One Carbon to Rule Them All: Formaldehyde is a One-Carbon Signal Connecting One-Carbon Metabolism and Epigenetic Methylation.

Formaldehyde is commonly thought of as an environmental toxin or laboratory fixation reagent, but there is a growing appreciation for its broader physiological contributions as a naturally generated one-carbon metabolite across all kingdoms of life. In this In Focus article, we summarize emerging advances in the field that show how formaldehyde plays diverse roles as a one-carbon signal in DNA damage, one-carbon metabolism, and epigenetic regulation.

In vivo imaging of cathepsin B in activated glia in the brain after orofacial formalin test.

PURPOSE Cathepsin B (Cat B) is a cysteine lysosomal protease that is upregulated in many inflammatory diseases and widely expressed in the brain. Here, we used a Cat B activatable near-infrared (NIR) imaging probe to measure glial activation in vivo in the formalin test, a standard orofacial inflammatory pain model. The probe's efficacy was quantified with immunohistochemical analysis of the somatosensory cortex. PROCEDURES Three different concentrations of Cat B imaging probe (30, 50, 100 pmol/200 g bodyweight) were injected intracisternally into the foramen magnum of rats under anesthesia. Four hours later formalin (1.5%, 50 µl) was injected into the upper lip and the animal's behaviors recorded for 45 min. Subsequently, animals were repeatedly scanned using the IVIS Spectrum (8, 10, and 28 h post imaging probe injection) to measure extracellular Cat B activity. Aldehyde fixed brain sections were immunostained with antibodies against microglial marker Iba1 or astrocytic GFAP and detected with fluorescently labeled secondary antibodies to quantify co-localization with the fluorescent probe. RESULTS The Cat B imaging probe only slightly altered the formalin test results. Nocifensive behavior was only reduced in phase 1 in the 100 pmol group. In vivo measured fluorescence efficiency was highest in the 100 pmol group 28 h post imaging probe injection. Post-mortem immunohistochemical analysis of the somatosensory cortex detected the greatest amount of NIR fluorescence localized on microglia and astrocytes in the 100 pmol imaging probe group. Sensory neuron neuropeptide and cell injury marker expression in ipsilateral trigeminal ganglia was not altered by the presence of fluorescent probe. CONCLUSIONS These data demonstrate a concentration- and time-dependent visualization of extracellular Cat B in activated glia in the formalin test using a NIR imaging probe. Intracisternal injections are well suited for extracellular CNS proteinase detection in conditions when the blood-brain barrier is intact.

Deciphering the molecular mechanism and regulation of formaldehyde detoxification in Mycobacterium smegmatis.

The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.

Exquisite exposure: Formaldehyde as a metabolic regulator.

Throughout life, whether through external consumption or internal production, we are exposed to different reactive metabolites considered toxic to the body. Pham et al.1 uncover metabolic regulation by one such harmful metabolite: formaldehyde.

Role and mechanism of miR-871-3p/Megf8 in regulating formaldehyde-induced cardiomyocyte inflammation and congenital heart disease.

OBJECTIVE AND DESIGN: We aimed to investigate the molecular mechanism underlying formaldehyde (FA)-induced congenital heart disease (CHD) using in vitro and in vivo models. MATERIALS AND SUBJECTS: Neonatal rat heart tissues and H9C2 cells were used for in vitro studies, while FA-exposed new-born rats were used for in vivo studies. TREATMENT: H9C2 cells were exposed to FA concentrations of 0, 50, 100 and 150 µM/mL for 24 h. METHODS: Whole transcriptome gene sequencing identified differentially expressed miRNAs in neonatal rat heart tissues, while Real-time quantitative PCR (RT-qPCR) assessed miR-871-3p and Megf8 expression. RNA pull-down and dual-luciferase reporter assays determined miR-871-3p and Megf8 relationships. Inflammatory cytokine expression was assessed by western blotting. A FA-induced CHD model was used to validate miR-871-3p regulatory effects in vivo. RESULTS: We identified 89 differentially expressed miRNAs, with 28 up-regulated and 61 down-regulated (fold change ≥ 2.0, P < 0.05). Inflammation (interleukin) and signalling pathways were found to control FA-induced cardiac dysplasia. miR-871-3p was upregulated in FA-exposed heart tissues, modulated inflammation, and directly targeted Megf8. In vivo experiments showed miR-871-3p knockdown inhibited FA-induced inflammation and CHD. CONCLUSION: We demonstrated miR-871-3p's role in FA-induced CHD by targeting Megf8, providing potential targets for CHD intervention and improved diagnosis and treatment strategies.

