Post-mortem Interval estimate based on dental pulp: A histomorphology approach.

Estimating the post-mortem interval (PMI) of human remains based on the histomorphology of dental pulp parameters is promising, but available evidence is scarce and sometimes contradictory without a scientific model. The aim of the study is to characterise the histomorphological changes of dental pulp associated with the decomposition of human remains by a qualitative and quantitative approach. The main aim is to establish a correlation based on post-mortem (PM) dental pulp histomorphology and the PMI, and whether pulp degradation could be an available medico-legal tool for PMI estimation beyond the first week after death (late PMI). The eligible sample consisted of 27 sound teeth from 16 healthy patients aged 16 to 72 years due to orthodontic or oral surgery treatment, to create PMI's simulating the death of the subject as the time elapsed from tooth avulsion. Data collected from patients (sex, date of birth, tooth position, date and hour of the avulsion, date and hour of pulp extraction) were anonymised in accordance with the requirements of Faculty of Dental Medicine of the University of Lisbon. The sample was divided into 9 groups of 3 teeth according to different PMI sets from T0 (baseline) up to 2 weeks (T0, 7, 12, 24, 36, 48, and 72 hours, 1 and 2 weeks). All the dental samples were stored at room temperature up to the time of pulp extraction and then prepared with haematoxylin and eosin stain. High-resolution microscopy was performed to obtain histological images. An operator performed the qualitative evaluation of blood vessels, collagen fibres, and the extra-cellular matrix (ECM) in PM pulps and measured the variation in cells/nuclei density by counting 6 different ROIs (Regions of Interest) for each pulp manually and automatically (quantitative analysis). Qualitative results showed that the degeneration of dental pulp appears 7 hours after death but histological changes in vessels, fibres, and ECM in PM dental pulp are characterised by high variability, consequently it is not possible to generalise the results for early PMIs. Quantitative measurements proved that cell count cannot be standardised due to the presence of superimposed layers of cells and nuclei fragmentation. Odontoblasts did not demonstrate evidence of cellular or nuclear lysis up to 14 PM suggesting their applicability in late PMIs. Future research will focus on late PMIs and different techniques of tooth preparation.

Effects of pre-treatment, historical age, and sample characteristics on the stable isotope analyses of killer whale (Orcinus orca) bone.

RATIONALE: Stable isotope analysis of bone provides insight into animal foraging and allows for ecological reconstructions over time, however pre-treatment is required to isolate collagen. Pre-treatments typically consist of demineralization to remove inorganic components and/or lipid extraction to remove fats, but these protocols can differentially affect stable carbon (δ13C) and nitrogen (δ15N) isotope values depending on the chemicals, tissues, and/or species involved. Species-specific methodologies create a standard for comparability across studies and enhance understanding of collagen isolation from modern cetacean bone. METHODS: Elemental analyzers coupled to isotope ratio mass spectrometers were used to measure the δ13C and δ15N values of powdered killer whale (Orcinus orca) bone that was intact (control) or subjected to one of three experimental conditions: demineralized, lipid-extracted, and both demineralized and lipid-extracted. Additionally, C:N ratios were evaluated as a proxy for collagen purity. Lastly, correlations were examined between control C:N ratios vs. historical age and control C:N ratios vs. sample characteristics. RESULTS: No significant differences in the δ15N values were observed for any of the experimental protocols. However, the δ13C values were significantly increased by all three experimental protocols: demineralization, lipid extraction, and both treatments combined. The most influential protocol was both demineralization and lipid extraction. Measures of the C:N ratios were also significantly lowered by demineralization and both treatments combined, indicating the material was closer to pure collagen after the treatments. Collagen purity as indicated via C:N ratio was not correlated with historical age nor sample characteristics. CONCLUSIONS: If only the δ15N values from killer whale bone are of interest for analysis, no pre-treatment seems necessary. If the δ13C values are of interest, samples should be both demineralized and lipid-extracted. As historical age and specimen characteristics are not correlated with sample contamination, all samples can be treated equally.

Optimizing tenderness of M. Semitendinosus steak for elderly people with the combination of ficin and sous-vide cooking.

