Pharmacogenetics of sodium-glucose co-transporter-2 inhibitors: Validation of a sex-agnostic pharmacodynamic biomarker.

AIM: To validate pharmacodynamic responses to sodium-glucose co-transporter-2 (SGLT2) inhibitors and test for association with genetic variants in SLC5A4, SLC5A9, and SLC2A9. METHODS: Canagliflozin (300 mg), a SGLT2 inhibitor, was administered to 30 healthy volunteers. Several endpoints were measured to assess clinically relevant responses, including drug-induced increases in urinary excretion of glucose, sodium and uric acid. RESULTS: This pilot study confirmed that canagliflozin (300 mg) triggered acute changes in mean levels of several biomarkers: fasting plasma glucose (-4.1 mg/dL; P = 6 × 10-5 ), serum creatinine (+0.05 mg/dL; P = 8 × 10-4 ) and serum uric acid (-0.90 mg/dL; P = 5 × 10-10 ). The effects of sex on glucosuria depended upon how data were normalized. Whereas males' responses were ~60% greater when data were normalized to body surface area, males and females exhibited similar responses when glucosuria was expressed as grams of urinary glucose per gram-creatinine. The magnitude of glucosuria was not significantly correlated with fasting plasma glucose, estimated glomerular filtration rate or age in those healthy individuals without diabetes with an estimated glomerular filtration rate of more than 60 mL/min/1.73m2 . CONCLUSIONS: Normalizing data relative to creatinine excretion will facilitate including data from males and females in a single analysis. Furthermore, because our ongoing pharmacogenomic study (NCT02891954) is conducted in healthy individuals, this will facilitate detection of genetic associations with limited confounding by other factors such as HbA1c and renal function.

MFSD2A potentiates gastric cancer response to anti-PD-1 immunotherapy by reprogramming the tumor microenvironment to activate T cell response.

BACKGROUND: The efficacy of anti-programmed cell death protein 1 (PD-1) immunotherapy in various cancers, including gastric cancer (GC), needs to be potentiated by more effective targeting to enhance therapeutic efficacy or identifying accurate biomarkers to predict clinical responses. Here, we attempted to identify molecules predicting or/and promoting anti-PD-1 therapeutic response in advanced GC (AGC). METHODS: The transcriptome of AGC tissues from patients with different clinical responses to anti-PD-1 immunotherapy and GC cells was analyzed by RNA sequencing. The protein and mRNA levels of the major facilitator superfamily domain containing 2A (MFSD2A) in GC cells were assessed via quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. Additionally, the regulation of anti-PD-1 response by MFSD2A was studied in tumor-bearing mice. Cytometry by Time-of-Flight, multiple immunohistochemistry, and flow cytometry assays were used to explore immunological responses. The effects of MFSD2A on lipid metabolism in mice cancer tissue and GC cells was detected by metabolomics. RESULTS: Higher expression of MFSD2A in tumor tissues of AGC patients was associated with better response to anti-PD-1 immunotherapy. Moreover, MFSD2A expression was lower in GC tissues compared to adjacent normal tissues, and its expression was inversely correlated with GC stage. The overexpression of MFSD2A in GC cells enhanced the efficacy of anti-PD-1 immunotherapy in vivo by reprogramming the tumor microenvironment (TME), characterized by increased CD8+ T cell activation and reduced its exhaustion. MFSD2A inhibited transforming growth factor ß1 (TGFß1) release from GC cells by suppressing cyclooxygenase 2 (COX2)-prostaglandin synthesis, which consequently reprogrammed TME to promote anti-tumor T cell activation. CONCLUSIONS: MFSD2A potentially serves as a predictive biomarker for anti-PD-1 immunotherapy response in AGC patients. MFSD2A may be a promising therapeutic target to potentiate the efficacy of anti-PD-1 immunotherapy by reprogramming the TME to promote T cells activation.

Naloxone could limit morphine hypersensitivity: Considering the molecular mechanisms.

