Search Results (36)

In colorectal cancer (CRC), cancer-associated fibroblasts (CAFs) and the fibrotic stroma generate form a dense stromal barrier that restricts the intratumoural exposure and spatial distribution of oxaliplatin. To enable local stromal remodelling of this pathological stromal compartment, we selected fibroblast activation protein (FAP) as a stromal target and co-assembled two amphiphilic conjugates, oncoFAP and retinoic acid (RA), into an FAP-directed RA nanoformulation termed LRA^FAP. LRA^FAP exhibited a uniform size distribution (107.1 ± 5.8 nm), remained stable for at least 7 d at 37 °C in PBS or serum-containing PBS, and showed accelerated esterase-responsive release. In a TGF-β-induced CAF-like model, LRA^FAP markedly suppressed the expression of CAF activation-associated markers, reducing Fap and Acta2 mRNA levels by approximately 70% and 60%, respectively. In vivo, LRA^FAP showed enhanced accumulation in CAF-enriched tumours and an increase in intratumoural oxaliplatin levels of approximately 2.5-fold relative to oxaliplatin alone. LRA^FAP also reduced collagen deposition and CAF activation markers, and enhanced the antitumour efficacy of oxaliplatin while maintaining good tolerability. Collectively, these findings indicate that LRA^FAP promotes local stromal remodelling and improves intratumoural oxaliplatin exposure, thereby enhancing the efficacy of oxaliplatin-based chemotherapy in CRC. Full article

Background/Objectives: Prodrug strategies are a vital aspect of drug development, with ester prodrugs particularly notable for modifying parent drug properties through ester functional groups to enhance oral absorption. However, ester prodrugs are prone to hydrolysis by water and enzymes, making stability in the gastrointestinal (GI) tract prior to absorption a key challenge. Few formulation strategies effectively address this degradation issue. We recently introduced binary lipid systems (BLS), comprising a lipid and a water-soluble surfactant only that form stable microemulsions. This study aimed to explore the application of BLS for enhancing the oral absorption of ester prodrugs by coating drug crystals with BLS in solid granules and study the impact of the compositions of BLS on oral absorption. Methods: Olmesartan medoxomil (OLM), a methyl ester prodrug of olmesartan (OL), was selected as a model drug. Various lipids were combined with TPGS to form BLS and used to prepare OLM solid granules containing OLM crystals. Results: Among the tested formulations, OLM MCM-TPGS granules significantly enhanced drug release and protected OLM from enzyme-mediated degradation in two-step dissolution studies with esterase. Pharmacokinetic and tissue distribution studies in rats confirmed that OLM MCM-TPGS granules improved oral absorption by 145% and increased tissue uptake compared to OLM powder. Conclusions: This approach overcomes solubility limitations when using lipids and surfactants as excipients, enabling high drug loading in solid dosage forms and expanding the utility of lipids and surfactants for water-insoluble drugs. This novel formulation strategy holds great potential for enhancing oral absorption of ester prodrugs, representing a significant advancement in formulation technologies and offering more effective and versatile drug delivery solutions. Full article

The small-spotted catshark and the smooth-hound are cartilaginous, carnivorous fish with similar depth ranges in their habitats. These two species are among the most abundant elasmobranchs in the Adriatic Sea and are frequently caught by local fishermen using longline fishing. Despite their ecological similarities, little is known about the physiological differences in their digestive processes. The study of enzymatic digestion in these ecologically relevant species helps to fill the knowledge gap in the understanding of nutrient processing in cartilaginous fish. Therefore, the aim of this study was to determine, measure and compare the enzymatic activity of alkaline phosphatase, acid phosphatase, non-specific esterase and aminopeptidase. Fish were caught in the central part of the Adriatic Sea between 2021 and 2023. A total of 60 adult individuals were analyzed, with samples taken from six parts of the digestive tract. Histochemical analysis of 1440 slides revealed clear differences in enzyme activity between the two species. In the small-spotted catshark, cellular protein degradation was most pronounced in esophagus, posterior stomach and rectum, whereas in the smooth-hound, it was concentrated in posterior stomach and spiral intestine. Cellular digestion of lipids in the small-spotted catshark appears to occur primarily in the stomach. The results of this study provide new insights into the distribution of cellular digestive enzymes in cartilaginous fish and emphasize the importance of studying the entire digestive tract as an integrated system rather than focusing on individual parts. This study fills an important knowledge gap and contributes to a deeper understanding of digestive physiology, which in turn has implications for species conservation and biological variability. Full article