Biodegradation enhancement of high concentrations formaldehyde waste gas and verification of the metabolic mechanism.

The enhanced effects of formaldehyde biodegradation in a biofilm packing tower are investigated in this study. Three experimental groups were established: a blank control group, a biochar addition group, and a lanthanum addition group. The inlet gas flow rate, the inlet gas concentration, and the structural succession characteristics of the microbial community in the tower were investigated by regular sampling. The intracellular metabolites and key enzymes of the dominant functional bacteria, Pseudomonas P1 and Methylobacterium Q1, in the tower were analyzed. The results indicated that with the biochar addition, the formaldehyde purification efficiency increased significantly from 91.67-94.67 % to 94.12 96.85 %, and the bio-elimination capacity increased with an increase in the inlet gas flow rate from 2.314 to 13.988 mg L-1h-1 to 2.697-15.051 mg L-1h-1. With the addition of lanthanum, the purification efficiency increased significantly from 90.80-93.98 % to 94.36-96.78 %, and the bio-elimination capacity increased with an increase in the inlet gas concentration from 1.099-11.284 mg L-1h-1 to 1.266-11.961 mg L-1h-1. The microbial community structure in the tower changed with system operation, and the formaldehyde degrading functional bacteria formed the dominant bacteria. It was verified that P1 and Q1 metabolized high concentrations of formaldehyde by the serine cycle and the ribulose monophosphate (RuMP) cycle.

Obtainment and Inoculation of Acinetobacter pittii Strain JJ-2, and Combined Action with Plants for Formaldehyde and CO2 Removal: A Research Study.

A formaldehyde-degrading bacterium JJ-2 was isolated from the rhizosphere of Chlorophytum and identified as Acinetobacter pittii by colony morphology and 16S rDNA sequence analysis. Further studies showed that under optimal conditions, JJ-2 could maintain activity for six cycles at an initial formaldehyde concentration of 450 mg L-1. At the same time, the complete degradation time was shortened from 12 to 6 h. When the JJ-2 strain was inoculated into sterile soil, the surface spray method had the best effect, and the removal efficiency of 5 ppm formaldehyde increased by 22.63%. In an actual potted plants system colonized with strain JJ-2, the first and second fumigations (without re-inoculation) increased removal by 1.36 times and 0.92 times during the day and 1.27 times and 2.07 times at night. In addition, in the second fumigation, the plant-bacteria combined system was 693.63 ppm and the plant system was 715.34 ppm, effectively reducing the CO2 concentration. This study provides an economical, ecological, and efficient approach to improve the combined system of plants and bacteria to remove gaseous formaldehyde from indoor air, with a positive impact on carbon neutrality.

Engineering a synthetic energy-efficient formaldehyde assimilation cycle in Escherichia coli.

One-carbon (C1) substrates, such as methanol or formate, are attractive feedstocks for circular bioeconomy. These substrates are typically converted into formaldehyde, serving as the entry point into metabolism. Here, we design an erythrulose monophosphate (EuMP) cycle for formaldehyde assimilation, leveraging a promiscuous dihydroxyacetone phosphate dependent aldolase as key enzyme. In silico modeling reveals that the cycle is highly energy-efficient, holding the potential for high bioproduct yields. Dissecting the EuMP into four modules, we use a stepwise strategy to demonstrate in vivo feasibility of the modules in E. coli sensor strains with sarcosine as formaldehyde source. From adaptive laboratory evolution for module integration, we identify key mutations enabling the accommodation of the EuMP reactions with endogenous metabolism. Overall, our study demonstrates the proof-of-concept for a highly efficient, new-to-nature formaldehyde assimilation pathway, opening a way for the development of a methylotrophic platform for a C1-fueled bioeconomy in the future.

