Search Results (89)

Background/Objectives: The aim of this research is to analyze the effect of paclitaxel on endoplasmic reticulum (ER) stress in spermatogenic cells. Methods: In the study, spermatogonium (GC1) and spermatocyte (GC2) cell lines were used. The IC50 dose of paclitaxel was calculated using an MTT assay. Each cell line was separated into two different groups: control (GC1-C, GC2-C) and paclitaxel-treated (GC1-P, GC2-P). The control cells were incubated under standard culture conditions. The paclitaxel group cells were incubated in culture medium containing the paclitaxel IC50 dose for 24 h. After the experiments, all groups were stained with GRP78, p-PERK, and p-eIF2α antibodies using semi-quantitative immunocytochemistry. Results: Paclitaxel showed cytotoxicity. In the experimental model of the paclitaxel-treated cells, all the markers showed elevated levels of immunoreactivity, indicating ER stress. Conclusions: Paclitaxel administration triggered ER stress in spermatogenic cells. Studies of ER-related stress mechanisms in spermatogenic cells with further advanced molecular analyses will be important for therapeutic strategies. Full article

Background: Unambiguous clinical interpretation of PCR results for urinary tract infections (UTIs) remains a challenge. Here we compare and correlate multiplex qPCR results (quantification cycle values) with traditional microbial culture results (colony forming units) for clinical samples. Methods: Serial dilutions [10⁸ to 10⁰ colony forming units (CFU)/mL] were performed on five Gram-negative and two Gram-positive UTI-causing bacterial pathogens. For each dilution, quantitative cultures on solid media to confirm CFU/mL values and a real-time PCR UTI panel employing a nanofluidic Open Array^TM platform producing quantification cycle (Cq) values were performed. Cq values were correlated with CFU/mL values, generating a semi-quantitative interpretive scale for clinical samples. The clinical utility of the scale was then assessed using PCR and culture data from 168 clinical urine samples. Results: For Gram-negative bacteria, Cq values of <23, 23 to 28, and >28 corresponded with ≥10⁵ CFU/mL, <10⁵ CFU/mL and negative cultures, respectively. For Gram-positive bacteria, Cq values of <26, 26 to 30, and >30 corresponded with ≥10⁵ CFU/mL, <10⁵ CFU/mL and negative cultures, respectively. Among 168 urine specimens (including 138 Gram-negative and 30 Gram-positive bacteria), there was 83.3% agreement (n = 140/168) and 16.6% non-agreement (n = 28/168) between culture CFU/mL and qPCR Cq. Gram-negative bacteria had higher agreement (87.6%, 121/138) than Gram-positive bacteria (63.3%, 19/30). Conclusions: This study demonstrates that qPCR Cq results can be directly correlated with traditional urine quantitative culture results and reliably identify the clinically relevant cutoff of 10⁵ CFU/mL for detected uropathogens. Full article

Conventional diagnostic methods (CDMs) for lower respiratory infections (LRIs) have limitations in detecting causative pathogens. This study evaluates the utility of shotgun metagenomic sequencing (SMS) as a complementary diagnostic tool using bronchoalveolar lavage (BAL) fluid. Sixteen BAL fluid samples from pneumonia patients with positive CDM results—including bacterial/fungal cultures; PCR for Mycobacterium tuberculosis or cytomegalovirus; and the BioFire^® FilmArray^® Pneumonia Panel (BioFire Diagnostics LLC, Salt Lake City, UT, USA)—underwent 10 Gb SMS on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Reads were aligned to the NCBI RefSeq database; with fungal identification further supported by internal transcribed spacer (ITS) analysis. Antibiotic resistance genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database. Microbial reads accounted for 0.00002–0.04971% per sample. SMS detected corresponding bacteria in 63% of cases, increasing to 69% when subdominant taxa were included. Fungal reads were low; however, Candida species were identified in four samples via ITS. No viral reads were detected. ARGs meeting perfect match criteria were found in two cases. This is the first real-world study comparing SMS with CDMs, including semiquantitative PCR, in BAL fluid for LRI. SMS shows promise as a supplementary diagnostic method, with further research needed to optimize its performance and cost-effectiveness. Full article

