Caffeine Decreases Topiramate Levels in Zebrafish Larvae in a Pentylenetetrazol-Induced Seizure Model.

Epilepsy ranks as the second-most prevalent neurological disease, and is characterized by seizures resulting in neurobiological and behavioral impairment. Naturally occurring in coffee beans or tea leaves, the alkaloid caffeine (CAF) is the most prevalent global stimulant. Caffeine has been observed to influence epileptic seizures and the efficacy of antiepileptic medications, with a notable impact on topiramate (TPM). This study aimed to explore the influence of CAF on TPM's anticonvulsant effects in zebrafish larvae within a PTZ-induced seizure model, concurrently determining TPM concentrations through a sophisticated analytical approach based on ultrahigh-performance liquid chromatography and subsequent mass spectrometric detection. Zebrafish larvae four days post-fertilization were incubated for 18 h with varying doses of TPM or combinations of CAF + TPM, and locomotor activity was then assessed. Seizures were induced by introducing a PTZ solution to achieve a final concentration of 20 mM. Utilizing liquid chromatography-mass spectrometry (LC-MS/MS), TPM levels in the larvae were quantified. CAF co-administration (especially in higher doses) with TPM caused a decrease in the average locomotor activity in the larvae compared to TPM alone. Moreover, CAF decreased TPM levels in the larvae at all investigated doses. In conclusion, these findings offer a novel perspective on the interplay between CAF and TPM, shedding light on previously unexplored facets. The potential impact of CAF consumption in assisting with epileptic seizure control, unless proven otherwise, suggests a noteworthy consideration for future research and clinical practices.

Optimization of the Zebrafish Larvae Pentylenetetrazol-Induced Seizure Model for the Study of Caffeine and Topiramate Interactions.

Epilepsy is a common neurological disorder characterized by seizures that cause neurobiological and behavioral impairment. Caffeine (CAF), which is the most widely consumed stimulant in the world, is reported to influence epileptic seizures and antiepileptic drugs, especially topiramate (TPM). The aim of the study was to optimize the zebrafish larvae pentylenetetrazol-induced seizure model for the study of CAF and TPM interactions, which include the determination of dose space, and the delivery of an analytical method for monitoring CAF, TPM, and CAF metabolite paraxanthine (PAR) in Zebrafish larvae. Methods: The zebrafish larvae, 4 days post-fertilization, were incubated for 18 h with CAF, TPM, or CAF + TPM, with subsequent locomotor activity assessment. Seizures were evoked by adding PTZ solution to obtain a final concentration of 20 mM. Subsequently, the liquid chromatography-mass spectrometry (LC-MS/MS) analytical method was used to simultaneously assess the levels of both CAF and TPM in the larvae. CAF (50 mg/L) and TPM (75 µM) given separately decreased the average larvae locomotor activity compared to the PTZ group but, however, were not able to lower it to the control level. Co-administration of 25 mg/L CAF and 50 µM TPM suppressed the activity to the same level. Adding 25 µM TPM to 50 mg/L CAF decrease the measured CAF level in the larvae. Until proven otherwise, CAF consumption should be regarded as a potential determinant in the modulation of TPM's efficacy in the management of epileptic seizures. The optimized model will contribute to the standardization of studying CAF and TPM interactions and building the understanding of the molecular bases of the interaction.

LC-MS Metabolomic Profiling of Five Types of Unrefined, Cold-Pressed Seed Oils to Identify Markers to Determine Oil Authenticity and to Test for Oil Adulteration.

