Digestive Tract Research Articles

Mechanistic insights into traditional Chinese medicine for digestive tract cancers: implications for gastric, hepatic, esophageal, intestinal, and pancreatic tumors

Abstract The increasing incidence of cancer-related deaths highlights the pressing need for effective treatment modalities, particularly in the context of digestive tract cancers, such as gastric, hepatic, esophageal, intestinal, and pancreatic tumors. While conventional drug therapies play a critical role in managing these malignancies, their associated side effects often pose significant challenges to patient quality of life. Thus, there is a growing focus on traditional Chinese medicine (TCM) and its compounds, which are safe, non-toxic, and reliable. During anti-tumor therapy, TCM compounds, based on their multi-target, multi-pathway, and multi-level regulatory effects, fully mobilize multiple mechanisms of the body, presenting significant advantages in inhibiting tumor development, boosting patient welfare, and increasing their lifespan. This article reviews the mechanisms by which TCM inhibits tumor cell proliferation, promotes tumor cell death, suppresses tumor cell invasion and metastasis, regulates the tumor microenvironment, inhibits angiogenesis, and enhances anti-tumor drug resistance. This knowledge might provide a theoretical and scientific basis for preventing and treating tumors using TCM.

In Vitro Cholesterol Uptake by the Microflora of Selected Kefir Starter Cultures

Kefir, a fermented milk beverage, is recognized for its potential health benefits, including its cholesterol-lowering properties. This study demonstrated that selected kefir starter cultures, including Lactococcus strains and yeasts, significantly reduce cholesterol-binding capacity under simulated gastrointestinal conditions, underscoring the challenges of probiotic delivery. We compared the performance of these cultures under laboratory conditions (growth broths) and simulated digestive juice models. Lactococcus strains showed significant differences in cholesterol binding between the two environments, highlighting the limitations of relying solely on laboratory testing. Yeast cultures also exhibited greater cholesterol binding in their native broths, but their survival was limited in digestive models. Our findings suggest that effective probiotic formulations should prioritize strains with high cholesterol-binding capacity and robust survival rates throughout the digestive tract. This study provides valuable insights for future research on the mechanisms behind these functionalities and the potential of kefir yeast strains for use in human digestive models. Our results can be used to inform the development of improved probiotic formulations for cholesterol management.

Aspects of Growth, Diet, and Reproduction of <i>Oreochromis niloticus</i> (Linnaeus, 1758) in a Hypertrophic Milieu, the Municipal Lake of Yaoundé, Cameroon

The Nile tilapia, <i>Oreochromis niloticus</i>, is a highly valued fish species in Africa for subsistence and commercial purposes. However, overfishing activities in non-supervised lakes or reservoirs threaten its availability. In the view of contributing to the database on the life history traits of this species in Cameroon and supporting its sustainable management, the present study investigated the growth patterns, food preferences, and reproductive aspects of <i>O. niloticus</i> in a hypertrophic milieu, the Yaoundé Municipal Lake, from May to October 2018. Fish specimens were sampled, measured, weighed, and sexed. The digestive tract of each specimen and the females’ gonads were collected. The intestines were measured, and the stomach contents were identified. The females’ reproductive aspects were determined. Overall, 101 specimens were collected and distributed among 48 (47.5%) males and 53 (52.5%) females, with a sex ratio of 1: 1.1. In this hypoxic (1.35 ± 0.4 mg/L) and highly conductive (210.5 ± 56.9 µS/cm) milieu, <i>O. niloticus</i> displayed a relatively good condition (<i>Kn</i> > 1) throughout the sampling period and negative allometric growth patterns for both sexes (<i>b </i>< 3; <i>p</i> < 0.001). Materials from plant origin were the preferred prey types, with an index of relative importance (IRI) of 78.20%; meanwhile, mud and materials from animal origin were secondary prey items (IRI: 13 ‒ 17%). The relative gut length (RGL) value above 3.0 and the foraging activity on detritus suggest that <i>O. niloticus</i> has an herbivorous or detritivorous dietary habit and is a bottom-feeder. Female length at first maturity (L<sub>50</sub>) was 17 cm. Their gonado-somatic index (GSI) showed sexual activity throughout the whole sampling period, with the peak spawning phase happening in August. The absolute fecundity of gravid females varied between 316 and 1816 oocytes (942 ± 430 oocytes), and the relative fecundity ranged from 1–5 oocytes/gram of body weight (3 ±1 oocytes/g). The results highlight the performance of the Nile tilapia in this ecosystem and provide information for its sustainable use and improvement of fish culture.