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism.

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

N-acetylcysteine modulates redox imbalance and inflammation in macrophages and mice exposed to formaldehyde.

This study aimed to evaluate the protective role of N-acetylcysteine (NAC) in cells and mice exposed to formaldehyde. For the in vitro study, J774A.1 macrophages cells were incubated for 8, 16 and 24 h with formaldehyde or NAC to assess cell viability and reactive oxygen species (ROS). In the in vivo study, C57BL/6 mice (n = 48) were divided into 6 groups: control (CG), vehicle (VG) that received saline by orogastric gavage, a group exposed to formaldehyde 1% (FG) and formaldehyde exposed groups that received NAC at doses of 100, 150 and 200 mg/Kg (FN100, FN150 and FN200) for a period of 5 days. In vitro, formaldehyde promoted a decrease in cell viability and increased ROS, while NAC reduced formaldehyde-induced ROS production. Animals exposed to formaldehyde presented higher leukocyte counts in the blood and in the bronchoalveolar lavage fluid, and promoted secretion of inflammatory markers IL-6, IL-15, and IL-10. The exposure to formaldehyde also promoted redox imbalance and oxidative damage characterized by increased activities of superoxide dismutase, catalase, decreased GSH/GSSG ratio, as well as it increased levels of protein carbonyls and lipid peroxidation. NAC administration after formaldehyde exposure attenuated oxidative stress markers, secretion of inflammatory mediators and lung inflammation. In conclusion, both in in vitro and in vivo models, NAC administration exerted protective effects, which modulated the inflammatory response and redox imbalance, thus preventing the development airway injury induced by formaldehyde exposure.

Feed intake and milk yield and composition of lactating dairy goats in response to partial substitution of soybean meal for formaldehyde-treated sesame meal in the diet.

This study was conducted to evaluate the effect of substitution of soybean meal (SBM) for formaldehyde-treated sesame meal (FTSM) on nutrient intake and digestibility, ruminal and blood parameters and milk production and composition in lactating Murciano-Granadina goats. Forty lactating goats were randomly assigned to one of the following four treatments: (1) diet with 16.5% CP, containing SBM (CON); (2) diet with 16.5% CP, containing untreated SM (USM); (3) diet with 16.5% CP, containing FTSM (FT); and (4) diet with 14.5% CP containing FTSM (LPFT). The results showed that nutrient intake was highest in the FT group (p < 0.001), while it was similar between the CON and LPFT groups, except for the intake of CP, which was higher in the CON group. The FT and LPFT had lower ruminal pH compared to CON and USM groups (p < 0.001), with goats in group FT having the highest volatile fatty acids (VFA) production (p < 0.001). The highest propionate concentration was observed in the LPFT treatment (p < 0.001), followed by the FT, CON, and USM treatments. Goats offered USM and LPFT treatments presented the highest and lowest acetate: propionate values, respectively, among the experimental groups (p < 0.001). The results also showed that LPFT goats had the lowest blood urea nitrogen (BUN) level (p = 0.004), while FT goats presented a lower non-esterified FA (NEFA) level compared with CON and LPFT goats (p = 0.01). Goats offered the FT diet had the highest milk yield (p = 0.002) and energy-corrected milk yield (p < 0.001) among all dietary groups. The highest milk fat (p < 0.001), protein (p = 0.001), lactose (p = 0.007), total solids (p = 0.003), and solids-not-fat (SNF) (p = 0.003) contents were observed in FT goats, which didn't differ from USM goats. The inclusion of formaldehyde-treated SM increased the percentage of C18:3 (p < 0.001) and C20:1 (p = 0.04) FAs compared with USM and CON treatments. Milk from USM, FT, and LPFT goats had lower levels of saturated (p < 0.001) and medium-chain FAs (p = 0.014) compared with CON goats, whereas milk from CON goats had lower levels of unsaturated, monounsaturated, and long-chain FAs compared to other groups (p < 0.001). The lowest and the highest concentrations of polyunsaturated FAs were observed in CON and LPFT goats, respectively (p = 0.001). It can be concluded that SBM can be advantageously replaced by formaldehyde-treated SM in the diet as a feasible alternative to improve feed intake and production performance of dairy goats.