The authenticity of food products marketed as health-promoting foods-especially unrefined, cold-pressed seed oils-should be controlled to ensure their quality and safeguard consumers and patients. Metabolomic profiling using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF) was employed to identify authenticity markers for five types of unrefined, cold-pressed seed oils: black seed oil (Nigella sativa L.), pumpkin seed oil (Cucurbita pepo L.), evening primrose oil (Oenothera biennis L.), hemp oil (Cannabis sativa L.) and milk thistle oil (Silybum marianum). Of the 36 oil-specific markers detected, 10 were established for black seed oil, 8 for evening primrose seed oil, 7 for hemp seed oil, 4 for milk thistle seed oil and 7 for pumpkin seed oil. In addition, the influence of matrix variability on the oil-specific metabolic markers was examined by studying binary oil mixtures containing varying volume percentages of each tested oil and each of three potential adulterants: sunflower, rapeseed and sesame oil. The presence of oil-specific markers was confirmed in 7 commercial oil mix products. The identified 36 oil-specific metabolic markers proved useful for confirming the authenticity of the five target seed oils. The ability to detect adulterations of these oils with sunflower, rapeseed and sesame oil was demonstrated.

The Involvement of the Endogenous Opioid System in the Gastrointestinal Aging in Mice and Humans.

Nearly 20% of elderly patients suffer from constipation, but the age-related changes in the gastrointestinal (GI) tract remain insufficiently elucidated. In this study, the alterations within the endogenous opioid system (EOS) as a potential cause of constipation in the elderly were evaluated. The GI functions were assessed in vitro and in vivo and compared between 6-, 12- and 18-month old mice. Moreover, the effect of opioid receptor (MOP, DOP, KOP) agonists on the mouse GI tract functions and the EOS components expression in mouse tissues and colonic biopsies from patients with functional constipation were determined. In the oldest mice, the GI peristalsis was significantly impaired as compared to the younger groups. The tissue response to MOP and DOP, but not KOP, agonists weakened with age in vitro; for DOP, it was confirmed in vivo. In the mouse upper GI tract, Oprm1, Oprd1, Oprk1 expression decreased with age; in the colon, Oprm1 expression increased. There were no differences in the expression of these genes in the colonic biopsies from patients >50 years old as compared to the younger group. In conclusion, the age-related impairment of the GI peristalsis may result from reduced MOP and DOP response to the activation with opioid agonists or the alterations in the EOS expression.

Characterization of the Synergistic Effect between Ligands of Opioid and Free Fatty Acid Receptors in the Mouse Model of Colitis.

BACKGROUND: Recent studies suggest that lipids, including free fatty acids (FFAs), are necessary for proper µ opioid receptor (MOR) binding and that activation of opioid receptors (ORs) improves intestinal inflammation. The objective of the study was to investigate a possible interaction between the ORs and FFA receptors (FFARs) ligands in the colitis. METHODS: The potential synergistic effect of ORs and FFARs ligands was evaluated using mouse model of acute colitis induced by dextran sulfate sodium (DSS, 4%). Compounds were injected intraperitoneally (i.p.) once or twice daily at the doses of 0.01 or 0.02 mg/kg body weight (BW) (DAMGO-an MOR agonist), 0.3 mg/kg BW (DPDPE-a δ OR (DOR) agonist) and 1 mg/kg BW (naloxone-a non-selective OR antagonist, GLPG 0974-a FFAR2 antagonist, GSK 137647-a FFAR4 agonist and AH 7614-a FFAR4 antagonist) for 4 days. RESULTS: Myeloperoxidase (MPO) activity was significantly decreased after DAMGO (0.02 mg/kg BW) and GSK 137647 (1 mg/kg BW) administration and co-administration as compared to DSS group. CONCLUSIONS: Treatment with ligands of ORs and FFARs may affect the immune cells in the inflammation; however, no significant influence on the severity of colitis and no synergistic effect were observed.

Activation of Free Fatty Acid Receptor 4 Affects Intestinal Inflammation and Improves Colon Permeability in Mice.