Pancreatic cancer – challenge for modern medicine

Introduction. Pancreatic cancer is one of the most dangerous carcinomas of the digestive tract, with a very poor prognosis for patients. Annually, more than three and a half thousand people in Poland are diagnosed with this tumor. Aim. The aim of the work was to assess and compile existing knowledge about pancreatic adenocarcinoma. Materials and methods. Data for the paper was gathered with the usage of Google Scholar. Moreover, articles from sources like PubMed or National Institutes of Health were used. Materials were published between 2015 and 2024. Results. Tumor morbidity is still increasing, the biggest group of patients are people who are more than 55 years old. Many modifiable and nonmodifiable factors play a role in the pathogenesis of pancreatic cancer. Lifestyle, stimulants, BMI or physical activity can be included into the first group. Nonmodifiable factors are sex, age, race, genetic predisposition or blood group. Typical symptoms of this tumor are loss of the body weight, fatigue, nausea, jaundice and abdominal pain. Clinicians use the following diagnostic methods: CT, MRI, ERCP, MRCP or USG. Only the radical chirurgical treatment gives a hope for total recovery, additionally chemotherapy can be admitted. Chemotherapy schemes are FOLFIRINOX or monotherapy with gemcitabine. Conclusions. Pancreatic cancer is still a challenge for clinicians, as its treatment is limited by poor effectiveness of possible ways of treatment. The majority of tumors are diagnosed at the advanced stage, when full patient recovery is not possible. New ways of treatment such as immunotherapy, monoclonal antibodies or gene therapy are a ray of hope. Keywords: pancreatic cancer, treatment of pancreatic cancer, epidemiology, diagnostic methods.

Reverse resistance to immune checkpoint inhibitor in a patient with recurrent cardia cancer by intratumoral injection of recombinant human adenovirus type 5: a case report and literature review

Advanced metastatic cardia cancer is an intractable malignance with poor prognosis. It is often accompanied by upper digestive tract obstruction, which seriously affects the quality of patients. Therefore, effective relief of eating obstruction is an important goal in the treatment of cardia cancer. Immune checkpoint inhibitors (ICIs) have shown significant efficacy in cardia cancer, but only a small percentage of patients will benefit from them due to immune resistance. Oncolytic viruses have been shown to enhance the efficacy of ICIs by altering the immune microenvironment. This indicates that oncolytic virus has the potential value of overcoming the immune resistance of cardia cancer. Here, we present a case with local recurrent and multiple metastatic cardia cancer accompanied by eating obstruction. After 4 cycles of chemotherapy plus ICI therapy, the patient´s metastases were significant shrink, but the recurrent carida lesion were almost unchanged. Then we implemented exploratory local injection of recombinant human adenovirus type 5(H101) into recurrent cardia lesion by painless gastroscopy. Surprisingly, the cardia lesion shrank significantly, and the eating obstruction was greatly relieved. We also observed a significant increase of infiltrated CD4+T cells in biopsy tissues after H101 treatment. Our study not only conformed the value of oncolytic viruses to reverse ICI resistance in patients with gastric cancer, but also revealed its underlying impact on immune microenvironment.

A Biochemistry Teaching Experiment That Demonstrates the Digestion of Carbohydrates, Proteins, and Lipids in the Digestive Tract

A Biochemistry Teaching Experiment That Demonstrates the Digestion of Carbohydrates, Proteins, and Lipids in the Digestive Tract

Dosing of Bacterial Phytase (EC 3.1.3.26) in Laying Hens’ Diets

Despite the acknowledged advancements in the utilization of phytase dosing, limited research has been conducted on commercial laying hens. To assess the effectiveness of doses of exogenous phytase, a total of 320 laying hens aged between 44 and 64 weeks were utilized. These hens were divided into five treatments, which were then distributed using a completely randomized design with eight replicates, each containing eight birds. The treatments consisted of phytase supplementation levels of 0, 500, 1000, 1500, and 3000 FTU/kg. Quadratic regression equations were utilized to determine the maximum and minimum values for the levels of phytase supplementation. A significant difference was observed in the levels of phytase supplementation, where a quadratic model was fitted to various variables including egg weight (g), egg mass (g/bird/day), feed conversion per egg mass (g/g), yolk concentration (%), albumen concentration (%), shell thickness (μm), shell strength (kgf), specific gravity, and yolk color. Further, an increased supplementation improved the digestive tract of the hens, thereby providing a better villus–crypt ratio. Considering the advantageous effects of phytase dosing, it is recommended to supplement corn and soybean meal-based diets with 1500 FTU/kg of bacterial phytase for laying hens during the 44- to 64-week production period.

