Intestinal activating transcription factor 4 regulates stress-related behavioral alterations via paraventricular thalamus in male mice.

Chronic stress induces depression- and anxiety-related behaviors, which are common mental disorders accompanied not only by dysfunction of the brain but also of the intestine. Activating transcription factor 4 (ATF4) is a stress-induced gene, and we previously show that it is important for gut functions; however, the contribution of the intestinal ATF4 to stress-related behaviors is not known. Here, we show that chronic stress inhibits the expression of ATF4 in gut epithelial cells. ATF4 overexpression in the colon relieves stress-related behavioral alterations in male mice, as measured by open-field test, elevated plus-maze test, and tail suspension test, whereas intestine-specific ATF4 knockout induces stress-related behavioral alterations in male mice. Furthermore, glutamatergic neurons are inhibited in the paraventricular thalamus (PVT) of two strains of intestinal ATF4-deficient mice, and selective activation of these neurons alleviates stress-related behavioral alterations in intestinal ATF4-deficient mice. The highly expressed gut-secreted peptide trefoil factor 3 (TFF3) is chosen from RNA-Seq data from ATF4 deletion mice and demonstrated decreased in gut epithelial cells, which is directly regulated by ATF4. Injection of TFF3 reverses stress-related behaviors in ATF4 knockout mice, and the beneficial effects of TFF3 are blocked by inhibiting PVT glutamatergic neurons using DREADDs. In summary, this study demonstrates the function of ATF4 in the gut-brain regulation of stress-related behavioral alterations, via TFF3 modulating PVT neural activity. This research provides evidence of gut signals regulating stress-related behavioral alterations and identifies possible drug targets for the treatment of stress-related behavioral disorders.

Bridging upstream and downstream for improved adenovirus 5 bioprocess.

Adenoviruses are well-known viral vectors that have been previously used in gene therapy and as a vaccine-delivery vehicle for humans and animals. During the COVID-19 pandemic, it gained renewed attention, but at the same time, it raised concerns due to side effects observed with some of the resulting vaccines administered to patients. It has been indicated that these side effects might be attributed to impurities present in the final product. Therefore, constant enhancement of the vaccine purity and further improvement of impurity detection methods are needed. In this work, we showcase an example of industry-relevant adenovirus bioprocess optimization. Our data show the effect of upstream parameters on the bioburden introduced to the downstream process. We provide an example of process optimization using a combination of the PATfix analytical method, ddPCR, infectivity, total DNA, and total protein analyses to optimize cell density, multiplicity of infection, and length of production. Additionally, we provide data illustrating the robustness of the convective interaction media quaternary amine monolithic chromatography step. This anion exchange strategy was shown to remove over 99% of protein and DNA impurities, including those unable to be addressed by tangential flow filtration, while maintaining high adenovirus recoveries.

The Effect of High-Salt Diet on Oxidative Stress Production and Vascular Function in Tff3-/-/C57BL/6N Knockout and Wild Type (C57BL/6N) Mice.

INTRODUCTION: It is well documented that high-salt (HS) diet increases systemic and vascular oxidative stress in various animal models and in humans, leading to impairment of vascular reactivity. The present study examined the interaction of genotype and HS diet intake and the potential effects of oxidative stress - antioxidative system balance on the flow-induced dilation (FID) in pressurized carotid arteries of normotensive Tff3-/-/C57BL/6N knockout mice and their wild-type (WT) controls. METHODS: Male, ten-week-old transgenic Tff3-/-/C57BL/6N (Tff3-/-) knockout mice and WT/C57BL/6N (WT) (parental strain) healthy mice were divided in LS (0.4% NaCl in rodent chow) and HS (4% NaCl in rodent chow fed for 1 week) groups. Additionally, LS and HS groups were treated with 1 mmol/L 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) dissolved in the drinking water. After anesthesia with ketamine chloride (100 mg/kg) and midazolam (5 mg/kg), blood pressure was measured, carotid arteries and aortas were isolated, and blood samples were collected. RESULTS: FID was decreased in WT_HS mice and restored by superoxide scavenger TEMPOL in vivo. On the other hand, attenuated FID of Tff3-/- mice was not further affected by HS diet or TEMPOL in vivo treatment. Vascular superoxide/reactive oxygen species levels were increased with HS diet in both strains and restored by TEMPOL. HS upregulated glutathione peroxidase 1 (GPx1) gene expression in WT_HS and Tff3-/-_HS mice, while GPx activity was significantly decreased only in WT_HS group. Systemic (serum) markers of oxidative stress (oxLDL and AOPP) and arterial blood pressure were similar among groups. CONCLUSION: HS diet increases vascular oxidative stress and impairs vasodilation in WT mice. Tff3 gene deficiency attenuates vasodilation per se, without further effects of HS intake. This can be attributed to vascular upregulation of antioxidative enzyme GPx1 in Tff3-/-/C57BL/6N mice conferring protection from oxidative stress.