Fungal communities in high plateau ecosystems remain understudied despite their crucial roles in soil ecosystems, and yeasts inhabiting extreme regions have potential for industrial and biotechnological applications. We studied the fungal diversity in soils across 14 Chilean Altiplano sites using amplicon-based metagenomics and isolation of yeasts to assess their growth under various conditions and hydrolytic enzyme secretion. Using the metagenomic approach, the Ascomycota and Basidiomycota phyla were found to be the most abundant (85% and 8%, respectively). Unclassified families and genera prevailed at six and ten sites, respectively. At the other sites, the most abundant families included Cladosporiaceae, Teratosphaeriaceae, and Sporormiaceae, and the genera Oleoguttula, Coniochaeta, and Peziza. Biodiversity indices did not correlate with the soil’s geographic origin, organic matter content, humidity, or pH. Most isolated yeasts belong to the Naganishia, Holtermanniella, and Vishniacozyma genera, growing at temperatures ranging from 4 °C to 26 °C. Most isolates could use glucose, sucrose, and maltose as carbon sources and exhibited amylase, esterase, pectinase, and protease activities at 30 °C and below. Our results indicate that the evaluated soil physicochemical parameters do not explain the fungal distribution in the Altiplano and highlight the region as a reservoir of unknown fungi, including yeasts with industrially relevant enzymes. Full article

Bacillus cereus is a spore-forming, Gram-positive bacterium that causes foodborne illness and dairy spoilage. This study analyzed B. cereus s.s. isolates from milking environments, raw milk, and dairy products to assess their genotypic and phenotypic traits. From 466 samples, 61 isolates were obtained: 27 from milking environments, 9 from dairy environments, 8 from raw milk, and 17 from dairy products. Genomic sequencing identified genes encoding proteolytic (BC5350, BC0666, BC2984, BC0598, BC5351, BC3383, BC2735), lipolytic (BC4862, BC2141, BC1027, BC4123, BC4345, BC5402, BC5401), and esterase (BC1954, BC4515, BC3413, BC3606) enzymes. Plate assays confirmed enzymatic activities. Proteolytic genes were more prevalent in environmental samples, followed by raw milk and dairy products. Lipolytic genes were most frequent in raw milk, followed by environmental samples and dairy products. Esterase genes were most common in dairy environments. These findings suggest that dairy processing influences the enzymatic profile of B. cereus s.s., potentially impacting food safety and quality in the dairy industry. Understanding the distribution of these genes may help develop strategies to mitigate spoilage and contamination risks in dairy products. Full article

Artificial lipids have become increasingly important in generating novel nanoenzymes and nanoparticles. Imidazole has been well established as a versatile catalyst in synthetic chemistry and through its related amino acid histidine in enzymes. By exploiting the transphosphatidylation reaction of phospholipase D, the choline headgroup of phosphatidyl choline was exchanged for the imidazole moiety containing histidinol. Here, we introduce a novel phosphatidylhistidinol (PtdHisOH) lipid and characterise it with respect to its catalytic abilities and its ability to modulate vesicle size. Our data reveal a zinc-dependent esterase activity that was strongest in vesicles and micelles, with slower catalytic rates being observed in flat lipid presentation systems and two-phase systems, indicating the importance of surface presentation and curvature effects on the catalytic activity of PtdHisOH. Such lipids offer the opportunity to impart de novo catalytic functionality to self-assembled lipid systems such as synthetic cells, leading to the development of new technologies for biocatalysis applications. Full article

In this study, we examined the gastrointestinal tract of the European barracuda (Sphyraena sphyraena) living in the Adriatic Sea near Dubrovnik, Croatia. The study aimed to identify the presence and distribution of four digestive enzymes: alkaline phosphatase, aminopeptidase, acid phosphatase, and non-specific esterase. We found that alkaline phosphatase activity was present in the brush border of the enterocytes in all the investigated intestinal segments. The activity of the alkaline phosphatase was the strongest in the pyloric caeca but strong only in the basal part of the intestinal villi in the anterior and middle intestinal segments. In the posterior intestinal segment, alkaline phosphatase had strong activity along the entire villi. The activity of acid phosphatase was weak in all the investigated parts of the intestine, except in the posterior part, where it was moderate. Aminopeptidase was detected in the brush border of the intestinal epithelium, with stronger activity in the pyloric caeca and anterior part of the intestine and weaker activity posteriorly. The activity of the non-specific esterase was moderate in the pyloric caeca and anterior part of the intestine, while it was weak in the posterior segment and the lamina propria in all parts of the digestive tract. Weak acid phosphatase activity was observed only in the lamina propria of the posterior part of the intestine. This study is the first to evaluate the activity of digestive enzymes in the European barracuda. Full article