Extracellular vesicles (EVs) have emerged as promising biomarkers and therapeutic agents, yet their quantification remains technically challenging due to the limitations of conventional methods. Here, a low-cost, fluorescence-based, paper-strip immunoassay is presented for rapid and semi-quantitative estimation of EV concentration, inspired by pH strips. The assay utilizes nitrocellulose membranes functionalized with capture antibodies (anti-CD63, CD9, CD81) and fluorescent dye (ExoBrite™) for EV detection. Systematic optimization of assay parameters—including dye application sequence, incubation time, antibody configuration, and dye concentration—revealed that labeling EVs with dye and incubating on the nitrocellulose paper strips for 20 min yielded the strongest and most reproducible signal. A 200× dilution of ExoBrite™ dye was determined to provide the best balance between sensitivity and specificity. A standard curve generated through twofold serial dilution of EVs from ovarian cancer cell culture medium confirmed a positive, concentration-dependent fluorescence response, establishing a usable dynamic range. Compared to existing technologies, this platform enables fast, simple-to-implement EV quantification using minimal sample volume and equipment. The simplicity and scalability of the method offer strong potential for use in clinical diagnostics and EV research applications. Full article

Folate deficiency is a widespread nutritional issue, and biofortifying dairy products through lactic acid bacteria (LAB) is a promising strategy to enhance natural folate levels. This study aimed to develop a reliable method for selecting Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains with enhanced folate production for use as functional starter cultures. Initially, a traditional microbiological assay (MA) was used to measure folate production in 36 LAB strains isolated from fermented milks. Due to MA’s limitations, an untargeted and semi-quantitative method combining ultra-high-performance liquid chromatography (UHPLC) with high-resolution mass spectrometry (HRMS) was developed for a more comprehensive folate screening. The MA showed higher folate production in S. thermophilus strains (309–639 µg/L) compared to L. delbrueckii subsp. bulgaricus (up to 48 µg/L). Subsequently, nine selected LAB strains were further analyzed using the UHPLC-HRMS approach, which enabled the identification and semi-quantification of six folate metabolites, namely dihydrofolate, tetrahydrofolate (THF), 10-formyl-THF, 5,10-methenyl-THF, 5,10-methylene-THF, and 5-methyl-THF. Lab-scale yogurt production using the top-performing strains, as identified through the HRMS method, demonstrated an increase in folate content over a 14-day shelf life. These findings revealed the potential of UHPLC-HRMS as a high-throughput alternative method for folates detection, offering a promising tool for screening folate-enhanced LAB strains for biofortification. Full article

Bacterial vaginosis (BV) is associated with various adverse pregnancy outcomes. It is usually diagnosed via microscopy. Semi-quantitative relative culture (SRC) was investigated as a complementary diagnostic method to determine the prevalence of BV and its association with preterm birth and preterm, premature rupture of membranes (PPROM) in pregnant women in Slovenia. We examined 3437 consecutive vaginal swabs from pregnant women during the five-year period and were able to link the results to 2531 pregnancy outcomes. The isolates were identified using MALDI-TOF, and the results were assessed by the relative amounts of Gardnerella vaginalis and lactobacilli according to two stringency criteria. The prevalence of BV was 6.5% via microscopy and was higher for SRC, 9.9% or 11.1%, depending on the stringency criteria. The association with adverse pregnancy outcomes was better when SRC was used, resulting in adjusted odds ratios of 1.76 (1.30 to 2.37) and 1.97 (1.38 to 2.82) for preterm birth and PPROM, respectively, with more stringent interpretation. Microscopically detected BV was not associated with either outcome. The clinical validity of SRC was demonstrated by its better correlation with adverse pregnancy outcomes in a large cohort of pregnant women. SRC with MALDI-TOF identification is a promising advancement of vaginal culture. Full article