Association of serum trefoil factor 3 and leptin levels with obesity: A case-control study.

BACKGROUND: Obesity has a detrimental impact on individuals, communities, and healthcare systems. Trefoil factor 3 is a secretory protein involved in metabolic processes related to weight regulation. However, its relation with obesity is not fully understood. OBJECTIVE: We aimed to assess the serum trefoil factor 3 level and to immunohistochemical detect the leptin in obese patients to evaluate their relation to obesity pathogenesis. METHODS: As a case-control study, we enrolled 83 non-obese persons as a control group with a BMI (18.5-24.9) and 83 obese persons as a patient group with a BMI > 30. All the study volunteers are subjected to anthropometric measurements, glucose, and lipid profile analysis by colorimetric methods. Serum trefoil factor 3 level was estimated by ELISA and leptin hormone was detected immunohistochemically in the blood using cell block technique. RESULTS: ROC curve analysis for TFF3 showed a good relation with obesity with an AUC of 0.891 and a cut-off value of > 96 ng/ml. There was a significant positive correlation between TFF3 and fasting blood sugar, total cholesterol, and triglycerides. The logistic regression analysis showed that TFF3 is a good risk factor for obesity incidence [p = 0.008; OR = 1.117; (95 % CI): 1.029-1.213]. This was confirmed by multiple linear regression that gave an equation for the possibility of predicting BMI using several factors including TFF3 [BMI = 0.821 + 0.051 × TFF3 + 0.044 × FBS + 0.85 × TC]. The more surprising was the ability of the immunohistochemistry cell block technique to detect leptin antigens associated with an obese person blood not only adipose tissue or serum. CONCLUSION: Leptin hormone and TFF3 could be good indicators for obesity incidence. Further research with a larger sample size and in different populations could completely approve our results.

Co-current filtrate flow in TFF perfusion processes: Decoupling transmembrane pressure from crossflow to improve product sieving.

Hollow fiber-based membrane filtration has emerged as the dominant technology for cell retention in perfusion processes yet significant challenges in alleviating filter fouling remain unsolved. In this work, the benefits of co-current filtrate flow applied to a tangential flow filtration (TFF) module to reduce or even completely remove Starling recirculation caused by the axial pressure drop within the module was studied by pressure characterization experiments and perfusion cell culture runs. Additionally, a novel concept to achieve alternating Starling flow within unidirectional TFF was investigated. Pressure profiles demonstrated that precise flow control can be achieved with both lab-scale and manufacturing-scale filters. TFF systems with co-current flow showed up to 40% higher product sieving compared to standard TFF. The decoupling of transmembrane pressure from crossflow velocity and filter characteristics in co-current TFF alleviates common challenges for hollow fiber-based systems such as limited crossflow rates and relatively short filter module lengths, both of which are currently used to avoid extensive pressure drop along the filtration module. Therefore, co-current filtrate flow in unidirectional TFF systems represents an interesting and scalable alternative to standard TFF or alternating TFF operation with additional possibilities to control Starling recirculation flow.

Evaluation of various membranes at different fluxes to enable large-volume single-use perfusion bioreactors.