Bladder Adenocarcinoma in a Constellation of Multiple Site Malignancies: An Unusual Case and Systematic Review

Background and Objectives: Multiple primary malignant tumors represent a small percentage of the total number of oncological cases and can involve either metachronous or synchronous development and represent challenges in diagnosis, staging, and treatment planning. Our purpose is to present a rare case of bladder adenocarcinoma in a female patient with multiple primary malignant tumors and to provide systematic review of the available literature. Materials and Methods: A 67-year-old female patient was admitted with altered general condition and anuria. The past medical history of the patient included malignant melanoma (2014), cervical cancer (2017), colon cancer (2021), obstructive anuria (2023), and liver metastasectomy (2023). Transurethral resection of bladder tumor was performed for bladder tumors. Results: Contrast CT highlighted multiple pulmonary metastases, a poly nodular liver conglomerate, retroperitoneal lymph node, II/III grade left ureterohydronephrosis, and no digestive tract tumor masses. The pathological result of the bladder resection showed an infiltrative adenocarcinoma. Conclusions: The difference between primary bladder adenocarcinoma tumor and metastatic colorectal adenocarcinoma is the key for the future therapeutic strategy. Identification and assessment of risk factors such as viral infection, radiotherapy, chemotherapy, smoking, and genetics are pivotal in understanding and managing multiple primary malignant tumors. Personalized prevention strategies and screening programs may facilitate the early detection of these tumors, whether synchronous or metachronous. The use of multicancer early detection (MCED) blood tests for early diagnosis appears promising. However, additional research is needed to standardize these techniques for cancer detection.

Insights Into the Adsorption Behavior of Polyethylene Microplastics Towards Lead(II) Ions

ABSTRACTMicroplastics and metals represent widespread contaminants which can cause significant damage to aquatic ecosystems and organisms. However, the complex interactions between microplastics and various metals in water environments remains to be understood. This study aims to investigate the interaction dynamics between lead(II) ions and polyethylene in freshwater environments. Adsorption trials were conducted systematically by adjusting operational parameters such as initial Pb(II) concentration, equilibrium pH value, and residence times. An in‐depth characterization study using scanning electron microscopy (SEM), x‐ray diffraction and Fourier transform infrared (FTIR) analysis was conducted to explain the adsorption mechanism of polyethylene microplastics (PEM). The data indicated the porous surface of PEM, highlighting the presence of diverse functional groups. According to the Langmuir model, the PEM exhibited maximum Pb(II) uptake of 3.69 mg/g at pH 4.5. The pseudo‐first‐order model demonstrated superior fitting to Pb(II)‐PEM kinetics. Desorption trials were performed to assess the release of Pb(II) from Pb(II)‐bounded PEM using various chemical agents. It was observed that 0.01 M HNO₃ desorbed Pb(II) ions optimally, achieving a desorption efficiency exceeding 99.9%. Consequently, desorption trials provided evidence that Pb(II)‐bounded PEM may release Pb(II) ions in acidic environments, facilitating the transfer of Pb(II) to the digestive tracts of aquatic organisms.

Vesical Varices: An Unusual Cause of Hematuria

AbstractPortal hypertension is a common complication of cirrhosis and may lead to the formation of venous collateral channels. Usually, varices due to portal hypertension develop in the lower esophagus, stomach, rectum, or other parts of the digestive tract. Extraintestinal ectopic varices are rare. Here, we present a patient with ectopic urinary bladder varices, who presented with hematuria and symptomatic anemia. The bleeding was managed with minimally invasive endovascular approach.