A novel approach to diagnosing crystal-storing histiocytosis: utility of scanning electron microscopy for formalin-fixed paraffin-embedded tissue specimens.

Crystal-storing histiocytosis (CSH) is a rare disorder that shows infiltration of histiocytes with an aberrant cytoplasmic accumulation of crystalline structures and is often accompanied by lymphoproliferative-plasma cell disorders (LP-PCD) as background diseases. The diagnosis of CSH requires identification of crystalline structures that accumulate in the infiltrating histiocytes, which may be challenging by optical microscopy alone. In this case report, we describe an atypical course of systemic CSH with multifocal fibrosclerosis of an unknown background disease that was diagnosed by ultrastructural observation, including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), in pathological autopsy. In addition, crystalline structures were successfully identified by scanning electron microscopic observations using formalin-fixed and paraffin-embedded (FFPE) tissue from biopsy specimens taken before death. Since CSH was identified by SEM in a tiny biopsy specimen, observation of histiocytic infiltrative lesions by SEM using FFPE tissue may lead to early detection of and initiation of treatment for CSH.

In Situ Proximity Ligation Assay to Visualize Protein-Protein Interactions in Tumor Specimens.

Protein-protein interactions (PPI) are the basis of various biological phenomena, such as intracellular signal transduction, gene transcription, and metabolism. PPI are also considered to be involved in the pathogenesis and development of various diseases, including cancer. PPI phenomenon and their functions have been elucidated by gene transfection and molecular detection technologies. On the other hand, in histopathological analysis, although immunohistochemical analyses provide information pertaining to protein expression and their localization in pathophysiological tissues, it has been difficult to visualize the PPI of these proteins. An in situ proximity ligation assay (PLA) was developed as a microscopic visualization technique for PPI in formalin-fixed, paraffin-embedded (FFPE) tissues as well as in cultured cells and frozen tissues. PLA using histopathological specimens enables cohort studies of PPI, which can clarify the significance of PPI in pathology. We have previously shown the dimerization pattern of estrogen receptors and significance of HER2-binding proteins using breast cancer FFPE tissues. In this chapter, we describe a methodology for the visualization of PPI using PLA in pathological specimens.

Simultaneous influence of light and CO2 on phytoremediation performance and physiological response of plants to formaldehyde.

Phytoremediation technology is an effective method to remove formaldehyde indoors, but the purification capacity and physiological response of plants to formaldehyde under the simultaneous influence of light and CO2 have not been examined in previous studies. In this study, formaldehyde fumigation experiments were conducted on the C3 plants Epipremnum aureum A. and Chlorophytum comosum L., and the crassulacean acid metabolism (CAM) plant Dieffenbachia maculate A. The phytoremediation performance and physiological response of plants were studied. The initial concentration of formaldehyde was established at 11.950 ± 1.442 [Formula: see text]; the light intensities were 448 ± 7 [Formula: see text], 1628 ± 22 [Formula: see text], and 3259 ± 22 [Formula: see text], respectively; and the concentrations of CO2 were 455 ± 29 [Formula: see text], 978 ± 50 [Formula: see text], 2020 ± 66 [Formula: see text], and 3006 ± 95 [Formula: see text], respectively. The results indicated that the highest purification rates of formaldehyde by E. aureum, D. maculata, and C. comosum were 55.8%, 43.7%, and 53.2%, respectively. The light intensity had a positive effect on the formaldehyde purification rates of all three plants and positively stimulated peroxidase (POD) activity, while the CO2 concentration had no significant impact on the formaldehyde purification capacity and plants' physiological characteristics. Exposure to formaldehyde inhibited formaldehyde dehydrogenase (FADH) activity and positively stimulated catalase (CAT) activity. The superoxide dismutase (SOD) activity positively correlated with the formaldehyde purification capacity of plants.