The culturable yeast communities in temperate forest soils under the ornithogenic influence were studied in a seasonal dynamic. To investigate the intense ornithogenic influence, conventional and “live” feeders were used, which were attached to trees in the forest and constantly replenished throughout the year. It was found that the yeast abundance in the soil under strong ornithogenic influence reached the highest values in winter compared to the other seasons and amounted to 4.8 lg (cfu/g). This was almost an order of magnitude higher than the minimum value of yeast abundance in ornithogenic soils determined for summer. A total of 44 yeast species, 21 ascomycetes and 23 basidiomycetes, were detected in ornithogenic soil samples during the year. These included soil-related species (Barnettozyma californica, Cyberlindnera misumaiensis, Cutaneotrichosporon moniliiforme, Goffeauzyma gastrica, Holtermanniella festucosa, Leucosporidium creatinivorum, L. yakuticum, Naganishia adeliensis, N. albidosimilis, N. globosa, Tausonia pullulans, and Vanrija albida), eurybionts (yeast-like fungus Aureobasidium pullulans, Debaryomyces hansenii, and Rhodotorula mucilaginosa), inhabitants of plant substrates and litter (Cystofilobasidium capitatum, Cys. infirmominiatum, Cys. macerans, Filobasidium magnum, Hanseniaspora uvarum, Metschnikowia pulcherrima, and Rh. babjevae) as well as a group of pathogenic and opportunistic yeast species (Arxiozyma bovina, Candida albicans, C. parapsilosis, C. tropicalis, Clavispora lusitaniae, and Nakaseomyces glabratus). Under an ornithogenic influence, the diversity of soil yeasts was higher compared to the control, confirming the uneven distribution of yeasts in temperate forest soils and their dependence on natural hosts and vectors. Interestingly, the absolute dominant species in ornithogenic soils in winter (when the topsoil temperature was below zero) was the basidiomycetous psychrotolerant yeast T. pullulans. It is regularly observed in various soils in different geographical regions. Screening of the hydrolytic activity of 50 strains of this species at different temperatures (2, 4, 10, 15 and 20 °C) showed that the activity of esterases, lipases and proteases was significantly higher at the cultivation temperature. Ornithogenic soils could be a source for the relatively easy isolation of a large number of strains of the psychrotolerant yeast T. pullulans to test, study and optimize their potential for the production of cold-adapted enzymes for industry. Full article

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts. Full article

Bovine coronavirus (BCoV), bovine rotavirus, bovine viral diarrhea virus, and bovine astrovirus are the most common intestinal pathogenic viruses causing diarrhea in cattle. We collected 1646 bovine fecal samples from January 2020 to August 2023. BCoV was the major pathogen detected, with a positive rate of 34.02% (560/1646). Of the 670 diarrheal samples and 976 asymptomatic samples, 209 and 351 were BCoV-positive, respectively. Studying the relevance of diarrhea associated with BCoV has shown that the onset of diarrheal symptoms post-infection is strongly correlated with the cattle’s age and may also be related to the breed. We amplified and sequenced the hemagglutinin esterase (HE), spike protein, and whole genomes of the partially positive samples and obtained six complete HE sequences, seven complete spike sequences, and six whole genomes. Molecular characterization revealed that six strains were branched Chinese strains, Japanese strains, and partial American strains from the GⅡb subgroup. Strains HBSJZ2202 and JSYZ2209 had four amino acid insertions on HE. We also analyzed ORF1a and found disparities across various regions within GIIb, which were positioned on separate branches within the phylogenetic tree. This work provides data for further investigating the epidemiology of BCoV and for understanding and analyzing BCoV distribution and dynamics. Full article