This study aimed to evaluate the sous-vide cooking and ficin treatment effects on the tenderness of beef steak and optimize it for the elderly using response surface methodology (RSM). The M. semitendinosus (ST) from Chikso cattle was shaped into 5 × 5 × 2.54 cm pieces. Ficin solution was injected into the ST steak at 10% of the meat weight, and sous-vide cooked in a water bath at 65 °C for 6 or 12 h. As ficin concentration increased, L*- and a*-value, shear force, and hardness decreased, while soluble peptides increased (P < 0.05). As cooking time increased, cooking loss and collagen solubility of the steak increased (P < 0.05). An interaction effect between ficin and sous-vide cooking was found in L*- and a*-value, shear force, hardness, and soluble peptides (P < 0.05). A model to optimize the hardness for elderly people was established (R2 = 0.7991). Optimization conditions by RSM were 0.86 U/L with 8.87 h (23 N/cm3) for tooth intake (grade 1), 16.31 U/L with 13.24 h (3 N/cm3) for gums intake (grade 2), according to KS H 4897 and Universal Design Foods concept for the elderly. These optimized conditions enable the production of customized products tailored to the oral conditions of elderly people.

Liver fibrosis analysis using digital pathology.

Digital pathology has enabled the noninvasive quantification of pathological parameters. In addition, the combination of digital pathology and artificial intelligence has enabled the analysis of a vast amount of information, leading to the sharing of much information and the elimination of knowledge gaps. Fibrosis, which reflects chronic inflammation, is the most important pathological parameter in chronic liver diseases, such as viral hepatitis and metabolic dysfunction-associated steatotic liver disease. It has been reported that the quantitative evaluation of various fibrotic parameters by digital pathology can predict the prognosis of liver disease and hepatocarcinogenesis. Liver fibrosis evaluation methods include 1 fiber quantification, 2 elastin and collagen quantification, 3 s harmonic generation/two photon excitation fluorescence (SHG/TPE) microscopy, and 4 Fibronest™..　In this review, we provide an overview of role of digital pathology on the evaluation of fibrosis in liver disease and the characteristics of recent methods to assess liver fibrosis.

Low-Invasive Sampling Method with Tape-Disc Sampling for the Taxonomic Identification of Archeological and Paleontological Bones by Proteomics.

Collagen from paleontological bones is an important organic material for isotopic measurement, radiocarbon analysis, and paleoproteomic analysis to provide information on diet, dating, taxonomy, and phylogeny. Current paleoproteomic methods are destructive and require from a few milligrams to several tens of milligrams of bone for analysis. In many cultures, bones are raw materials for artifacts that are conserved in museums, which hampers damage to these precious objects during sampling. Here, we describe a low-invasive sampling method that identifies collagen, taxonomy, and post-translational modifications from Holocene and Upper Pleistocene bones dated to 130,000 and 150 BC using dermatological skin tape discs for sampling. The sampled bone micropowders were digested following our highly optimized enhanced filter-aided sample preparation protocol and then analyzed by MALDI FTICR MS and LC-MS/MS for identifying the genus taxa of the bones. We show that this low-invasive sampling does not deteriorate the bones and achieves results similar to those obtained by more destructive sampling. Moreover, this sampling method can be carried out at archeological sites or in museums.

Effect of combined red and infrared wavelengths on inflammation, hemorrhage, and muscle damage caused by Bothrops leucurus snake venom.

To evaluate the effects of red and infrared wavelengths, separately and combined, on the inflammatory process and collagen deposition in muscle damage caused by B. leucurus venom. 112 mice were inoculated with diluted venom (0.6mg/kg) in the gastrocnemius muscle. The animals were divided into four groups: one control (CG) and three treatments, namely: 1) red laser (λ=660 nm) (RG), 2) infrared laser (λ=808 nm) (IG) and 3) red laser (λ=660 nm) + infrared (λ=808 nm) (RIG). Each group was subdivided into four subgroups, according to the duration of treatment application (applications every 24 hours over evaluation times of up to 144 hours). A diode laser was used (0.1 W, CW, 1J/point, ED: 10 J/cm2). Both wavelengths reduced the intensity of inflammation and the combination between them significantly intensified the anti-inflammatory response. Photobiomodulation also changed the type of inflammatory infiltrate observed and RIG had the highest percentage of mononuclear cells in relation to the other groups. Hemorrhage intensity was significantly lower in treated animals and RIG had the highest number of individuals in which this variable was classified as mild. As for collagen deposition, there was a significant increase in RG in relation to CG, in RIG in relation to CG and in RIG in relation to IG. Photobiomodulation proved to be effective in the treatment of inflammation and hemorrhage caused by B. leucurus venom and stimulated collagen deposition. Better results were obtained with the combined wavelengths.