BACKGROUND: Naloxone has been used as an opioid antagonist to prevent multiple adverse side effects of opioid-like tolerance and hyperalgesia. This study has investigated naloxone combined with morphine to limit pain hypersensitivity. In addition, the expression of brain-derived neurotrophic factor (BDNF) and K+ Cl- cotransporter2 (KCC2) were also studied. METHODS: Forty-eight adult male Wistar rats (180-220 g) were divided into eight groups, with six rats in each group. Rats were divided into two tolerance and hyperalgesia groups; the sham group, the morphine group, the treatment group (naloxone along with morphine), and the sham group (naloxone along with saline) for eight consecutive days. Tail-flick test was performed on days 1, 5, and 8, and the plantar test on days 1 and 10. On days 8 and 10, the lumbar segments of the spinal cord were collected, and BDNF and KCC2 expression were analyzed using western blotting and immunohistochemistry, respectively. RESULTS: Results showed that tolerance and hyperalgesia developed following eight days of repeated morphine injection. BDNF expression significantly increased, but KCC2 was downregulated. Co-administration of naloxone and morphine decreased tolerance and hyperalgesia by decreasing BDNF and increasing KCC2 expression, respectively. CONCLUSION: This study suggests that BDNF and KCC2 may be candidate molecules for decreased morphine tolerance and hyperalgesia.

The sodium glucose co-transporter 2 inhibitor ertugliflozin for Alzheimer's disease: Inhibition of brain insulin signaling disruption-induced tau hyperphosphorylation.

An antidiabetic agent sodium glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin has been revealed to bind to catalytic anionic site of acetylcholinesterase (AChE), which is considered to be associated with the cognitive decline in neurodegenerative diseases, such as Alzheimer's disease (AD). The aim of the present study was thus to probe the effect of ertugliflozin on AD. Intracerebroventricular injection of streptozotocin (STZ/i.c.v) (3 mg/kg) was done bilaterally in male Wistar rats at 7-8 weeks of age. Two treatment doses (5 mg/kg and 10 mg/kg) of ertugliflozin were given intragastrically to STZ/i.c.v-induced rats for 20 days daily for behavioral assessment. Biochemical estimations of cholinergic activity, neuronal apoptosis, mitochondrial function and synaptic plasticity were performed. Behavioral results with ertugliflozin treatment revealed attenuation of cognitive deficit. Ertugliflozin also inhibited hippocampal AChE activity, downregulated pro-apoptotic marker expression, as well as mitigated mitochondrial dysfunction and synaptic damage in STZ/i.c.v rats. Importantly, we found that the hyperphosphorylation of tau in the hippocampus of STZ/i.c.v rats was decreased after oral administration of ertugliflozin, which was accompanied by decreased Phospho.IRS-1Ser307/Total.IRS-1 ratio and increased Phospho.AktSer473/Total.Akt and Phospho.GSK3ßSer9/Total.GSK3ß ratios. Our results indicated that treatment with ertugliflozin reversed AD pathology, which may be associated with inhibition of insulin signaling disruption-induced tau hyperphosphorylation.

The effects of postnatal erythropoietin and nano-erythropoietin on behavioral alterations by mediating K-Cl co-transporter 2 in the valproic acid-induced rat model of autism.

In this study, based on the excitatory/inhibitory imbalance theory of autism, the time window of GABA switch, the role of K-Cl co-transporter 2 (KCC2) in adjustment GABA switch, and brain permeability to erythropoietin (EPO), the effects of postnatal -EPO and- nano- erythropoietin (NEPO) have been evaluated in the valproic acid (VPA) rat model of autism. The VPA was administered for animal modeling of autism at gestational day (GD) 12.5 (600 mg/kg). Male offsprings were injected with EPO and NEPO in a clinically proper postnatal dosing regimen on postnatal days (PND) 1-5, and autistic-like behaviors were tested at the end of the first month. Then animals were sacrificed, and neuron morphology and KCC2 expression were examined by Nissl staining and Western blot. According to our findings, high-dose NEPO improved autism-associated phenotypes. Neuroprotective effects of EPO and NEPO have been shown in the hippocampus. Postnatal NEPO treatment reversed KCC2 expression abnormalities induced by prenatal VPA. Our results might support the role of KCC2 in ASD and the excitatory/inhibitory imbalance hypothesis. We suggested Nano- erythropoietin and other KCC2 interventions as a new approach to the early treatment and prevention of autism.