Introduction: Hirschsprung disease (HD) manifests as a developmental anomaly affecting the enteric nervous system, where there is an absence of ganglion cells in the lower part of the intestine. This deficiency leads to functional blockages within the intestines. HD is usually confirmed or ruled out through rectal biopsy. The identification of any ganglion cells through hematoxylin and eosin (H&E) staining rules out HD. If ganglion cells are absent, further staining with acetylcholine-esterase (AChE) histochemistry or calretinin immunohistochemistry (IHC) forms part of the standard procedure for determining a diagnosis of HD. In 2017, our Institute of Pathology at University Hospital of Heidelberg changed our HD diagnostic procedure from AChE histochemistry to calretinin IHC. In this paper, we report the impact of the diagnostic procedure change on surgical HD therapy procedures and on the clinical outcome of HD patients. Methods: We conducted a retrospective review of the diagnostic procedures, clinical data, and postoperative progress of 29 patients who underwent surgical treatment for HD in the Department of Pediatric Surgery, University of Heidelberg, between 2012 and 2021. The patient sample was divided into two groups, each covering a treatment period of 5 years. In 2012–2016, HD diagnosis was performed exclusively using AChE histochemistry (AChE group, n = 17). In 2017–2021, HD diagnosis was performed exclusively using calretinin IHC (CR group, n = 12). Results: There were no significant differences between the groups in sex distribution, weeks of gestation, birth weight, length of the aganglionic segment, or associated congenital anomalies. Almost half of the children in the AChE group, twice as many as in the CR group, required an enterostomy before transanal endorectal pull-through procedure (TERPT). In the AChE group, 4 patients (23.5%) required repeat bowel sampling to confirm the diagnosis. Compared to the AChE group, more children in the CR group suffered from constipation post TERPT. Discussion: Elevated AChE expression is linked to hypertrophied extrinsic cholinergic nerve fibers in the aganglionic segment in the majority of patients with HD. The manifestation of increased AChE expression develops over time. Therefore, in neonatal patients with HD, especially those in the first 3 weeks of life, an increase in AChE reaction is not detected. Calretinin IHC reliably identifies the presence or absence of ganglion cells and offers multiple benefits over AChE histochemistry. These include the ability to perform the test on paraffin-embedded tissue sections, a straightforward staining pattern, a clear binary interpretation (negative or positive), cost-effectiveness, and utility regardless of patient age. Conclusions: The ability of calretinin IHC to diagnose HD early and time-independently prevented repeated intestinal biopsies in our patient population and allowed us to perform a one-stage TERPT in the first months of life, reducing the number of enterostomies and restoring colonic continuity early. Patients undergoing transanal pull-through under the age of 3 months require a close follow-up to detect cases with bowel movement problems. Full article

Spotted sea bass (Lateolabrax maculatus) is a high-economic-value aquacultural fish widely distributed in the coastal and estuarine areas of East Asia. In August 2020, a sudden outbreak of disease accompanied by significant mortality was documented in L. maculatus reared in marine cage cultures located in Nanhuang island, Yantai, China. Two coinfected bacterial strains, namely, NH-LM1 and NH-LM2, were isolated from the diseased L. maculatus for the first time. Through phylogenetic tree analysis, biochemical characterization, and genomic investigation, the isolated bacterial strains were identified as Vibrio harveyi and Photobacterium damselae subsp. piscicida, respectively. The genomic analysis revealed that V. harveyi possesses two circular chromosomes and six plasmids, while P. damselae subsp. piscicida possesses two circular chromosomes and two plasmids. Furthermore, pathogenic genes analysis identified 587 and 484 genes in V. harveyi and P. damselae subsp. piscicida, respectively. Additionally, drug-sensitivity testing demonstrated both V. harveyi and P. damselae subsp. piscicida exhibited sensitivity to chloramphenicol, ciprofloxacin, ofloxacin, orfloxacin, minocycline, doxycycline, tetracycline, and ceftriaxone. Moreover, antibiotic resistance genes were detected in the plasmids of both strains. Extracellular product (ECP) analysis demonstrated that both V. harveyi and P. damselae subsp. piscicida can produce hemolysin and amylase, while V. harveyi additionally can produce caseinase and esterase. Furthermore, infected fish displayed severe histopathological alterations, including infiltration of lymphocytes, cellular degeneration and necrosis, and loose aggregation of cells. Artificial infection assays determined that the LD₅₀ of P. damselae subsp. piscicida was 3 × 10⁵ CFU/g, while the LD₅₀ of V. harveyi was too low to be accurately evaluated. Furthermore, the dual infection of V. harveyi and P. damselae subsp. piscicida elicits a more rapid and pronounced mortality rate compared to single challenge, thereby potentially exacerbating the severity of the disease through synergistic effects. Ultimately, our findings offer compelling evidence for the occurrence of coinfections involving V. harveyi and P. damselae subsp. piscicida in L. maculatus, thereby contributing to the advancement of diagnostic and preventative measures for the associated disease. Full article