Mechanisms and perspectives of B vitamins associated one carbon metabolism on colorectal cancer risk

Colorectal cancer (CRC) represents a significant global health challenge as a common malignancy of the digestive tract. The involvement of B vitamins-specifically folic acid (B9), riboflavin (B2), pyridoxine (B6), and cobalamin (B12)-is crucial in metabolic processes by mediating the transfer of one-carbon (1C) units, which plays a fundamental role in cellular functions and tumor growth. 1C metabolism is involved in synthesis of proteins, lipids, nucleic acids, and other cofactors. 1C metabolism, intertwined with the metabolism of other nutrients, forms complex pathways where B vitamins act as precursors or coenzymes, influencing the production of various intermediates. These vitamins, as essential nutrients, are implicated to varying the pathogenesis and progression of colorectal cancer such as epigenetics. Furthermore, 1C metabolism affects tumor cell fate through multiple aspects including nucleotide synthesis, redox homeostasis, and the interaction with gut microbiota. Given these roles, understanding and monitoring B vitamin levels and their metabolic pathways are essential for colorectal cancer prevention and management. This approach not only helps in reducing tumor-related mortality but also opens new avenues for research into CRC mechanisms and potential therapeutic strategies.

Past and Recent Progress on Metallic Digestive Tract Stents.

The implantation of digestive tract stents at various lesion sites can effectively improve digestive tract patency, opening up an excellent treatment method for diseases that are currently incurable or resistant to conventional surgery. Digestive tract stents have been extensively studied and widely used worldwide due to their unique advantages of simple implantation, low trauma, satisfactory effect, and low complication rate. Among the various types of stents, metallic stents have been developed to improve surgical efficacy due to their excellent mechanical properties and are constantly being improved. This review provides an overview of the design and development of conventional nonbiodegradable metallic digestive tract stents such as nitinol (NiTi alloy), stainless steel, and cobalt-based alloy stents. Furthermore, biodegradable metallic stents for the digestive tract, such as magnesium-based, iron-based, and zinc-based stents, are described. This paper also evaluates the advantages and disadvantages of existing metallic digestive stents as well as future research directions and challenges in the development of metallic digestive tract stents.

The Sternocleidomastoid Muscle Reverse Pad: A “Safety Net” in Catastrophic Tracheal Surgery Situation

Background: This paper presents the outcomes of employing the inferiorly based rotated sternocleidomastoid muscle flap in complex tracheal reconstruction/repair scenarios, focusing on the key objectives of ensuring stable airway, functional digestive tract and patient survival. Methods: A retrospective analysis was performed for patients treated at two medical centers (A.O. San Camillo Forlanini, Rome, and A.O.U. Careggi, Florence) from 2011 to 2023, in which the sternocleidomastoid muscle (SCM) flap, detached from the mastoid and basicranium, was rotated on the lower pivot directly onto the repair site and pedicled to the sternal origin to ensure the continuity of the airway. Average postoperative hospital stay, follow-up period and patient survival were analyzed. Follow-up assessments encompassed bronchoscopies and CT scans conducted at intervals of 15 and 28 days, and subsequently at 3 and 9 months. Results: A total of five patients were enrolled in this study. These cases included one patient with anterior tracheal wall lesions with abundant tissue loss, one patient with an anterior wall necrosis due to descending cervical mediastinitis and three patients with extra-long tracheoesophageal fistulas (TEFs) (greater than 4.5 cm or >30% of the total tracheal length). In the case of the direct repair of a TEF with a proximal tracheal stenosis, the sternocleidomastoid muscle was used to reconstruct the tissue deficit caused by extensive loss of substance in the left lateral side of the tracheal wall. In case of repair through exclusion of the TEF, the sternocleidomastoid muscle was interposed between the visceral sutures after exclusion of the TEF by an endomechanical device, in one case even substituting the membranous part of the tracheal wall. Our technique allows rotation on the sternal head of the sternocleidomastoid muscle with the lowest rotation radius, pedicled to the sternal origin, detached from the mastoid process and superior nuchal line, thus providing optimal vascularization from the superior thyroid artery/external carotid artery and accessory vasculature from the suprascapular artery. Patients exhibited uneventful postoperative recovery concerning airway and digestive patency. The mean postoperative hospitalization duration was 41 days. The follow-up assessments were negative for postoperative complications. Conclusions: The use of sternocleidomastoid muscle flap was proposed to ensure repair and protection of the suture margin or to constitute a portion, as a scaffold, of the wall by leveraging the muscle’s vascularization and thickness. This technique may be considered a leading component in managing complex situations in tracheal surgery.