The growing demand for biological therapeutics has increased interest in large-volume perfusion bioreactors, but the operation and scalability of perfusion membranes remain a challenge. This study evaluates perfusion cell culture performance and monoclonal antibody (mAb) productivity at various membrane fluxes (1.5-5 LMH), utilizing polyvinylidene difluoride (PVDF), polyethersulfone (PES), or polysulfone (PS) membranes in tangential flow filtration mode. At low flux, culture with PVDF membrane maintained higher cell culture growth, permeate titer (1.06-1.34 g/L) and sieving coefficients (≥83%) but showed lower permeate volumetric throughput and higher transmembrane pressure (TMP) (>1.50 psi) in the later part of the run compared to cultures with PES and PS membrane. However, as permeate flux increased, the total mass of product decreased by around 30% for cultures with PVDF membrane, while it remained consistent with PES and PS membrane, and at the highest flux studied, PES membrane generated 12% more product than PVDF membrane. This highlights that membrane selection for large-volume perfusion bioreactors depends on the productivity and permeate flux required. Since operating large-volume perfusion bioreactors at low flux would require several cell retention devices and a complex setup, PVDF membranes are suitable for low-volume operations at low fluxes whereas PES membranes can be a desirable alternative for large-volume higher demand products at higher fluxes.

Cost-effective identification of Barrett's esophagus in the community: A first step towards screening.

BACKGROUND AND AIM: The first step towards developing a screening strategy for Barrett's esophagus (BE) is the identification of individuals in the community. Currently available tools include endoscopy, less-invasive non-endoscopic devices, and non-invasive risk stratification models. We evaluated the cost of potential strategies for identification of BE as a first step towards screening. METHODS: Two hypothetical cohorts of the general population aged ≥ 50 years with BE prevalence rates of 1.9% and 6.8% were modeled. Four potential screening tools were evaluated: (i) risk stratification based on non-weighted clinical factors according to US/European guidelines, (ii) weighted risk stratification using algorithmic models, (iii) less-invasive devices such as Cytosponge + trefoil factor 3 (TFF3), and (iv) endoscopy. Using a decision-analytic model, the cost per BE case identified and the cost-effectiveness were compared for six potential BE screening strategies based on combinations of the four screening tools; (i) + (iv), (ii) + (iv), (iii) + (iv), (i) + (iii) + (iv), (ii) + (iii) + (iv), and only (iv). RESULTS: The cost per BE case identified was lowest for the weighted risk stratification followed by Cytosponge-TFF3 then endoscopy strategy at both 1.9% and 6.8% BE prevalences (US$9282 and US$3406, respectively) although it was sensitive to the cost of less-invasive devices. This strategy was also most cost-effective for a BE prevalence of 1.9%. At BE prevalence of 6.8%, the Cytosponge-TFF3 followed by endoscopy strategy was most cost-effective. CONCLUSIONS: Incorporating weighted risk stratification and less-invasive devices such as Cytosponge-TFF3 into BE screening strategies has a potential to cost-effectively identify BE in the community although device cost and the community prevalence of BE will impact the optimal strategy.

Different Forms of TFF3 in the Human Endocervix, including a Complex with IgG Fc Binding Protein (FCGBP), and Further Aspects of the Cervico-Vaginal Innate Immune Barrier.

TFF3 is a typical secretory poplypeptide of mucous epithelia belonging to the trefoil factor family (TFF) of lectins. In the intestine, respiratory tract, and saliva, TFF3 mainly exists as a high-molecular-mass complex with IgG Fc binding protein (FCGBP), which is indicative of a role in mucosal innate immunity. For the first time, we identified different forms of TFF3 in the endocervix, i.e., monomeric and homodimeric TFF3, as well as a high-molecular-mass TFF3-FCGBP complex; the latter also exists in a hardly soluble form. Immunohistochemistry co-localized TFF3 and FCGBP. Expression analyses of endocervical and post-menopausal vaginal specimens revealed a lack of mucin and TFF3 transcripts in the vaginal specimens. In contrast, genes encoding other typical components of the innate immune defense were expressed in both the endocervix and vagina. Of note, FCGBP is possibly fucosylated. Endocervical specimens from transgender individuals after hormonal therapy showed diminished expression, particularly of FCGBP. Furthermore, mucus swabs from the endocervix and vagina were analyzed concerning TFF3, FCGBP, and lysozyme. It was the aim of this study to illuminate several aspects of the cervico-vaginal innate immune barrier, which is clinically relevant as bacterial and viral infections are also linked to infertility, pre-term birth and cervical cancer.