Diet is considered an important trigger in inflammatory bowel diseases (IBD), as feeding habits can affect intestinal permeability and clearance of bacterial antigens, consequently influencing the immune system. Free fatty acid receptors (FFARs), expressed on the intestinal epithelial cells, belong to the family of luminal-facing receptors that are responsive to nutrients. The objective of this study was to characterize the anti-inflammatory activity and the effect on intestinal barrier function of synthetic FFAR agonists in mouse models of colitis. Therapeutic activity of GW9508 (FFAR1 agonist), 4-CMTB (FFAR2 agonist), AR420626 (FFAR3 agonist), and GSK137647 (FFAR4 agonist) was investigated in two models of semi-chronic colitis: induced by trinitrobenzenesulfonic acid (TNBS), mimicking Crohn's disease, as well as induced by dextran sulfate sodium (DSS), which recapitulates ulcerative colitis in humans. Moreover, we assessed the influence of FFARs agonists on epithelial ion transport and measured the ion flow stimulated by forskolin and veratridine. Administration of FFAR4 agonist GSK137647 attenuated both TNBS-induced and DSS-induced colitis in mice, as indicated by macroscopic parameters and myeloperoxidase activity. The action of FFAR4 agonist GSK137647 was significantly blocked by pretreatment with selective FFAR4 antagonist AH7614. Moreover, FFAR1 and FFAR4 agonists reversed the increase in the colon permeability caused by inflammation. FFAR4 restored the tight junction genes expression in mouse colon. This is the first evaluation of the anti-inflammatory activity of selective FFAR agonists, showing that pharmacological intervention targeting FFAR4, which is a sensor of medium and long chain fatty acids, attenuates intestinal inflammation.

Attitude and Behaviors towards SARS-CoV-2 Vaccination among Healthcare Workers: A Cross-Sectional Study from Poland.

Healthcare workers are particularly exposed to biological risk during their daily occupational activities. Nowadays, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become one of the most widespread infectious agents. In the current study, we performed a survey on the attitude and behavior of Polish healthcare workers (HCW), which comprise physicians (MD) and administrative healthcare assistants (HA) towards the Coronavirus Disease 2019 (COVID-19) vaccination. Our study involved 2300 subjects (42.17% female; 10.96% MD; 5.87% HA). The evaluation was conducted using a Google Forms survey based on original questions and the Depression, Anxiety and Stress Scale-21 Items questionnaire. HCW significantly more often demonstrated their willingness to get vaccinated against the SARS-CoV-2 as compared to the control group (82.95% vs. 54.31%, respectively). The main concern, as regards all groups, was the development of long-term side effects after getting COVID-19 vaccine. The study revealed that depression significantly affects the willingness to get vaccinated. The readiness was significantly strengthened by positive medical history of recommended vaccinations, fear of catching COVID-19, as well as fear of passing on the disease to the relatives. Overall, the percentage of HCW, who want to be vaccinated against COVID-19 remains unsatisfactory. Further works exploring this subject are needed to take a step closer to achieving the herd immunity in the era of the COVID-19 pandemic.

Medical Students and SARS-CoV-2 Vaccination: Attitude and Behaviors.

Since physicians play a key role in vaccination, the initial training of medical students (MS) should aim to help shape their attitude in this regard. The beginning of vaccination programs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an excellent time to assess the attitudes held by both medical and non-medical students regarding vaccination. A 51- to 53-item questionnaire including the Depression, Anxiety and Stress Scale was administered to 1971 students (49.21% male; 34.86% MS); two career-related questions were also addressed to the MS. The majority of surveyed students indicated a desire to get vaccinated against SARS-CoV-2, with more medical than non-medical students planning to get vaccinated (91.99% vs. 59.42%). The most common concern about SARS-CoV-2 infection was the risk of passing on the disease to elderly relatives. While conspiracy theories regarding the COVID-19 vaccine are less popular among MS, both groups indicated concerns that vaccines may cause autism is equally common (~5%). Further studies exploring social attitudes towards the SARS-CoV-2 vaccine are a necessary first step to optimizing vaccination programs and achieving herd immunity.

Mental Well-Being (Depression, Loneliness, Insomnia, Daily Life Fatigue) during COVID-19 Related Home-Confinement-A Study from Poland.