Background/Objectives: Potency testing of clinical-grade lentiviral vectors (LVVs) is critical to support a drug’s commercial approval. Careful consideration should be paid to the development of a suitable potency test during the drug’s clinical development. We aimed to develop an affordable, quantitative test for our CAR19-LVV, based on a measure of transgene’s functional activity. Methods: Several indicators of functional activity of CAR19-LVV were explored in a co-culture setting of CAR-transduced Jurkat cells and CD19-expressing target cells. The selected assay was further developed and subjected to validation. Assay’s adaptability to other CAR-encoding LVV and autologous CAR-T cell products was also investigated. Results: Measure of CD69 expression on the membrane of Jurkat-CAR-expressing cells is a specific indicator of CAR functionality. Quantification of CD69 in terms of mean fluorescence intensity (MFI), coupled with an intra-assay standard curve calibration, allows for a quantitative assay with high precision, specificity, robustness, linearity and accuracy. The assay has also shown optimal performance for a CARBCMA-LVV product. Importantly, we show that in primary T cells, CD69 expression reflects CAR-T cell cytotoxicity. After adaptation, we have applied a CD69-based potency test, with simultaneous measurement of CAR-T cell cytotoxicity, to autologous CAR-T cell products, demonstrating the assay’s specificity also in this context. Conclusions: We developed a validated, in vitro cell-based potency test, using a quantitative flow-cytometry method, for our CAR19-LVV. The assay is based on the detection of T-cell activation upon CAR binding to antigen, which is a measure of transgene functionality. The assay was easily adapted to another CAR-encoding LVV, targeting a different molecule. Furthermore, the same assay principle can be applied in the context of autologous CAR-T cell products. The quantitative CD69 potency assay shows reduced variability among autologous products compared to the IFNγ assay and allows for simultaneous evaluation of traditional semi-quantitative cytotoxicity, thereby directly evaluating the drug’s mechanism of action (MoA) in the same assay. Full article

Background and Objectives: In previous studies, we reported that the assessment of the cumulative thermal dose in the crystalline lens, conducted through computational modeling utilizing a supercomputer and the biothermal transport equation, exhibited a significant association with the incidence of nuclear cataracts. In this study, we have investigated the types of proteins that expressed underlying 35.0 °C (normal-temp) and 37.5 °C (warming-temp) by using the shotgun liquid chromatography (LC) with tandem mass spectrometry (MS/MS)-based global proteomic approach. Materials and Methods: We have discussed the changes in protein expression in warmed iHLEC-NY2 cells using Gene Ontology analysis and a label-free semiquantitative method based on spectral counting. Results: In iHLEC-NY2, 615 proteins were detected, including 307 (49.9%) present in both lenses cultured at normal-temp and warming-temp, 130 (21.1%) unique to the lens cultured at normal-temp, and 178 (29.0%) unique to the lens cultured at warming-temp. Furthermore, LC–MS/MS analysis showed that warming decreased the expression of actin, alpha cardiac muscle 1, actin-related protein 2, putative tubulin-like protein alpha-4B, ubiquitin carboxyl-terminal hydrolase 17-like protein 1, ubiquitin-ribosomal protein eL40 fusion protein, ribosome biogenesis protein BMS1 homolog, histone H2B type 1-M, and histone H2A.J. in iHLEC-NY2. Conclusions: The decreases in the specific protein levels of actin, tubulin, ubiquitin, ribosomes, and histones may be related to cataract development under warming conditions. This investigation could provide a critical framework for understanding the correlation between temperature dynamics and the development of nuclear cataracts. Full article

Since 2012, the Joint Safety Team (JST) has led initiatives aimed at increasing safety awareness and improving safety culture in research laboratory environments at the University of Minnesota. Specifically, its Analysis and Compliance committee has implemented biannual lab safety walkthroughs and safety surveys to monitor safety culture, sentiments, and compliance. Despite several lab safety teams (LSTs) using similar initiatives, the logistics and effectiveness of these initiatives have yet to be reported. Hence, JST has analyzed the decade of overall lab safety walkthrough scores and scores of individual safety items in the context of the evolution of our rubrics and external factors. Similarly, the changes to the safety survey content have been analyzed to demonstrate the dynamic approach of gauging sentiments towards arising safety issues. Generally, the results suggest that these initiatives improved laboratory safety. Furthermore, safety compliance was observed to vary by semester, with fewer safety issues in the spring semester compared to the fall semester, and to be affected by the COVID-19 pandemic. These observations support the effectiveness of these student-led initiatives, despite the initiatives being designed for internal use. Additionally, the initiative logistics are described to inform LSTs of strategies for creating, sustaining, and improving their own initiatives. Full article