A Rapid Self-Assembling Peptide Hydrogel for Delivery of TFF3 to Promote Gastric Mucosal Injury Repair.

Self-assembled peptide-based nanobiomaterials exhibit promising prospects for drug delivery applications owing to their commendable biocompatibility and biodegradability, facile tissue uptake and utilization, and minimal or negligible unexpected toxicity. TFF3 is an active peptide autonomously secreted by gastric mucosal cells, possessing multiple biological functions. It acts on the surface of the gastric mucosa, facilitating the repair process of gastric mucosal damage. However, when used as a drug, TFF3 faces significant challenges, including short retention time in the gastric mucosal cavity and deactivation due to degradation by stomach acid. In response to this challenge, we developed a self-assembled short peptide hydrogel, Rqdl10, designed as a delivery vehicle for TFF3. Our investigation encompasses an assessment of its properties, biocompatibility, controlled release of TFF3, and the mechanism underlying the promotion of gastric mucosal injury repair. Congo red/aniline blue staining revealed that Rqdl10 promptly self-assembled in PBS, forming hydrogels. Circular dichroism spectra indicated the presence of a stable ß-sheet secondary structure in the Rqdl10 hydrogel. Cryo-scanning electron microscopy and atomic force microscopy observations demonstrated that the Rqdl10 formed vesicle-like structures in the PBS, which were interconnected to construct a three-dimensional nanostructure. Moreover, the Rqdl10 hydrogel exhibited outstanding biocompatibility and could sustainably and slowly release TFF3. The utilization of the Rqdl10 hydrogel as a carrier for TFF3 substantially augmented its proliferative and migratory capabilities, while concurrently bolstering its anti-inflammatory and anti-apoptotic attributes following gastric mucosal injury. Our findings underscore the immense potential of the self-assembled peptide hydrogel Rqdl10 for biomedical applications, promising significant contributions to healthcare science.

PTEN, ERG, SPINK1, and TFF3 Status and Relationship in a Prostate Cancer Cohort from Jordanian Arab Population.

Background and Objectives: Prognostic biomarkers in prostate cancer (PCa) include PTEN, ERG, SPINK1, and TFF3. Their relationships and patterns of expression in PCa in developing countries, including Jordan, have not yet been investigated. Materials and Methods: A tissue microarray (TMA) of PCa patients was taken from paraffin-embedded tissue blocks for 130 patients. PTEN, ERG, SPINK1, and TFF3 expression profiles were examined using immunohistochemistry (IHC) and correlated with each other and other clinicopathological factors. Results: PTEN loss of any degree was observed in 42.9% of PCa cases. ERG and TFF3 were expressed in 59.3% and 46.5% of PCa cases, respectively. SPINK1 expression was observed in 6 out of 104 PCa cases (5.4%). Among all PCa cases (n = 104), 3.8% (n = 4) showed SPINK1+/ERG+ phenotype, 1.9% (n = 2) showed SPINK1+/ERG- phenotype, 56.7% (n = 59) showed SPINK1-/ERG+ phenotype, and 37.5% showed SPINK1-/ERG- phenotype (n = 39). Among ERG positive cases (n = 63), 6.3% were SPINK1 positive. Among SPINK1 positive cases (n = 6), 66.7% were ERG positive. SPINK1 expression was predominantly observed in a subgroup of cancers that expressed TFF3 (6/6). Additionally, a statistically significant loss of PTEN expression was observed from Gleason Score 6 (GS6) (Grade Group 1 (GG1)) to GS9-10 (GG5); (p-value 0.019). Conclusions: This is the first study to look at the status of the PTEN, ERG, SPINK1, and TFF3 genes in a Jordanian Arab population. Loss of PTEN has been linked to more aggressive prostate cancer with high GSs/GGs. SPINK1 expression was predominantly observed in a subgroup of cancers that expressed TFF3. Our results call for screening these biomarkers for grading and molecular subtyping of the disease.

Novel WEE2 homozygous mutations c.1346C>T and c.949A>T identified in primary infertile women due to unexplained fertilization failure.