Regulatory mechanism of formaldehyde release in heme degradation catalyzed by Staphylococcus aureus IsdG.

IsdG-type enzymes catalyze the noncanonical degradation of heme to iron, staphylobilin (SB), and formaldehyde (HCHO), presumably by binding heme in an unusually distorted conformation. Their unique mechanism has been elucidated for MhuD from Mycobacterium tuberculosis, revealing an unusual ring opening of hydroxyheme by dioxygenation. A similar mechanism has been postulated for other IsdG enzymes; however, MhuD, which is special as an IsdG-type enzyme, retains a formyl group in the linearized tetrapyrrole. Recent reports on Staphylococcus aureus IsdG have suggested the formation of SB retaining a formyl group (formyl-SB), but its identification is preliminary. Furthermore, the reaction properties of formyl-SB and the mechanism of HCHO release remain unclear. In this study, the complex reaction of S. aureus IsdG was reexamined to elucidate its mechanism, including the identification of reaction products and their control mechanisms. Depending on the reaction conditions, IsdG produced both SB and formyl-SB as the main product, the latter of which was isolated and characterized by MS and NMR measurements. The formyl-SB product was generated upon the reaction between hydroxyheme-IsdG and O2 without reduction, indicating the dioxygenation mechanism as found for MhuD. Under reducing conditions, hydroxyheme-IsdG was converted also to SB and HCHO by activating another O2 molecule. These results provide the first overview of the complicated IsdG reaction. The heme distortion in the IsdG-type enzymes is shown to generally promote ring cleavage by dioxygenation. The presence or absence of HCHO release can be influenced by many factors, and the direct identification of S. aureus heme catabolites is of interest.

Immunohistochemical Detection of the Chaperone-Mediated Autophagy Markers LAMP2A and HSPA8 in Formalin-Fixed and Paraffin-Embedded Tissues.

Autophagy is crucial for maintaining cellular homeostasis and its deregulation is involved in disease development, including cancer. The key players of chaperone-mediated autophagy (CMA), a particular selective subtype of autophagy, are HSPA8 and LAMP2A. Both proteins can be immunohistochemically detected in formalin-fixed paraffin-embedded (FFPE) tissue. LAMP2A is frequently overexpressed in a variety of cancers where it likely supports cancer cell survival and resistance to anti-cancer therapies in a context-dependent manner. Here we present the immunohistochemical staining protocol of antibodies against LAMP2A and HSPA8, using an automated staining system, suitable for routine diagnostics. Additionally, we also suggest a staining evaluation method.

The formaldehyde treated guar meal and prill fat, agro industrial by-products as dietary rumen protected protein and energy source: improves growth performance, feed conversion, nutrient utilization, microbial protein synthesis, blood metabolites and economic efficiency in growing dairy buffalo (Bubalus bubalis) calves.