Development and optimization of amphiphilic self-assembly into nanostructured liquid crystals for transdermal delivery of an antidiabetic SGLT2 inhibitor.

The anti-hyperglycemic sodium glucose co-transporter 2 inhibitor Canagliflozin (CFZ) represents a recent antihyperglycemic modality, yet it suffers from low oral bioavailability. The current work aims to formulate CFZ-loaded transdermal nanostructured liquid crystal gel matrix (NLCG) to improve its therapeutic efficiency. Pre-formulation study included the construction of pseudoternary phase diagrams to explore the effect of two conventional amphiphiles against amphiphilic tri-block copolymer in the formulation of NLCG. The influence of different co-solvents was also investigated with the use of monooleine as the oil. Physical characterization, morphological examination and skin permeation were performed for the optimized formulations. The formula of choice was further investigated for skin irritation and chemical stability. Pharmacodynamic evaluation of the successful formula was conducted on hyperglycemic as well as normoglycemic mice. In addition, oral glucose tolerance test was conducted. Results revealed the supremacy of Poloxamer for stabilizing and maximizing liquid crystal gel (LCG) area percentage that reached up to 12.6%. CFZ-NLCG2 isotropic formula showed the highest permeation parameters; maximum flux value of 7460 µg/cm2 h and Q24 of 5327 µg/cm2. Pharmacodynamic evaluation revealed the superiority of the antihyperglycemic activity of CFZ-NLCG2 in fasting mice and its equivalence in the oral glucose tolerance test (OGTT) compared to the oral one. The obtained results confirmed the success of CFZ-NLCG2 in the transdermal delivery of CFZ in therapeutically effective concentration compared to the oral route, bypassing first pass effect; in addition, eliminates the possible gastrointestinal side effects related to the inhibition of intestinal sodium glucose co-transporter (SGLT) and maximizes its selectivity to the desired inhibition of renal SGLT.

Optimization of an Efficient Cell Culture Hepatitis B Infection System for Assessment of Hepatitis B Virus Neutralizing Monoclonal Antibodies.

BACKGROUND: Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection. OBJECTIVE: This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs. METHODS: Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications. RESULTS: Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system. CONCLUSION: Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics.

Continuous 17α-ethinylestradiol exposure impairs the sperm quality of marine medaka (Oryzias melastigma).

17α-ethinylestradiol (EE2) is an anthropogenic estrogen that is widely used for hormone therapy and oral contraceptives. It was reported that EE2 exposure induced reproductive impairments through processes affecting reproduction behavior and inducing ovotestis. However, the effects of continuous EE2 exposure on the reproductive performance remain largely unknown. In this study, adult marine medaka fish (Oryzias melastigma) were exposed to EE2 (85 ng/L) for one (F0) and two (F1) generations. Our results indicate that continuous EE2 exposure reduced fecundity and sperm motility. The testicular transcriptome, followed by bioinformatic analysis revealed the dysregulation of pathways related to steroidogenesis, sperm motility, and reproductive system development. Collectively, our findings indicate that continuous EE2 exposure directly affected sperm quality via the alteration of steroidogenesis and dysregulation of reproductive system development. The identified key factors including DNM1, PINK1, PDE7B, and SLC12A7 can serve as biomarkers to assess EE2-reduced sperm motility.

Effect of Empagliflozin and Dapagliflozin on Ambulatory Arterial Stiffness in Patients with Type 2 Diabetes Mellitus and Cardiovascular Co-Morbidities: A Prospective, Observational Study.