The occurrence of unexplained fertilization failure can have profound psychological and financial consequences for couples struggling with infertility, and its pathogenesis remains unclear. Increasing evidence highlights genetic basis of unexplained fertilization failure occurrence. Here, we identified one novel homozygous nonsense mutation (c.949A>T), one novel homozygous missense mutation (c.1346C>T), and three reported homozygous mutations (c.585G>C, c.1006_1007insTA, c.1221G>A) in six unrelated probands, showing similar manifestations of unexplained fertilization failure. This finding expands the spectrum of WEE2 mutations, highlighting the critical role of WEE2 in fertilization process, and provides a basis for the prognostic value of testing for WEE2 mutations in primary infertile couples with unexplained fertilization failure.

The Forms of the Lectin Tff2 Differ in the Murine Stomach and Pancreas: Indications for Different Molecular Functions.

The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum (RT-PCR analysis). The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms.

Expression Profiling along the Murine Intestine: Different Mucosal Protection Systems and Alterations in Tff1-Deficient Animals.

Tff1 is a typical gastric peptide secreted together with the mucin, Muc5ac. Tff1-deficient (Tff1KO) mice are well known for their prominent gastric phenotype and represent a recognized model for antral tumorigenesis. Notably, intestinal abnormalities have also been reported in the past in these animals. Here, we have compared the expression of selected genes in Tff1KO mice and their corresponding wild-type littermates (RT-PCR analyses), focusing on different mucosal protection systems along the murine intestine. As hallmarks, genes were identified with maximum expression in the proximal colon and/or the duodenum: Agr2, Muc6/A4gnt/Tff2, Tff1, Fut2, Gkn2, Gkn3, Duox2/Lpo, Nox1. This is indicative of different protection systems such as Tff2/Muc6, Tff1-Fcgbp, gastrokines, fucosylation, and reactive oxygen species (ROS) in the proximal colon and/or duodenum. Few significant transcriptional changes were observed in the intestine of Tff1KO mice when compared with wild-type littermates, Clca1 (Gob5), Gkn1, Gkn2, Nox1, Tff2. We also analyzed the expression of Tff1, Tff2, and Tff3 in the pancreas, liver, and lung of Tff1KO and wild-type animals, indicating a cross-regulation of Tff gene expression. Furthermore, on the protein level, heteromeric Tff1-Fcgbp and various monomeric Tff1 forms were identified in the duodenum and a high-molecular-mass Tff2/Muc6 complex was identified in the proximal colon (FPLC, proteomics).

Human TFF2-Fc fusion protein alleviates DSS-induced ulcerative colitis in C57BL/6 mice by promoting intestinal epithelial cells repair and inhibiting macrophage inflammation.

The clinical drugs for ulcerative colitis mainly affect the inflammatory symposiums with limited outcomes and various side effects. Repairing the damaged intestinal mucosa is a promising and alternative strategy to treat ulcerative colitis. Trefoil factor family 2 (TFF2) could repair the intestinal mucosa, however, it has a short half-life in vivo. To improve the stability of TFF2, we have prepared a new fusion protein TFF2-Fc with much stability, investigated the therapeutic effect of TFF2-Fc on ulcerative colitis, and further illustrated the related mechanisms. We found that intrarectally administered TFF2-Fc alleviated the weight loss, the colon shortening, the disease activity index, the intestinal tissue injury, and the lymphocyte infiltration in dextran sulfate sodium (DSS)-induced colitis mice. In vitro, TFF2-Fc inhibited Caco2 cells injury and apoptosis, promoted cellular migration, and increased the expression of Occludin and ZO-1 by activating P-ERK in the presence of H2O2 or inflammatory conditioned medium (LPS-RAW264.7/CM). Moreover, TFF2-Fc could reduce lipopolysaccharide (LPS)-induced production of inflammation cytokines and reactive oxygen species in RAW264.7 cells, and also inhibits the polarization of RAW264.7 cells to M1 phenotype by reducing glucose consumption and lactate production. Taken together, in this work, we have prepared a novel fusion protein TFF2-Fc, which could alleviate ulcerative colitis in vivo via promoting intestinal epithelial cells repair and inhibiting macrophage inflammation, and TFF2-Fc might serve as a promising ulcerative colitis therapeutic agent.