The study investigated the effects of protein replacement with formaldehyde-treated guar meal (FTGM) and prill fat (PF) in the diet on performance of growing dairy buffalo calves. Thirty-two feedlots Surti breed dairy buffalo calves (age, 7.31 ± 0.34 months and body weight, 90.69 ± 6.19 kg) were assigned into four dietary treatments (n-8 calves/each): (1) control group, supplied basal diet as per ICAR (2013) nutrient requirements; (2) FTGM group, 30% crude protein (CP) requirement of concentrate mixture (dry matter basis (DMB)) replaced with FTGM in basal diet; (3) PF group, supplied basal diet + 100 g PF; and (4) FTGM + PF group, 30% CP requirement of concentrate mixture (DMB) replaced with FTGM in the basal diet + 100 g PF for 280 days. All the treatment diets were isonitrogenous. Growth performance was improved in FTGM + PF and FTGM groups. Apparent digestibility (%) of CP was increased in FTGM and FTGM + PF diet, while digestibility (%) of ether extract (EE) was increased in PF group. Serum total protein, albumen, urea nitrogen, and creatinine concentrations were higher in FTGM + PF and FTGM groups, whereas total cholesterol and triglycerides levels were greater in FTGM + PF and PF groups. Calculated methane emission had a discernible influence of treatment in FTGM and FTGM + PF. The overall cost of feeding per kilogram gain was lowest in FTGM and FTGM + PF groups. In conclusion, 30% CP replacement with FTGM with or without PF improved the growth performance, feed conversion ratio, and nutrient utilization; supported efficient utilization of resources; and economized the rearing of growing dairy buffalo calves.

Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor ß1 (TGFß) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors. ß-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFß signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in ß-catenin-positive cells. In vitro, activation of Wnt-ß-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of ß-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFß increased ß-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFß up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a ß-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFß signalling pathways in CD intestinal fibrosis.

Assessing immuno-expression of p53 protein and TP 53 gene amplification in histologically negative surgical margins of oral squamous cell carcinoma patients and normal oral mucosa.

OBJECTIVE: To assess and compare the immuno-expression of p53 and TP 53 gene amplification and correlate local recurrence and survival in histologically negative surgical margins of oral squamous cell carcinoma (OSCC) with normal oral mucosa. METHODS: Forty formalin-fixed paraffin-embedded tissue blocks of HNMs of OSCC and 40 normal oral mucosa samples were analyzed for p53 immunostaining and TP 53 gene amplification by PCR. RESULTS: Significantly, higher positivity was noted with p53 immuno-expression, TP53 gene amplification, and combined p53 and TP53 expression in the study group compared to the control group (C0.05). Most cases that were positive for p53 immuno-expression, TP 53 gene amplification, and combined p53 and TP53 expression showed local recurrence and poor survival. Kaplan-Meier survival analysis showed that subjects with TP53 and combined p53 and TP53 positivity had decreased survival rate than their negative counterparts. CONCLUSION: Detection of p53 in HNMs of OSCC can be used as a biomarker to identify patients at a higher risk of developing local recurrence and to predict survival. CLINICAL RELEVANCE: Combined p53 and TP 53 assessment may be more reliable for predicting LR to help clinicians and surgeons in treatment planning.

Formaldehyde exposure induces differentiation of regulatory T cells via the NFAT-mediated T cell receptor signalling pathway in Yucatan minipigs.

The use of minipigs (Sus scrofa) as a platform for toxicological and pharmacological research is well established. In the present study, we investigated the effect of formaldehyde (FA) exposure on helper T cell-mediated splenic immune responses in Yucatan minipigs. The minipigs were exposed to different inhaled concentrations of FA (0, 2.16, 4.62, or 10.48 mg/m3) for a period of 2 weeks. Immune responses elicited by exposure to FA were determined by assessing physiological parameters, mRNA expression, and cytokine production. Additionally, the distribution of helper T cells and regulatory T (Treg) cells and expression of NFAT families, which are well-known T cell receptor signalling proteins associated with regulatory T cell development, were evaluated. Exposure to FA suppressed the expression of genes associated with Th1 and Th2 cells in minipigs in a concentration-dependent manner. The subsequent production of cytokines also declined post-FA exposure. Furthermore, exposure to FA induced the differentiation of CD4+ Foxp3+ Treg cells with divergent expression levels of NFAT1 and NFAT2. These results indicated that exposure to FA increased the Treg cell population via the NFAT-mediated T cell receptor signalling pathway, leading to suppression of effector T cell activity with a decline in T cell-related cytokine production.