Influenza D virus (IDV) is the most recent addition to the Orthomyxoviridae family and cattle serve as the primary reservoir. IDV has been implicated in Bovine Respiratory Disease Complex (BRDC), and there is serological evidence of human infection of IDV. Evolutionary changes in the IDV genome have resulted in the expansion of genetic diversity and the emergence of multiple lineages that might expand the host tropism and potentially increase the pathogenicity to animals and humans. Therefore, there is an urgent need for automated, accurate and rapid typing tools for IDV lineage typing. Currently, IDV lineage typing is carried out using BLAST-based searches and alignment-based molecular phylogeny of the hemagglutinin-esterase fusion (HEF) gene sequences, and lineage is assigned to query sequences based on sequence similarity (BLAST search) and proximity to the reference lineages in the tree topology, respectively. To minimize human intervention and lineage typing time, we developed IDV Typer server, implementing alignment-free method based on return time distribution (RTD) of k-mers. Lineages are assigned using HEF gene sequences. The server performs with 100% sensitivity and specificity. The IDV Typer server is the first application of an RTD-based alignment-free method for typing animal viruses. Full article

The analysis of the secretome allows us to identify the proteins, especially carbohydrate-active enzymes (CAZymes), secreted by different microorganisms cultivated under different conditions. The CAZymes are divided into five classes containing different protein families. Thermothelomyces thermophilus is a thermophilic ascomycete, a source of many glycoside hydrolases and oxidative enzymes that aid in the breakdown of lignocellulosic materials. The secretome analysis of T. thermophilus LMBC 162 cultivated with submerged fermentation using tamarind seeds as a carbon source revealed 79 proteins distributed between the five diverse classes of CAZymes: 5.55% auxiliary activity (AAs); 2.58% carbohydrate esterases (CEs); 20.58% polysaccharide lyases (PLs); and 71.29% glycoside hydrolases (GHs). In the identified GH families, 54.97% are cellulolytic, 16.27% are hemicellulolytic, and 0.05 are classified as other. Furthermore, 48.74% of CAZymes have carbohydrate-binding modules (CBMs). Observing the relative abundance, it is possible to state that only thirteen proteins comprise 92.19% of the identified proteins secreted and are probably the main proteins responsible for the efficient degradation of the bulk of the biomass: cellulose, hemicellulose, and pectin. Full article

The presence of esterase enzymes in human skin and their role in drug metabolism has been reported, but their distribution in the various skin layers and the relative contributions of those layers to metabolism is poorly defined. To gain further insight into esterase distribution, we performed in vitro skin permeation of a commercial 28.3% methyl salicylate (MeSA) cream (Metsal™) in Franz diffusion cells, using a range of human skin membranes, all from the same donor. The membranes were viable epidermis separated by a dispase II enzymatic method, heat separated epidermis, dermatomed skin, and dermis separated by a dispase II enzymatic method. Methyl salicylate and its metabolite, salicylic acid (SA), were measured by high-performance liquid chromatography. Alpha naphthyl acetate and Hematoxylin and Eosin staining provided qualitative estimations of esterase distribution in these membranes. The permeation of methyl salicylate after 24 h was similar across all membranes. Salicylic acid formation and permeation were found to be similar in dermatomed skin and dermis, suggesting dermal esterase activity. These results were supported by the staining studies, which showed strong esterase activity in the dermal–epidermal junction region of the dermis. In contrast with high staining of esterase activity in the stratum corneum and viable epidermis, minimal stained and functional esterase activity was found in heat-separated and dispase II-prepared epidermal membranes. The results are consistent with dispase II digesting hemidesmosomes, penetrating the epidermis, and affecting epidermal esterases but not those in the dermis. Accordingly, whilst the resulting dispase II-generated dermal membranes may be used for in vitro permeation tests (IVPT) involving esterase-based metabolic studies, the dispase II-generated epidermal membranes are not suitable for this purpose. Full article