Augmented leptin-induced trefoil factor 3 expression and epidermal growth factor receptor transactivation differentially influences neoplasia progression in the stomach and colorectum of dietary fat-induced obese mice.

Obesity is a risk factor for gastrointestinal malignancies and tumors. However, which factors either protect or predispose the gastrointestinal organs to high-fat diet (HFD)-induced neoplasia remains unclear. Here, we demonstrate that HFD impacts the stomach to a greater extent as compared to the colorectum, resulting in leptin receptor (LepR) signaling-mediated neoplasia in the tissues. HFD activated leptin signaling, which in turn, accelerates the pathogenesis in the gastric mucosa more than that in the colorectum along with ectopic TFF3 expression. Moreover, in the stomach, higher levels of phosphorylated epidermal growth factor receptor (EGFR) in addition to the activation of STAT3 and Akt were observed as compared to the colorectum. The mice with LepR deletion in the gastrointestinal epithelium exhibited a suppressed induction of leptin, TFF3, and phosphorylated EGFR in the stomach, whereas the levels in the colorectum were insignificant. In co-transfected COS-7 cells with LepR and EGFR plasmid DNA, leptin transactivated EGFR to accelerate TFF3 induction along with activation of STAT3, ERK1/2, Akt, and PI3K p85/p55. Furthermore, TFF3 could bind to EGFR but did not transactivate LepR. Leptin-induced TFF3 induction was markedly suppressed by inhibitors of PI3K (LY294002) and EGFR (Erlotinib). Together, these results suggest a novel role of LepR-mediated signaling in transactivating EGFR that leads to TFF3 expression via the PI3K-Akt pathway. Therefore, this study sheds light on the identification of potentially new therapeutic targets for the treatment of pre-cancerous symptoms in stomach and colorectum.

Runx1 regulates Tff1 expression to expedite viability of retinal microvascular endothelial cells in mice with diabetic retinopathy.

Diabetic retinopathy (DR) represents a major complication of diabetes, and molecular mechanisms related to vascular dysfunction, particularly endothelial dysfunction, in DR remains unclear. In the present work, we generated a DR animal model using mice and a cell model in mouse retinal microvascular endothelial cells (mRMECs) to examine the role of Trefoil factor family 1 (Tff1) in DR. Tff1 was poorly expressed in DR mice and high glucose (HG)-treated mRMECs. Overexpression of Tff1 significantly attenuated streptozotocin-induced retinal proliferation and angiogenesis in DR mice and reduced the secretion of inflammatory factors. In HG-treated mRMECs, overexpression of Tff1 remarkably reduced the proliferation and angiogenesis of mRMECs. In further experiments, we found that Tff1 was transcriptionally repressed by Runt-related transcription factor 1 (Runx1) directly, and Tff1 expression was indirectly modulated by Runx1 via the core-binding factor subunit beta (CBF-ß)/nuclear factor, erythroid 2/microRNA-423-5p axis and the CBF-ß/estrogen receptor 1 (ESR1) axis. Moreover, Tff1 could inhibit the activation of NF-κB signaling pathway, which in turn attenuated retinal endothelial cell proliferation and angiogenesis. It was thus proposed that Runx1/Tff1/NF-κB axis may be a potential target for the treatment strategy of DR, and further studies are needed.

Scalable purification of recombinant structural proteins, hagfish intermediate filament α and É£, from inclusion bodies for fiber formation.

The purpose of this study was to determine a method to purify recombinant hagfish intermediate filament proteins, alpha and gamma, in a scalable manner. The study succeeded by having an increase in protein recovery of up to 35% when comparing centrifuge purification and the developed tangential flow purification. The proteins were approximately the same purity of 70% pure but further purification increased the purity of the proteins by 16%, based on ImageJ analysis. The developed tangential flow filtration purification and final purification methods could be easily scaled up to meet industry scale purification needs. The scaled-up processes described in this study did not interfere with fiber production or formation, indicating the methods can produce usable proteins for material development.

TFF1 gene single nucleotide polymorphism (rs3761376) and colorectal cancer risk.