A portable dry film FTIR instrument for industrial food and bioprocess applications.

The main objective of this study was to design, build, and test a compact, multi-well, portable dry film FTIR system for industrial food and bioprocess applications. The system features dry film sampling on a circular rotating disc comprising 31 wells, a design that was chosen to simplify potential automation and robotic sample handling at a later stage. Calibration models for average molecular weight (AMW, 200 samples) and collagen content (68 samples) were developed from the measurements of industrially produced protein hydrolysate samples in a controlled laboratory environment. Similarly, calibration models for the prediction of lactate content in samples from cultivation media (59 samples) were also developed. The portable dry film FTIR system showed reliable model characteristics which were benchmarked with a benchtop FTIR system. Subsequently, the portable dry film FTIR system was deployed in a bioprocessing plant, and protein hydrolysate samples were measured at-line in an industrial environment. This industrial testing involved building a calibration model for predicting AMW using 60 protein hydrolysate samples measured at-line using the portable dry film FTIR system and subsequent model validation using a test set of 26 samples. The industrial calibration in terms of coefficient of determination (R2 = 0.94), root mean square of cross-validation (RMSECV = 194 g mol-1), and root mean square of prediction (RMSEP = 162 g mol-1) demonstrated low prediction errors as compared to benchtop FTIR measurements, with no statistical difference between the calibration models of the two FTIR systems. This is to the authors' knowledge the first study for developing and employing a portable dry film FTIR system in the enzymatic protein hydrolysis industry for successful at-line measurements of protein hydrolysate samples. The study therefore suggests that the portable dry film FTIR instrument has huge potential for in/at-line applications in the food and bioprocessing industries.

Bioactive Peptide Profiling in Collagen Hydrolysates: Comparative Analysis Using Targeted and Untargeted Liquid Chromatography-Tandem Mass Spectrometry Quantification.

The investigation of collagen hydrolysates (CHs) is essential due to their widespread use in health, cosmetic, and therapeutic industries, attributing to the presence of bioactive dipeptides (DPs) and tripeptides (TPs). This study developed a novel targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with propyl chloroformate (PCF) derivatization to measure three bioactive peptides-Hydroxyprolyl-glycine (Hyp-Gly), Glycyl-prolyl-hydroxyproline (Gly-Pro-Hyp), and Prolyl-hydroxyproline (Pro-Hyp)-in CHs, with strong correlation coefficients (0.992, 1.000, and 0.995, respectively) and low limits of detection (LODs) of 1.40, 0.14, and 1.16 µM, respectively. Untargeted data-dependent acquisition (DDA) analyses measured peptide size distribution, while amino acid analysis assessed nutritional content. The analysis of ten commercial CHs revealed similar amino acid profiles but varied peptide lengths, indicating diverse hydrolysis conditions. Products with higher proportions of smaller peptides showed elevated levels of the targeted bioactive peptides, suggesting that a smaller peptide size may increase bioactivity. These findings can inform the optimization of CH supplements, providing consumers with detailed peptide content for more informed choices. Data are available via ProteomeXchange with the identifier PXD051699.

Denatured collagen in keratin layers and smooth muscles of teats with low or high teat apex scores in Holstein dairy cows.

We hypothesized that teats with a teat apex score (TAS) of 4 on a 4-point scale would exhibit elevated levels of denatured collagen compared with teats with lower TAS. We procured keratin layer and smooth muscle samples from Holsteins with TAS ranging from 1 to 4, as well as from crossbred heifers (Japanese Black male and Holstein female) with TAS of 1. Teats with a TAS of 4 demonstrated increased total collagen content, higher amounts of type I collagen (the harder, thicker variant), and reduced amounts of type III collagen (the softer, thinner variant) compared with teats with lower TAS. Teats with TAS of 3 and 4 exhibited evidence of damaged collagen in smooth muscle layers compared with teats with TAS of 1. Additionally, we identified 47-kDa heat shock protein-positive fibroblasts in the smooth muscles of teats with TAS of 3 and 4. Therefore, the smooth muscle of teats with a TAS of 4 exhibited increased amounts of denatured collagen in comparison to teats with lower TAS.