Background and Objectives: Individuals with type 2 diabetes mellitus (T2DM) have an increased risk of cardiovascular disease. Arterial stiffness is an independent prognostic marker for cardiovascular disease development. We aimed at determining the effect of two different sodium-glucose co-transporter-2 (SGLT-2) inhibitors on ambulatory arterial stiffness in individuals with T2DM. Materials and Methods: In this single-center, single-arm, prospective study performed from January 2020 to August 2021, we planned to enroll adult subjects with T2DM and stable antidiabetic and antihypertensive treatment, assigned either to empagliflozin or dapagliflozin for 6 months. All eligible subjects underwent ambulatory blood pressure monitoring. We set as the primary efficacy outcome the change in ambulatory pulse wave velocity (PWV) from baseline to week 24. Results: We finally enrolled 46 diabetic subjects, with a mean age of 62.89 (8.53) years and mean T2DM duration of 9.72 (6.37) years. Thirty patients received dapagliflozin, while sixteen patients received empagliflozin. Due to COVID-19 pandemic restrictive measures during the study, the mean follow-up period extended from 6 months to 9.98 (3.27) months. Regarding the prespecified primary efficacy outcome, we found that the SGLT-2 inhibitor treatment did not have a significant effect on PWV (p = 0.65). Prior history of cardiovascular disease did not significantly affect the observed effects. Other indices of arterial stiffness, such as augmentation index and central pulse pressure, were not significantly affected, neither by empagliflozin nor by dapagliflozin. Conclusions: SGLT-2 inhibitor treatment with empagliflozin or dapagliflozin in subjects with T2DM failed to improve ambulatory PWV over a mean follow-up of 10 months. Registration number: ISRCTN88851713.

Impact of SGLT2 inhibitors on the kidney in people with type 2 diabetes and severely increased albuminuria.

INTRODUCTION: Diabetes is the most common cause of end-stage kidney disease. Therapies such as sodium-glucose co-transporter-2 inhibitors have been identified over the last decade as effective oral hypoglycemic agents that also confer additional cardio and kidney protection. Knowledge of their mechanism of action and impact on patients with diabetes and albuminuria is vital in galvanizing prescriber confidence and increasing clinical uptake. AREAS COVERED: This manuscript discusses the pathophysiology of diabetic kidney disease, patho-physiological mechanisms for sodium-glucose co-transporter-2 inhibitors, and their impact on patients with type 2 diabetes mellitus and albuminuric kidney disease. EXPERT OPINION: Sodium-glucose co-transporter-2 inhibitors reduce albuminuria with consequent benefits on cardiovascular and kidney outcomes in patients with diabetes and severe albuminuria. While they have been incorporated into guidelines, the uptake of these agents into clinical practice has been slow. Increasing the uptake of these agents into clinical practice is necessary to improve outcomes for the large number of patients with diabetic kidney disease globally.P LAIN LANGUAGE SUMMARYPeople with type 2 diabetes and severe urinary protein loss are at high risk of progression to kidney failure requiring dialysis or transplantation. Preventing or slowing down loss of kidney function is crucial to preventing kidney failure. This review will discuss how diabetic kidney disease occurs, how a new family of glucose-lowering agents, the sodium-glucose co-transporter-2 inhibitors, work and how they affect people with type 2 diabetes mellitus who also have protein leaking from their kidneys. It will also detail the current data that underpins the guideline recommendations for use of these agents in the management of patients with and without diabetes.

Renoprotective effects of dapagliflozin in an iron overload non-diabetic rat model.

PURPOSE: Sodium glucose co-transporter (SGLT) 2 inhibitors are oral anti-diabetic drugs with proven kidney protective effects. Renal protective effects in non-diabetic individuals have also been shown in recent studies. The aim of this study was to determine the renal protective effects of dapagliflozin by evaluating the oxidative stress markers in the kidney tissue and demonstrating it in renal histological sections in an iron-overloaded rat model. METHODS: A total of 24 Wistar Albino rats were separated into 3 groups of 8 rats. Iron sucrose (60 â€‹mg/kg/day) was administered intraperitoneally to the first group (Group Fe) (n â€‹= â€‹8), iron sucrose and dapagliflozin (0.1 â€‹mg/kg/day) to the second group (Group Fe â€‹+ â€‹D) (n â€‹= â€‹8) and intraperitoneal saline as placebo to the control group (Group C) (n â€‹= â€‹8) for 4 weeks. The glomerular changes were semi-quantitatively scored with Oxford Classification. Oxidative stress was analyzed from the tissue fluorescent oxidation product (FLOP), malondialdehyde (MDA) and total sulfhydryl (T-SH) levels. RESULTS: Dapagliflozin prevented glomerular and mesangial damage of iron overload in the non-diabetic rat model. MDA levels were significantly higher in Group Fe compared to the Group C, and there was no significant difference between the Fe â€‹+ â€‹D group and Group C. T-SH levels were preserved in the Fe â€‹+ â€‹D group and were significantly higher than in the Fe group. CONCLUSIONS: The results of this study showed that dapagliflozin helped preserve the glomerular and mesangial structure histologically and reduced oxidative stress markers in a non-diabetic iron overload rat model.