INTRODUCTION: Trefoil Factor 1 (TFF1) is a secretory peptide with gastrointestinal protective functions. Abnormal TFF1 expression is reported in some cancers and functional promoter polymorphism in TFF1 is believed to be associated with risk of gastric cancer. We evaluated rs3761376 in a sample of Iranian patients with colorectal cancer. METHODS: Peripheral blood samples were taken from pathology confirmed cases of colorectal cancer and healthy volunteers. Genotyping was carried out using Restriction Fragment Length Polymorphism (RFLP) PCR. Any association with clinicopathologic data was assessed by SPSS version 19. RESULTS: A total of 245 participants, including 122 patients with cancer and 123 non-cancer subjects were enrolled. Age, body mass index, and smoking habits were not significantly different between the two groups (P > 0.05). Distribution of TFF1 genotypes was not found to be associated with colorectal cancer. However, distant metastasis was more prevalent in carriers of the mutant allele. CONCLUSION: TFF1 rs3761376 was not associated with colorectal cancer but it may be involved in metastasis. Therefore, further investigation is warranted to determine this relationship.

Low expression of TFF3 in papillary thyroid carcinoma may correlate with poor prognosis but high immune cell infiltration.

Background: Papillary thyroid carcinoma (PTC) is one of the most common endocrine malignancies and has a favorable prognosis. However, optimal treatments and prognostic markers have not been clearly identified. Methods: Gene expression data from primary PTC were downloaded from the Gene Expression Omnibus database and subjected to two analyses of differentially expressed genes (DEGs), followed by intersecting individual and integrated DEGs analyses as well as gene set enrichment analysis. Analysis of data from Sequence Read Archive and The Cancer Genome Atlas, immunohistochemistry and qRT-PCR of TFF3 were performed to validate the results. Finally, the relationship between gene expression and disease-free survival as well as immune cell infiltration were investigated. Results: Six critical DEGs and several tumor-enriched signaling pathways were identified. Immunohistochemistry and qRT-PCR validated the low expression of TFF3 in PTC. TFF3 and FCGBP are coexpressed in PTC, and patients with lower gene expression had worse disease-free survival but higher immune cell infiltration. Conclusion: TFF3 was significantly underexpressed and may function with FCGBP synergistically in PTC.

Lay abstract Thyroid cancers are some of the most common endocrine malignancies. However, the optimal treatments and prognostic markers have not been clearly identified. We identified six critical differentially expressed genes and several tumor-enriched signaling pathways in papillary thyroid carcinoma, and found that TFF3 was the most underexpressed gene, as validated by experiment. In addition, TFF3 and FCGBP worked synergistically and may mark prognosis and tumor immune cell infiltration, which may benefit patients with papillary thyroid carcinoma by providing early indication and prompting further basic investigation.

Association of functional genetic variants in TFF1 and nephrolithiasis risk in a Chinese population.

Trefoil Factor 1 (TFF1) is considered to be able to inhibit the formation of kidney stone. However, genetic variants in TFF1 and corresponding function in kidney stone development are still not well studied. In this study, the discovery set including 230 cases and 250 controls was used to analyze the association between seven tagSNPs of TFF1 gene and the nephrolithiasis risk. Further evaluation was confirmed by the validation set comprising 307 cases and 461 controls. The consequences of the two-stage case-control study indicated that individuals with the rs3761376 A allele have significantly increased nephrolithiasis risk than those with the GG genotypes [adjusted odds ratio (OR) = 1.35, 95% confidence interval (CI) = 1.05-1.73]. Moreover, we also carried out a stratified analysis and found the increased nephrolithiasis risks at A allele among males, overweight individuals, no hypertensive individuals, nondiabetic individuals, smokers, and drinkers. In the following functional experiments, the notably lower expression of TFF1 was exhibited by the vectors carrying A allele compared with those carrying G allele in both luciferase (P = 0.022) and expression vectors (P = 0.041). In addition to tissue detection, we confirmed a significant inverse association of rs3761376 G > A and TFF1 gene expression (P < 0.001). These results suggest that TFF1 rs3761376 may serve as a potential biomarker to predict the risk of nephrolithiasis.