Brillouin spectroscopy of medically relevant samples of bovine jugular vein and pericardium.

There is the rapid growth in application of Brillouin scattering spectroscopy to biomedical objects in order to characterize their mechanoelastic properties in this way. However, the possibilities and limitations of the method when applied to tissues have not yet been clarified. Here, applicability of Brillouin spectroscopy for testing the elastic response of medically relevant tissues of bovine jugular vein and pericardium was considered. Parameters of the Brillouin peak were studied for samples untreated, diepoxide-fixed, and preserved after treatment in alcohol solutions. It was found that diepoxide cross-linking resulted to a slight tendency to increase the Brillouin position for hydrated tissues. The variations in the position and width of the Brillouin peaks, associated with local fluctuations in water concentration, were reduced after diepoxide treatment in the case of the pericardium, but not in the case of the vein wall. To obtain more information about the elastic response of the protein scaffold without the participation of water, dried samples were also studied. Brillouin spectra of the dried pericardium and vein wall revealed a significant increase in the Brillouin peak position (elastic modulus) after conservation in alcohol. In the case of the vein wall, this effect was found for both collagen and elastin-related peaks, which were identified in the Brillouin spectrum. This result corresponds to a denser packing of fibrous proteins after preservation in alcohol solutions. The ability of Brillouin spectroscopy to independently characterize the effect of treatment on the instantaneous elastic modulus of various tissue components is also attractive for its application in the development of new materials for bioimplants. A comparison of the Brillouin longitudinal and Young's elastic moduli determined for the hydrated samples of the vein and pericardium showed that there is no clear correspondence between these material parameters. The usefulness of using both experimental methods to obtain new information about the elastic response of the material is discussed.

Disaster victim identification: Stable isotope analysis and the identification of unknown decedents.

Within the complex world of disaster victim identification, or DVI, forensic science practitioners use a variety of investigative techniques to work toward a common goal: identification of the decedents, bringing closure to the affected communities. Identification is a complex undertaking; the event (disaster) also can be extraordinarily complex, as it may be an acute event, or one that spans months or years. Compounding this time issue, remains may be heavily fragmented, dispersed, commingled, or otherwise disrupted by either the perpetrators or the disaster itself. To help solve these complexities, we explore the use of stable isotope analysis (SIA) in DVI events. SIA can be used with a variety of body tissues (hair, nail, bone, and teeth), and each represents different time depths in a decedent's life. Bone collagen and tooth enamel carbonate are useful to reconstruct an individual's diet and source water intakes, respectively, leading to likely population or geographic origin determinations. Additionally, the carbon and nitrogen isotopic signatures of bone collagen have calculated intraperson ranges. These facts allow investigators to determine likely origin of remains using isotopic data and can be used to link skeletal elements (to an individual), or perhaps more importantly, show that remains are not linked. Application of SIA can thus speed remains identification by eliminating individuals from short lists for identification, linking or decoupling remains, and reducing the need for some DNA testing. These strategies and hypothesis tests should commence early in the DVI process to achieve maximum effectiveness.

Fibrostricturing Crohn's Disease Is Marked by an Increase in Active Eosinophils in the Deeper Layers.

INTRODUCTION: Approximately 50% of patients with Crohn's disease (CD) develop intestinal strictures necessitating surgery. The immune cell distribution in these strictures remains uncharacterized. We aimed to identify the immune cells in intestinal strictures of patients with CD. METHODS: During ileocolonic resections, transmural sections of terminal ileum were sampled from 25 patients with CD and 10 non-inflammatory bowel disease controls. Macroscopically unaffected, fibrostenotic, and inflamed ileum was collected and analyzed for immune cell distribution (flow cytometry) and protein expression. Collagen deposition was assessed through a Masson Trichrome staining. Eosinophil and fibroblast colocalization was assessed through immunohistochemistry. RESULTS: The Masson Trichrome staining confirmed augmented collagen deposition in both the fibrotic and the inflamed regions, though with a significant increased collagen deposition in the fibrotic compared with inflamed tissue. Distinct Th1, Th2, regulatory T cells, dendritic cells, and monocytes were identified in fibrotic and inflamed CD ileum compared with unaffected ileum of patients with CD as non-inflammatory bowel disease controls. Only minor differences were observed between fibrotic and inflamed tissue, with more active eosinophils in fibrotic deeper layers and increased eosinophil cationic protein expression in inflamed deeper layers. Last, no differences in eosinophil and fibroblast colocalization were observed between the different regions. DISCUSSION: This study characterized immune cell distribution and protein expression in fibrotic and inflamed ileal tissue of patients with CD. Immunologic, proteomic, and histological data suggest inflammation and fibrosis are intertwined, with a large overlap between both tissue types. However strikingly, we did identify an increased presence of active eosinophils only in the fibrotic deeper layers, suggesting their potential role in fibrosis development.