Antibacterial Activity of Endophytic Fungi Isolated from Parang Romang (Boehmeria virgata (G.Forst) Guill) Leaves Against Gastrointestinal Pathogens

Parang romang (Boehmeria virgata (G.Forst) Guill) is a plant traditionally used for treating various digestive tract infections. However, obtaining bioactive compounds directly from the plant requires significant biomass. Therefore, endophytic fungi, sourced from specific plant parts, are explored for their ability to produce similar bioactive compounds. The research examined the potential antibacterial activity of endophytic fungi isolated from parang romang leaves against common gastrointestinal pathogens. The methods used in was, characterization of endophytic fungal isolates, and antagonistic activity was evaluated using the agar diffusion method. The results showed that the endophytic fungal strains, namely isolates IFBV 6, IFBV 7, and IFBV 10 could inhibit the bacteria Escherichia coli, Salmonella typhi, Shigella dysenteriae, and Vibrio cholera. With an average zone of inhibition for each strains isolate against Esherichia coli respectively 11.53 mm, 10.49 mm, and 14.85 mm; Salmonella typhi 14.47 mm, 8.66 mm, and 15.51 mm; Shigella dysenteriae 22.28 mm, 13.76 mm, and 15.33 mm; and Vibrio cholera 16.91mm, 11.94mm, 15.16mm. In conclusion, isolated endophytic fungal strains hold promise as potential as antibacterialn agents.

Spatiotemporal Ultrastructural, Histological, and Morphometric Changes in the Buccal Cavity of Grass Carp (Ctenopharyngodon idella) During Fingerling, Yearling, and Adult Stages.

Grass carp (Ctenopharyngodon idella) is a Cyprinid fish of aquacultural and research importance. The buccal cavity represents the gateway of the digestive tract. The present study investigated the adaptational changes involving various components of the buccal cavity of fingerling (three months posthatching, mph), yearling (12 mph), and adult (48 mph) C. idella using scanning electron microscopy, histology, and morphometry. The upper and lower lips appeared uninterrupted at the three studied stages and were limited caudally by the upper and lower jaws. The jaw epithelium was the thickest part of the buccal epithelium; ultrastructurally, it appeared smooth with ridge-like borders in fingerlings that transformed into microgrooves in yearlings. In adult C. idella, the jaw mucosa was organized into dome-shaped masses separated from each other by tight furrows. Each mass was formed from several keratinocytes with corrugated surfaces that featured numerous microdepressions. Except for the jaws, taste buds, mainly of type I, and acidic and neutral goblet cells were observed throughout the mucosa of the buccal cavity, and their densities were highest along the mucosal folds of the palate and oral floor, suggesting a principal role for these parts in both gustation and food lubrication. This study is the first to report age-associated spatiotemporal changes in the buccal cavity of grass carp and will serve as a fundamental reference during the interpretation of various types of oral pathologies in teleost.

What We Know So Far About ECG for Pancreatic Pseudocysts

Background: Endoscopic cysto-gastrostomy (ECG) has become the treatment of choice for pancreatic pseudocysts (PPCs). Endoscopic drainage of PPCs requires the creation of an anastomosis between the lumen of the PPCs and the lumen of the gastrointestinal tract. Various types of stents are used for this purpose. The aim of the study is to compare the indications, quantity, and results of using double pigtail plastic stents (DPPSs) and lumen-apposing fully covered metal stents (LAMSs) in ECG. Methods: A retrospective analysis was conducted of 39 patients (24 men, 15 women) treated for PPCs in the Department of Digestive Tract Surgery and the Department of Gastroenterology and Hepatology between October 2018 and February 2023. The mean age of patients was 51.13 (28–77). Data about etiology, cyst diameter, type, and complications of the stents were collected. Results: DPPSs were placed in smaller cysts (108 vs. 140 millimeters, p = 0.04) and were maintained for a longer duration compared to LAMSs (106 vs. 34 days, p = 0.001). Cyst recurrence was reported less frequently in patients with a LAMS (0 (0%) vs. 4 (19.05%), p = 0.05) and the therapeutic success was non-significantly higher in the LAMS group compared to the DPPS group (100% vs. 85.71%), p = 0.095. Conclusions: Both DPPSs and LAMSs are characterized by high therapeutic success and low complication rates in patients undergoing ECG for PPCs.

A case of meningitis caused by Arcobacter butzleri

AbstractA 73‐year‐old male who was a compromised host developed bacteremia, pyelonephritis, and bacterial meningitis due to Arcobacter butzleri. To the best of our knowledge, this is the first report of meningitis caused by A. butzleri. The outcome of treatment with meropenem was excellent. We speculate that the ingestion of contaminated chicken meat caused bacteremia via the digestive tract, leading to pyelonephritis and meningitis. A. butzleri is a possible cause of bacterial meningitis even in the absence of severe gastrointestinal symptoms.