Bile Acid Conjugation on Solid Nanoparticles Enhances ASBT-Mediated Endocytosis and Chylomicron Pathway but Weakens the Transcytosis by Inducing Transport Flow in a Cellular Negative Feedback Loop.

Bile acid-modified nanoparticles provide a convenient strategy to improve oral bioavailability of poorly permeable drugs by exploiting specific interactions with bile acid transporters. However, the underlying mechanisms are unknown, especially considering the different absorption sites of free bile acids (ileum) and digested fat molecules from bile acid-emulsified fat droplets (duodenum). Here, glycocholic acid (GCA)-conjugated polystyrene nanoparticles (GCPNs) are synthesized and their transport in Caco-2 cell models is studied. GCA conjugation enhances the uptake by interactions with apical sodium-dependent bile acid transporter (ASBT). A new pathway correlated with both ASBT and chylomicron pathways is identified. Meanwhile, the higher uptake of GCPNs does not lead to higher transcytosis to the same degree compared with unmodified nanoparticles (CPNs). The pharmacological and genomics study confirm that GCA conjugation changes the endocytosis mechanisms and downregulates the cellular response to the transport at gene levels, which works as a negative feedback loop and explains the higher cellular retention of GCPNs. These findings offer a solid foundation in the bile acid-based nanomedicine design, with utilizing advantages of the ASBT-mediated uptake, as well as inspiration to take comprehensive consideration of the cellular response with more developed technologies.

Sodium-glucose co-transporter-2 inhibitors in heart failure with reduced ejection fraction: Current evidence and future perspectives.

BACKGROUND: The sodium-glucose co-transporter-2 (SGLT2) inhibitors were developed as glucose-lowering drugs to treat type 2 diabetes (T2D). However, significant reductions in clinical outcomes have now been demonstrated in patients with heart failure with reduced ejection fraction (HFrEF), irrespective of the presence of T2D. Multiple hypotheses have been proposed for the underlying mechanisms, and the data to support these proposals are emerging. OBJECTIVES: To review the clinical outcome data with SGLT2 inhibitors in HFrEF and the data to support the mechanisms for these clinical effects. METHODS: Literature review was supported by a PubMed search for relevant articles up to 19 April 2022. FINDINGS: Current data support increased diuresis and reverse cardiac remodelling as important mechanisms for the reductions in heart failure hospitalizations and mortality observed with SGLT2 inhibitors (empagliflozin or dapagliflozin) in patients with HFrEF. Alteration in intrarenal haemodynamic is likely contributing to the observed renoprotective effect of SGLT2 inhibitors. CONCLUSIONS: Solid clinical data support the current recommendations to use empagliflozin or dapagliflozin in HFrEF. The underlying mechanisms likely include changes in cardiac and intrarenal haemodynamic. Yet, these mechanisms do not seem to solely explain the observed magnitude of clinical effect with SGLT2 inhibitors in HFrEF, and other mechanisms may contribute.

The effect of empagliflozin on contractile reserve in heart failure: Prespecified sub-study of a randomized, double-blind, and placebo-controlled trial.