The COVID-19 pandemic is a great threat to both physical and mental health as it may lead to psychological stress connected with an economic crisis, threat of unemployment, or fear of losing family members. Emerging data shows that the general public may be vulnerable to the pandemic-related stress and experience frequently prevalent anxiety. A study involving 471 subjects (85.6% female) was conducted online during the COVID-19 pandemic. We used the following scales: Insomnia Severity Index (ISI), Beck Depression Inventory (BDI), Revised University of California, Los Angeles (R-UCLA) Loneliness Scale, and Daily Life Fatigue scale (DLF). Women had higher mean scores of depression, loneliness, and daily life fatigue and more often than males started exercising. Among people professionally active before the pandemic, there were more cases of increased alcohol consumption than among students. No differences in alcohol consumption patterns were found between genders. People living alone had higher scores of loneliness and daily life fatigue compared to those living with someone. Respondents who started taking any new drugs during COVID-19 home confinement had higher outcomes in all questionnaires. During home confinement, high scores of depression, insomnia, loneliness, and everyday fatigue were observed.

Walnut Oil Alleviates Intestinal Inflammation and Restores Intestinal Barrier Function in Mice.

Ulcerative colitis belongs to inflammatory bowel diseases, which is a group of chronic disorders of the gastrointestinal tract. It is a debilitating condition with a wide range of symptoms including rectal bleeding, diarrhea, and visceral pain. Current dietary habits often lead to imbalance in n-6/n-3 polyunsaturated fatty acids (PUFA) in favor of n-6 PUFA. Recent data showed the potential anti-inflammatory advantage of n-3 PUFA. Walnut oil (WO) is rich in those fatty acids and mainly consists of linoleic and linolenic acids that may act via free fatty acids receptors (FFARs). We assessed the anti-inflammatory effect of WO in the mouse model of dextran sulfate sodium (DSS)-induced colitis. Moreover, we examined changes in the expression of tight junction proteins (TJ), pro-inflammatory cytokines, and FFAR proteins in the inflamed mouse colon. WO improves the damage score in inflamed tissue, significantly restoring ion transport and colonic wall permeability. Inflammation caused changes in TJ, FFAR, and pro-inflammatory gene proteins expression, which WO was able to partially reverse. WO has anti-inflammatory properties; however, its exact mechanism of action remains unclear. This stems from the pleiotropic effects of n-3 PUFA ligands associated with receptor distribution and targeted signaling pathways.

Expression of FFAR3 and FFAR4 Is Increased in Gastroesophageal Reflux Disease.

BACKGROUND: The negative impact of a high-fat diet on the course of gastroesophageal reflux disease (GERD) has been previously reported. Free fatty acid receptors (FFARs) may be mediators of this phenomenon. The aim of this study was to characterize the role of FFARs in the course of nonerosive (NERD) and erosive (ERD) reflux disease. METHODS: Collectively, 73 patients (62 with GERD and 11 healthy controls (HCs)) were recruited to the study. Esophageal biopsies were drawn from the lower third of the esophagus and kept for further experiments. Quantitative, real-time polymerase chain reaction was used to assess the expression of FFAR1, FFAR2, FFAR3, and FFAR4 in biopsies. Histological evaluation of dilated intracellular spaces (DISs) was also performed. RESULTS: FFAR3 exhibited the highest expression, and FFAR4 exhibited the lowest expression in all esophageal samples. Higher relative expression of FFAR1 and FFAR2 and significantly higher expression of FFAR3 (p = 0.04) was noted in patients with GERD compared to respective HCs. Patients with nonerosive GERD (NERD) presented higher expression of all FFARs compared to patients with erosive GERD (ERD) and respective HCs. Interestingly, in patients with ERD, the expression of FFAR3 was lower than in HCs. Significant, weak, positive correlation was found for FFAR3 and FFAR4 expression and DIS scores (r = 0.36, p < 0.05 for FFAR 3, and r = 0.39, p < 0.05 for FFAR4). CONCLUSIONS: In this study, we show that FFARs may play a role in GERD pathogenesis, particularly in the NERD type. It may be assumed that FFARs, in particular FFAR3 and FFAR4, may have diagnostic and therapeutic potential in GERD.

Free Fatty Acid Receptors as new potential therapeutic target in inflammatory bowel diseases.