Unravelling social status in the first medieval military order of the Iberian Peninsula using isotope analysis.

Medieval Iberia witnessed the complex negotiation of religious, social, and economic identities, including the formation of religious orders that played a major role in border disputes and conflicts. While archival records provide insights into the compositions of these orders, there have been few direct dietary or osteoarchaeological studies to date. Here, we analysed 25 individuals discovered at the Zorita de los Canes Castle church cemetery, Guadalajara, Spain, where members of one of the first religious orders, the Order of Calatrava knights, were buried between the 12th to 15th centuries CE. Stable carbon (δ13C) and nitrogen (δ15N) isotope analyses of bone collagen reveal dietary patterns typical of the Medieval social elite, with the Bayesian R model, 'Simmr' suggesting a diet rich in poultry and marine fish in this inland population. Social comparisons and statistical analyses further support the idea that the order predominantly comprised the lower nobility and urban elite in agreement with historical sources. Our study suggests that while the cemetery primarily served the order's elite, the presence of individuals with diverse dietary patterns may indicate complexities of temporal use or wider social interaction of the medieval military order.

Diets, stress, and disease in the Etruscan society: Isotope analysis and infantile skeletal palaeopathology from Pontecagnano (Campania, southern Italy, 730-580 BCE).

Susceptibility to morbidity and mortality is increased in early life, yet proactive measures, such as breastfeeding and weaning practices, can be taken through specific investments from parents and wider society. The extent to which such biosocialcultural investment was achieved within 1st millennium BCE Etruscan society, of whom little written sources are available, is unkown. This research investigates life histories in non-adults and adults from Pontecagnano (southern Italy, 730-580 BCE) in order to track cross-sectional and longitudinal breastfeeding and weaning patterns and to characterize the diet more broadly. Stable carbon and nitrogen isotope analysis of incrementally-sampled deciduous and permanent dentine (n = 15), bulk bone collagen (n = 38), and tooth enamel bioapatite (n = 21) reveal the diet was largely based on C3 staple crops with marginal contributions of animal protein. Millet was found to play a role for maternal diet and trajectories of breastfeeding and feeding for some infants and children at the site. The combination of multiple isotope systems and tissues demonstrates exclusive breastfeeding was pursued until 0.6 years, followed by progressive introduction of proteanocius supplementary foods during weaning that lasted between approximately 0.7 and 2.6 years. The combination of biochemical data with macroscopic skeletal lesions of infantile metabolic diseases and physiological stress markers showed high δ15Ndentine in the months prior to death consistent with the isotopic pattern of opposing covariance.

Early Colonial Diet in El Japón, Xochimilco, Mexico: Examining dietary continuity through stable isotope analysis of bone collagen and bioapatite.

OBJECTIVES: Early colonial documents from central Mesoamerica detail raising and planting of European livestock and crops alongside native ones. The extent to which Indigenous people, especially of the rural commoner class, consumed newly introduced foods is less known. This gap in knowledge is addressed through stable isotope analysis and comparison to published archaeological botanical, human, and faunal data. MATERIALS AND METHODS: Stable isotope analysis of bone collagen and bioapatite is applied to 74 skeletal samples of Indigenous human remains representing Colonial period individuals from El Japón-a farming hamlet in the Xochimilco area-to provide insight into long-term individual dietary practices in the context of a rapidly transforming Mesoamerican world. RESULTS: Carbon isotope ratios in collagen (δ13Ccollagen) average -8.10/00 VPDB (SD 0.55), while δ15N averages 8.90/00 AIR (SD 0.50). δ13Cbioapatite averages -2.90/00 VPDB (SD 0.60). Modest increase in carbon isotopic diversity is observed among more recent males from El Japón when compared to earlier males and females. DISCUSSION: Based on the isotopic results, it is estimated that the individuals of El Japón consumed maize or other C4 plants as a central source of carbohydrates. Dietary protein was largely supplied through domestic maize-fed fauna but potentially supplemented by wild terrestrial and aquatic fauna and fowl. Similarity in skeletal isotopic composition between precontact Mesoamericans from other sites and El Japón individuals of both earlier and later stratigraphy is interpreted as continuity in local diets and foodways despite potentially available European alternatives. Colonial taxation demands on preexisting agricultural regimes may have incentivized maize production, thus indirectly contributing to the maize-centered aspect of local foodways.