Histochemical Characterisation of the Turquoise-Fronted Parrot (Amazona aestiva) Digestive Tract.

Amazona aestiva, a member of the Psittacidae family, belongs to the genus Amazona. These animals are endowed with adaptations in their digestive systems that allow a natural diet composed of seeds and fruits, with anatomical characteristics that facilitate the acquisition and use of nutrients from these food groups. Although it is an important species, no previous information is available regarding the histology and histochemistry of its digestive tract. This study aimed to describe the morphological and histochemical characteristics of the digestive tract of this species. Fragments of the tongue, oesophagus, crop, proventriculus, ventriculus, small intestine, large intestine, liver and pancreas were collected from seven specimens without any clinical alterations in the digestive tract. A. aestiva's digestive tract observed the presence of an extremely developed tongue, a proventriculus with more delicate walls and a ventricle with less-developed musculature. Here, we present a detailed morphological and histochemical description of the Turquoise-fronted Parrot's digestive tract.

Finerenone: A Novel Drug Discovery for the Treatment of Chronic Kidney Disease.

The most common cause of chronic kidney disease (CKD) is diabetic nephropathy (DN). Primarilymineralocorticoid receptor antagonists (MRAs) (spironolactone and eplerenone), angiotensin-converting enzyme inhibitors or angiotensin receptor blockers were used for the treatment of CKD, but due to the high risk of hyperkalaemia, the combination was infrequently used. Currently after approval by FDA in 2021, finerenone was found to be effective in the treatment of CKD. Finerenone slowdowns the progression of diabetic nephropathy and lessens the cardiovascular morbidity in DN patients. The main objective of this review article is to provide a comprehensive and insightful overview of the role of finerenone by mainly focusing on its pharmacological properties, toxicity, uses, bioanalytical technique used for determination, and treatment options. Finerenone works by inhibiting the action of the mineralocorticoid receptor. Finerenone is quickly absorbed from the digestive tract after oral treatment and achieves peak plasma concentrations in 1-2 hours. Finerenone is actively metabolized through oxidation, epoxidation substitution, and direct hydroxylation. Elimination of finerenone is done through urine and feces. Determination of finerenone can be done through HPLC-MS and LSC. The present review covers the complete picture of ADME properties, bioanalytical techniques, clinical trials, toxicity, and possible avenues in this arena. Finerenone is effective compared to other mineralocorticoid receptor-like spironolactone and eplerenone, for the treatment of chronic kidney disease.

Identifying in vitro toxicity testing approaches for (novel) proteins in the context of food and feed risk assessment

Abstract This report documents the outcomes of the EFSA procurement (OC/EFSA/NIF/2022/01) aimed at identifying in vitro toxicity testing approaches for (novel) proteins in the context of food and feed safety assessment. In the present report, we present an integrated testing strategy for the evaluation of toxicity of novel/toxic proteins. A text‐mining approach was used to create a literature database of toxic outcomes associated with toxic proteins retrieved from the UniProt KB database using the search term “Toxin activity”. It was shown that toxic proteins are produced by a relatively limited phylogenetic subset, including, among others, bacteria, insects, serpents, molluscs, and fungi. Toxicological effects of these proteins are generally conserved within phylogenetic groups. Analysis of toxic effects from these proteins was performed using GO term analysis as well as a text‐mining based approach. Relevant tests to address and quantify these toxicity effects were identified and evaluated for their applicability in an in vitro based toxicity testing strategy. A stepwise approach was developed. As a first step, an initial in silico prediction of toxicity is carried out (Step 1). This is followed by a battery of in vitro assays to address the primary mechanisms of toxicity associated with toxic proteins (Step 2). If concern arises in the Step 2 battery of tests, the use of relevant in vitro model systems to explore potential target organ toxicity are required (Step 3). Knowledge gaps have been identified and recommendations are provided in in vitro toxicity testing strategies, in particular for (novel) proteins. Some of these gaps involve the selection and integration of a standardized, relevant in vitro digestion step, reflective of passage through the digestive tract, within the testing strategy, as well as a thorough assessment of the suitability and applicability of in vitro tests and new approach methodologies for regulatory toxicity assessment of (novel) proteins. To accelerate the incorporation of NAMs in the assessment of protein safety, case studies and proof of concept projects are needed to demonstrate the utility and effectiveness of in vitro toxicity testing strategies in the safety assessment of (novel) proteins.