Family of Free Fatty Acid Receptors (FFARs), specific G protein-coupled receptors comprises of four members: FFAR1-4, where each responds to different chain length of fatty acids (FAs). Over the years, FFARs have become attractive pharmacological targets in the treatment of type 2 diabetes, metabolic syndrome, cardiovascular diseases and asthma; recent studies also point to their role in inflammation. It is now well-established that activation of FFAR1 and FFAR4 by long and medium chain FAs may lead to reduction of inflammatory state; FFAR2 and FFAR3 are activated by short chain FAs, but only FFAR2 was shown to alleviate inflammation, mostly by neutrophil inhibition. All FFARs have thus been proposed as targets in inflammatory bowel diseases (IBD). Here we discuss current knowledge and future directions in FFAR research related to IBD.

Free Fatty Acid Receptors as New Potential Targets in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide. In developed countries, its mortality remains high, yet the prevalence has established owing to effective screening programs; however due to the westernization of lifestyle, the incidences in many other countries have increased. Although the treatment of CRC has improved in the last few years, the side effects of these approaches cannot be neglected. Recently, members of the family of free fatty acid receptors (FFARs) have become attractive pharmacological targets in many diseases, including asthma; studies also point to their role in carcinogenesis. Here, we discuss current knowledge and future directions in FFAR research related to CRC. Contradictory results of FFARs modulation may derive from the pleiotropic effects of FFAR ligands, receptor distribution and different signal transduction. Hence, we indicate directions of further studies to fully use the potential of FFARs in CRC.

Diagnostic challenges in interstitial lung diseases using the example of microscopic usual interstitial pneumonia image in a patient with the clinical hypothesis of idiopathic pulmonary fibrosis - a case report.

Idiopathic pulmonary fibrosis (IPF) is the most common of the idiopathic interstitial pneumonias. The high-resolution tomography (HRCT) is highly recommended among patients with suspected IPF. Usual interstitial pneumonia (UIP) is the histopathological and radiological pattern of IPF. IPF is diagnosed if the appropriate combination of HRCT patterns and histopathological patterns are present. CASE REPORT: The authors present a case of a 49-years-old woman who was hospitalised due to the detection of a focal lesion in the left lung in an X-ray examination. A CT scan of the chest with contrast revealed a solid infiltration of the same area. The histopathology from a transbronchial lung biopsy from this abnormality showed microscopic honeycomb, fibroblast fibrosis, macrophage clusters loaded with hemosiderin and widened bronchioles - an image suggesting Usual Interstitial Pneumonia (UIP). Following that, a HRCT showed considerable differences in the lung image compared to the previous CT examination - no polycyclic lesion in the left lung was detected and the fibrous changes decreased. The patient had substantial formal features of UIP from the histopathological examination from TBLB. Therefore, there was a clinicalradiological discrepancy in the histopathology report. After a multidisciplinary consultation by renowned experts in pulmonology, radiology and histopathology, regardless of the patient's diagnostic path, the primary cause of the disease could not be determined. The analysis of this case strongly suggests that the histopathological examination alone is not sufficient in the diagnosis of IPF.

Dietary fatty acid content influences the expression of genes involved in the lipid turnover and inflammation in mouse colon and spleen.

BACKGROUND: Dietary interventions can improve gastrointestinal (GI) symptoms. We determined the effects of fatty acids (FAs) supplementation with medium- and long-chain saturated FAs on mouse GI motility and correlated them with the expression of genes for free FA receptors (FFAR)1-4, FA binding protein 4 (FABP4) and inflammation. METHODS: Forty-eight BalbC were assigned to: standard diet (STD), diet rich in medium-chain saturated FAs (COCO) and long-chain saturated FAs (HF) (7% by weight). Body weight (BW) and food intake (FI) were monitored for 8-weeks. GI motility was determined by fecal pellet output (FPO) and colon bead expulsion tests. FABP4 inhibitor, BMS309403 (1mg/kg, ip) was injected to half of each group 2 days/week. mRNA expression of FABP4, (FFAR)1-4, and pro-inflammatory cytokines were measured in colonic and splenic tissues using real-time PCR. RESULTS: COCO and HF decreased FI. COCO accelerated overall GI transit (p<0.05). COCO increased the mRNA expression of FFAR2 (p<0.001) and TNFα (p<0.01); HF increased the expression of FABP4 and FFAR4 (p<0.05), and FFAR2 (p<0.001) in the colon, and decreased FFAR1 and FFAR4 (p<0.001), TNFα (p<0.01) and IL-1ß (p<0.05) in splenic tissues. BMS309403 decreased the FI and delayed colonic transit in STD+BMS and COCO+BMS vs. STD (p<0.05). HF+BMS increased colonic expression of FFAR3 (p<0.01), TNFα (p<0.01), IL-6 (p<0.01), and reduced FFAR4 (p<0.05); COCO+BMS decreased TNFα (p<0.01). CONCLUSION: Diversification in the dietary lipid content affected GI motility in mice and the expression of FFARs and pro-inflammatory cytokines in vivo.