OBJETIVOS: Los textos de la época colonial temprana del centro de México documentan la producción de cultivos y ganado europeo a la par de los productos agropecuarios nativos. La magnitud a la cuál las comunidades indígenas consumieron estos productos se conoce con menos certeza en especial dentro de los asentamientos rurales. En este trabajo, se analiza la variabilidad de datos de isótopos estables en el sitio El Japón, Xochimilco y los resultados se comparan con respecto al sexo biológico y la cronología; así como también con datos publicados de muestras humanas y faunísticas. MATERIALES Y MÉTODOS: Se aplican los estudios de isotopos estables en colágeno y bioapatita a 74 muestras esqueléticas de El Japón de la época colonial temprana, una aldea agrícola del área de Xochimilco, con tal de abordar las practicas dietéticas en el contexto de un mundo Mesoamericano en transformación tras el contacto europeo. RESULTADOS: Los isótopos estables de carbono en colágeno (δ13Ccollagen) producen un promedio de −8.10/00 VPDB (DE 0.55), mientras tanto los isótopos estables de nitrógeno en el mismo tejido producen un promedio de 8.90/00 AIR (DE 0.50). Los isótopos estables de carbono en la bioapatita (δ13Cbioapatite) producen un promedio de −2.90/00 VPDB (DE 0.60). Se observa un incremento mínimo en la diversidad isotópica entre los individuos de sexo masculino en comparación a los individuos de sexo femenino de la etapa temprana y tardía del sitio. DISCUSIÓN: Con base en los resultados isotópicos, y con base en comparación a muestras humanas de contextos arqueológicos europeos y norteamericanos se estima que los individuos de El Japón consumieron maíz u otros cultivos tipo C4 como fuentes principales de carbohidratos. Las fuentes de proteína dietética posiblemente fueron fauna alimentada con maíz, pero también se pudieron haber suplementado con alimentos silvestres incluyendo aves silvestres, y fauna terrestre o acuática. La similitud en variación isotópica entre sitios mesoamericanos que preceden el contacto europeo y El Japón de ambas etapas (temprana y tardía) se interpretan como persistencia en fuentes de alimentación y tradiciones culinarias a pesar de las posibles alternativas europeas. Las demandas tributarias coloniales sobre la producción agrícola chinampera pudiesen haber contribuido indirectamente a la continuidad del maíz como fuente alimenticia principal.

Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources.

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers. The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 µm, 160 Å) using isocratic elution and MS-compatible mobile phase. The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

Treatment with synchronized radiofrequency and facial muscle stimulation: Histologic analysis of human skin for changes in collagen and elastin fibers.

BACKGROUND: Skin's exposure to intrinsic and extrinsic factors causes age-related changes, leading to a lower amount of dermal collagen and elastin. AIM: This study investigated the effects of a novel facial muscle stimulation technology combined with radiofrequency (RF) heating on dermal collagen and elastin content for the treatment of facial wrinkles and skin laxity. METHODS: The active group subjects (N = 6) received four 20-min facial treatments with simultaneous RF and facial muscle stimulation, once weekly. The control subject (N = 1) was untreated. Skin biopsies obtained at baseline, 1-month and 3-month follow-up were evaluated histologically to determine collagen and elastin fibers content. A group of independent aestheticians evaluated facial skin appearance and wrinkle severity. Patient safety was followed. RESULTS: In the active group, collagen-occupied area reached 11.91 ± 1.80 × 106 µm2 (+25.32%, p < 0.05) and 12.35 ± 1.44 × 105 µm2 (+30.00%, p < 0.05) at 1-month and 3-month follow-up visits. Elastin-occupied area at 1-month and 3-month follow-up was 1.64 ± 0.14 × 105 µm2 (+67.23%, p < 0.05), and 1.99 ± 0.21 × 105 µm2 (+102.80%, p < 0.05). In the control group, there was no significant difference (p > 0.05) in collagen and elastin fibers. Active group wrinkle scores decreased from 5 (moderate, class II) to 3 (mild, class I). All subjects, except the control, improved in appearance posttreatment. No adverse events or side effects occurred. CONCLUSION: Decreased dermal collagen and elastin levels contributes to a gradual decline in skin elasticity, leading to facial wrinkles and unfirm skin. Study results showed noticeable improvement in facial appearance and increased dermal collagen and elastin content subsequent to simultaneous, noninvasive RF, and facial muscle stimulation treatments.

