene modules, including 26 lncRNAs and 55 mRNAs as hub genes in the blue module, 36 lncRNAs and 31 mRNAs as hub genes in the turquoise module, and 56 mRNAs and 30 lncRNAs as hub genes in the brown module. JUN and ACSL5 were subsequently identified in the modular gene network. After functional pathway enrichment, 40 lncRNAs and 16 mRNAs were found to be related to differences in BMD. All three modules were enriched in metabolic pathways. Finally, mRNA/lncRNA/pathway networks were constructed using the identified regulatory networks of lncRNAs/mRNAs and pathway enrichment relationships. Conclusion: The mRNAs and lncRNAs identified in this WGCNA could be novel clinical targets in the diagnosis and management of osteoporosis. Our findings may help elucidate the complex interactions between transcripts and non-coding RNAs and provide novel perspectives on the regulatory mechanisms of osteoporosis.

Tissue Phantoms for Biomedical Applications in Raman Spectroscopy: A Review.

Raman spectroscopy is a group of analytical techniques, currently applied in several research fields, including clinical diagnostics. Tissue-mimicking optical phantoms have been established as an essential intermediate stage for medical applications with their employment from spectroscopic techniques to be constantly growing. This review outlines the types of tissue phantoms currently employed in different biomedical applications of Raman spectroscopy, focusing on their composition and optical properties. It is therefore an attempt to present an informed range of options for potential use to the researchers.

Integrated Analysis of DEAD-Box Helicase 56: A Potential Oncogene in Osteosarcoma.

Background: Osteosarcoma is a solid tumor common in the musculoskeletal system. The DEAD-box helicase (DDX) families play an important role in tumor genesis and proliferation. Objective:To screen potential molecular targets in osteosarcoma and elucidate its relationship with DDX56. Methods: We employed the Gene Expression Omnibus and The Cancer Genome Atlas datasets for preliminary screening. DDX56 expression was measured by RT-qPCR in three osteosarcoma cell lines. Biological roles of DDX56 were explored by Gene ontology, Kyoto Encyclopedia of Genes and Genomes and Ingenuity Pathway Analysis. Cell proliferation, cycle, and apoptosis assays were performed using Lentivirus™ knockdown technique. Results: It was found that DDX56 expression was regularly upregulated in osteosarcoma tissue and cell lines, while DDX56 knockdown inhibited cell proliferation and promoted cell apoptosis. Conclusions: The findings suggest DDX56 as a potential therapeutic target for the treatment of osteosarcoma.