Exploring the Reliability and Validity of the Huntington's Disease Quality of Life Battery for Carers (HDQoL-C) within A Polish Population.

Huntington's disease (HD) is a rare genetic neurodegenerative disorder that causes motor disorders, neuropsychiatric symptoms and a progressing deterioration of cognitive functions. Complex issues resulting from the hereditary nature of HD, the complexity of symptoms and the concealed onset of the disease have a great impact on the quality of life of family carers. The caregivers are called the "forgotten people" in HD, especially with relation to genetic counseling. This study aims to explore the reliability and validity of the Huntington's Disease Quality of Life Battery for carers (HDQoL-C) within a Polish population. A total of 90 carers recruited from the Enroll-HD study in Polish research centers of the European Huntington's Disease Network completed a polish translation of the HDQoL-C. Data were subjected to Principle Components Analysis (PCA) and reliability measures. The Polish version of the shortened versions of the HDQoL-C is similarly valid compared to the original English version and suitable for use within this population. The HDQoL-C has previously demonstrated a wide range of benefits for practitioners in capturing and understanding carer experience and these benefits can now be extended to Polish speaking populations.

Possible application of trefoil factor family peptides in gastroesophageal reflux and Barrett's esophagus.

Gastroesophageal reflux disease (GERD) is a chronic disorder of the digestive tract characterised mainly by a heartburn. Being one of the most common gastrointestinal diseases, the prevalence of GERD reaches up to 25.9% in Europe. Barrett's esophagus (BE) is an acquired condition characterized by the replacement of the normal stratified squamous epithelium with metaplastic columnar epithelium. BE is believed to develop mainly from chronic GERD and is the most important risk factor of esophageal adenocarcinoma. Despite the availability of drugs such as proton pomp inhibitors and antacids, GERD is still a burden to local economy and impairs health-related quality of life in patients. Also, the endoscopic surveillance in patients with BE is burdensome and expensive what drives the need for biomarker of intestinal metaplasia and dysplasia. Trefoil factor family (TFF), consisting of TFF1, TFF2 and TFF3 peptides is gaining more and more attention due to its unique biochemical features and numerous functions. In this review the role of TFF1, TFF2 and TFF3 as potential treatment option and/or biomarker in the upper GI tract is discussed with particular focus on GERD and BE.

New Trends in Liposome-based Drug Delivery in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common cancers in both men and women. Approximately one-third of patients do not survive five years from diagnosis, which indicates the need for treatment improvement, also through new ways of drug delivery. A possible strategy to increase treatment efficacy is the use of liposomal formulation, which allows delivering both hydrophobic and hydrophilic compounds with better biocompatibility and reduced side-effects. Liposomal formulations showed better antitumor activity, longer drug accumulation and no cytotoxic effect on normal cells when compared to free drugs. In this review, we will present liposomal preparations studied in CRC in vitro and in vivo. We will focus on the advantages of liposomal delivery over conventional therapy as well as modifications which increase specificity, drug accumulation and efficacy. Moreover, we will discuss formulations investigated in clinical trials. Liposomal delivery has a great potential in overcoming current limitations of cancer therapy and development of this system gives new perspectives in CRC treatment.