Distinct spectral signatures unfold ECM stiffness-triggered biochemical changes in breast cancer cells.

Cancer progression often accompanies the stiffening of extracellular matrix (ECM) in and around the tumor, owing to extra deposition and cross-linking of collagen. Stiff ECM has been linked with poor prognosis and is known to fuel invasion and metastasis, notably in breast cancer. However, the underlying biochemical or metabolic changes and the cognate molecular signatures remain elusive. Here, we explored Raman spectroscopy to unveil the spectral fingerprints of breast cancer cells in response to extracellular mechanical cues. Using stiffness-tuneable hydrogels, we showed that cells grown on stiff ECM displayed morphological changes with high proliferation. We further demonstrated that Raman Spectroscopy, a label-free and non-invasive technique, could provide comprehensive information about the biochemical environment of breast cancer cells in response to varying ECM stiffness. Raman spectroscopic analysis classified the cells into distinct clusters based on principal component-based linear discriminant analysis (PC-LDA). Multivariate curve resolution-alternating least squares (MCR-ALS) analysis indicated that cells cultured on stiff ECM exhibited elevated nucleic acid content and lesser lipids. Interestingly, increased intensity of Raman bands corresponding to cytochrome-c was also observed in stiff ECM conditions, suggesting mitochondrial modulation. The key findings harboured by spectral profiles were also corroborated by transmission electron microscopy, confirming altered metabolic status as reflected by increased mitochondria number and decreased lipid droplets in response to ECM stiffening. Collectively, these findings not only give the spectral signatures for mechanoresponse but also provide the landscape of biochemical changes in response to ECM stiffening.

Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics.

BACKGROUND: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown. PURPOSE: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. STUDY DESIGN: Controlled laboratory study. METHODS: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed. RESULTS: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020). CONCLUSION: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. This raises concern for the risk of mechanical failure of the cartilage tissue after CHG exposure. CLINICAL RELEVANCE: Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Coexisting Th1 and Th2 cytokines in patients with collagenous gastritis and implications for its pathogenesis.

OBJECTIVES: Collagenous gastritis (CG) is a rare cause of refractory dyspepsia and anemia that frequently affects children and young adults and whose histological hallmark is chronic mucosal inflammation with a subepithelial collagen band. The etiology remains obscure, and no established treatments exist. We investigated the pathogenesis of CG by determining the expression profiles of genes related to immunity and inflammation in index biopsies. METHODS: Gastric biopsies from 10 newly diagnosed patients with CG were evaluated using the NanoString nCounter assay. Gastric biopsies from 14 normal individuals served as controls. The gene expression ratios for CG versus controls were determined in pooled samples and confirmed in individual samples by quantitative reverse transcription polymerase chain reaction. The results were compared with previously reported expression data from a cohort of patients with collagenous colitis, a colonic disorder with similar morphology, including subepithelial collagen band. RESULTS: CG biopsies featured enhanced expression of key genes encoding both Th1 (IFNγ, TNF-α, IL-2, IL-10, IL-12A, IL-12B, and IL-18) and Th2 cytokines (IL-3, IL-4, IL-5, IL-6, and IL-13). In contrast, biopsies from patients with CC exhibited upregulated Th1 cytokines only. CONCLUSIONS: We show in this first published gene expression profiling study that CG involves simultaneous upregulation of Th1 and Th2 cytokines. This finding is unique, contrasting with other types of chronic gastritis as well as with collagenous colitis, which shares the presence of a collagen band. Involvement of Th2 immunity in CG would support further investigation of potential dietary, environmental, or allergic factors to guide future therapeutic trials.