Spices and aromatic herbs are important components of everyday nutrition in several countries and cultures, thanks to their capability to enhance the flavor of many dishes and convey significant emotional contributions by themselves. Indeed, spices as well as aromatic herbs are to be considered not only for their important values of antimicrobial agents or flavor enhancers everybody knows, but also, thanks to their olfactory and gustatory spectrum, as drivers to stimulate the consumers’ memories and, in a stronger way, emotions. Considering these unique characteristics, spices and aromatic herbs have caught the attention of consumer scientists and experts in sensory analysis for their evaluation using semi-quantitative approaches, with interesting evidence. In this pilot study as a first step, each studied botanical, belonging to Piperaceae or aromatic herbs, has been subjected to headspace solid phase micro-extraction (HS-SPME) coupled with gas-chromatography mass spectrometry (GC-MS) analysis to assess their spontaneous volatile emission, representing the complex chemical pattern, which encounters the consumers’ olfactory perception. Furthermore, the present investigation, performed on 12 individuals, outlines the administration of a pilot study, merging the typical sensory analysis with emotional data collection and the innovative contribution related to the study around the Autonomic and Central Nervous System activation in consumers, performed using wearable technologies and related signal processing. The results obtained by our study, beyond demonstrating the feasibility of the approach, confirmed, both in terms of emotional responses and biomedical signals, the significant emotional potential of spices and aromatic herbs, most of which featuring an overall positive valence, yet with inter-subjects’ variations. Future investigations should aim to increase the number of volunteers evaluated with such an approach to draw more stable conclusions and attempting a customization of product preferences based on both implicit and explicit sensory responses. Full article

Despite various opportunities, urban development projects have been causing significant challenges to the preservation and conservation of historic cultural heritage. In particular, an increasing number of World Heritage (WH) properties are impacted by the direct and indirect effects of development projects, reflecting the existing uprising conflict. Grasping the challenge, Heritage Impact Assessment (HIA) has been developed as a proactive assessment tool to identify and predict potential impacts, mitigate the negative impacts on heritage values, and sustain the attributes conveying OUVs in WH assets. The growing demand for urban development and its potential impacts on cultural heritage properties underscores the necessity for the development of a targeted Heritage Impact Assessment methodology for urban development threats. To adequately address multiple impacts, this paper proposes a triangulation of a qualitative matrix for impact identification and a semi-quantitative indicator-based index for impact analysis and evaluation. The methodology is applied to the World Heritage property of Masjed-e Jame of Isfahan in Iran. In drawing upon this example, a systematic and integrated impact assessment procedure is developed to capture a broad category of potential impacts and their significance that is crucial for determining site-specific mitigation strategies and informed decision-making within the context of heritage management and sustainable urban development. Full article

Background: The aim of our study was to examine the combined effects of tranexamic acid (TXA) and vancomycin powder (VP) on chondrocytes in vitro. Despite the use of TXA and VP being linked to a reduced risk of extensive postoperative blood loss and periprosthetic joint infections (PJIs) in TKA, the possible cytotoxic side effects on periarticular cell types remain unclear. Methods: Human chondrocytes were harvested from hyaline cartilage and expanded in monolayer culture before being simultaneously exposed to different concentrations of TXA and VP for varying exposure times. Cell viability and proliferation were assessed using an ATP assay and an Annexin 5 assay, respectively, while changes in the relative expression of chondrogenic marker genes were examined using semiquantitative RT-PCR. Results: The simultaneous exposure of chondrocytes to TXA and VP for more than 48 h led to a reduction in both cell viability and proliferation rates. When exposing chondrocytes to the lowest examined concentrations of both TXA (10 mg/mL) and VP (3 mg/mL), the observed effects were delayed until 96 h. However, our study found no dependencies of the observed effects on the concentrations tested. Further, we found no effects on the expression of chondrogenic marker genes. Conclusions: Consequently, limiting the exposure time of chondrocytes to TXA and VP in an in vitro setting to 24 h may be considered safe and could help to further improve the understanding of the safe use of substances in vivo. However, further in vitro research is required to develop a comprehensive understanding of the effects of both VP and TXA on important periarticular cell types in TKA, including chondrocytes, osteocytes, and tenocytes. Full article

Geomorphosite inventory remains an urgent task for many geographical domains where it can facilitate the exploration of geoheritage resources. The present study reports a new and sufficient portion of information from Mountainous Adygeya in southwestern Russia, which is a geoheritage-rich area in the western part of the Greater Caucasus Mountains. The Cossack, Monk, and Three Teeth mountains were analyzed qualitatively and semi-quantitatively. Their morphology and geological settings were described, their geoheritage properties were scored by several criteria, and their cultural meanings were considered. It was established that the total heritage values of these geomorphosites are comparable. The challenging accessibility of these landforms is offset by the availability of sites for distant observation. Each proposed geomorphosite has its own cultural meaning reflected by its name. The proposed geomorphosites are constituents of the geoheritage resources of Mountainous Adygeya, and they can be exploited via local product branding and georoute functioning. Full article

The production of polyhydroxyalkanoates using submerged cultures of Cupriavidus necator DSM 428 was evaluated using low-cost substrates from agroindustry: (i) dextrose from cassava starch and (ii) a mixture of sugarcane vinasse from the bioethanol industry and dextrose from cassava starch. The effects of vinasse composition (2.5, 5.0, 7.5, 25, 50, and 75% v/v) and the use of raw and activated carbon-pre-treated vinasse were assessed. The results indicate that cultivations using only cassava starch dextrose reached 4.33 g/L of biomass as the dry cell weight and a poly(3-hydroxybutyrate) (PHB) production of 47.1%. Raw vinasse proportions of 25, 50, and 75% in the culture medium resulted in total inhibition. Vinasse treated at the same ratios led to biomass production in the range 1.7–4.44 g/L. The higher PHB production scenario was obtained in a medium containing dextrose and treated vinasse (7.5%), yielding 5.9 g/L of biomass and 51% of PHB accumulation. The produced PHB was characterized by XRD and FTIR for an analysis of crystalline structure and chemical functional groups, respectively. EDS was employed for a semi-quantitative analysis of the chemical composition, and SEM was used to analyze the morphology of the microgranules. The results of DSC and TGA analyses demonstrated the thermal stability of the obtained PHB. Full article

Trade-offs in ecosystem services (ESs) manifest when the enhancement of one service leads to the diminishment of another. These trade-offs pose a notable challenge, impacting the sustainability of particular socioecological system peri-urban landscapes (PULs). This issue arises from the dynamic processes associated with peri-urbanization, which threaten natural ecosystems and their services in peri-urban areas. Additionally, the escalating demand for ecosystem services in PULs contributes to these trade-offs. Policymaking and planning concerning ES trade-offs in PULs should prioritize promoting a balance between conflicting services and fostering synergies among them. However, it is noteworthy that ES trade-offs in PULs are not given high priority in policy and planning agendas. Knowledge regarding policy development and planning for ES trade-offs in PULs often remains concealed within specific country and regional case studies. Consequently, this research seeks to characterize the ES trade-offs in selected PUL case studies, with the objective of identifying potential commonalities among them. Furthermore, this study aims to identify (i) the factors driving ES trade-offs, (ii) challenges related to how policymaking and planning address ES trade-offs in PULs, and (iii) recommendations for enhancing governance practices to better manage peri-urban ES trade-offs. We designed a semi-quantitative survey and collected information about 24 case studies located across the world. The answers from this survey were analyzed using principal component analysis. The results showed that the most common trade-offs occurred between “cultural and provisioning” and “regulating and provisioning” ESs. It was found that urban development is the primary driver behind the emergence of the examined trade-offs. To address this issue at the governance level, this study recommends establishing mechanisms to facilitate collaboration among stakeholders. This should be accompanied by robust dissemination efforts and the promotion of awareness among actors regarding the fundamental concepts of ESs and PULs. Full article