BACKGROUND: Sodium-glucose co-transporter-2 inhibitors improve cardiac structure but most studies suggest no change in left ventricular (LV) systolic function at rest. Whether sodium-glucose co-transporter-2 inhibitors improve LV contractile reserve is unknown. We investigated the effect of empagliflozin on LV contractile reserve in patients with heart failure (HF) and reduced ejection fraction. METHODS: Prespecified sub-study of the Empire HF trial, a double-blind, placebo-controlled, and randomized trial. Patients with LV ejection fraction (LVEF) ≤ 40% on guideline-directed HF therapy were randomized (1:1) to empagliflozin 10 mg or placebo for 12 weeks. The treatment effect on contractile reserve was assessed by low dose dobutamine stress echocardiography. RESULTS: In total, 120 patients were included. The mean age was 68 (SD 10) years, 83% were male, and the mean LVEF was 38 (SD 10) %. Respectively 60 (100%) and 59 (98%) patients in the empagliflozin and placebo groups completed stress echocardiography. No statistically significant effect of empagliflozin was observed for the contractile reserve assessed by LV-GLS (adjusted mean absolute change, empagliflozin vs placebo, 0.7% [95% confidence interval {CI} -0.5 to 2.0, P = .25]) or LVEF (adjusted mean absolute change, empagliflozin vs placebo, 2.2% [95% CI -1.4 to 5.8, P = .22]) from baseline to 12 weeks. LV-GLS contractile reserve was associated with accelerometer-measured daily activity level (coefficient -24 accelerometer counts [95% CI -46 to -1.8, P = .03]). CONCLUSIONS: Empagliflozin for 12 weeks added to guideline-directed HF therapy did not improve LV contractile reserve in patients with HF and reduced ejection fraction.

Partial ablation of frontal cortical subplate leads to developmental abnormalities in KCC2 in the prefrontal cortex.

During early brain development, the subplate relays thalamocortical afferents to the overlying cortex. Disconnection of thalamic inputs to the prefrontal cortex by lesions of the subplate of the developing prefrontal cortex at early neonatal periods result in adult-onset behavioral abnormalities reminiscent of positive, negative, and cognitive symptoms of schizophrenia. Delayed maturation of γ-amino butyric acid (GABA) function may contribute to certain abnormalities of the prefrontal cortex and clinical manifestations of schizophrenia. Lesions to the subplate have also been implicated in developmental abnormalities of GABA neurotransmission in somatosensory and visual cortices. Therefore, we sought to examine the effects of subplate lesions in the developing prefrontal cortex of rats on the expression of GABA markers [parvalbumin and glutamic acid decarboxylase (GAD67)] and proteins responsible for GABAergic synaptic maturation [potassium-chloride cotransporter (KCC2) and sodium­potassium-chloride cotransporter (NKCC1)]. Lesioned and control rats were sacrificed between postnatal days (P) 5 and 90 and immunolabeled for parvalbumin, GAD67, KCC2, and NKCC1 in the prelimbic area of the prefrontal cortex. We found decreased immunoreactivity of KCC2 on neuronal cell membranes at P11 compared to control rats. However, the overall immunoreactivity of KCC2 and NKCC1 did not differ between lesion and control animals at all time points studied. Lesioned rats also showed decreased expression of parvalbumin, but not GAD67. Our results indicate that mechanisms underlying trafficking and membrane binding of KCC2 may contribute to altered GABA receptor function during development in schizophrenia.

Effects of in utero exposure to monosodium glutamate on locomotion, anxiety, depression, memory and KCC2 expression in offspring.

In pregnancy, there is a significant risk for developing embryos to be adversely affected by everyday chemicals such as food additives and environmental toxins. In recent times, several studies have documented the detrimental effect of exposure to such chemicals on the behaviour and neurodevelopment of the offspring. This study evaluated the influence of the food additive, monosodium glutamate (MSG), on behaviour and development in mice. Pregnant dams were exposed to MSG 2 or 4 g/kg or distilled water from gestation day 10-20. On delivery, postnatal day 1 (PN 1), 3 pups were sacrificed and whole brain samples assayed for KCC2 expression by western blot. The remaining pups were housed until PN 43 before commencing behavioural assessment. Their weights were measured at birth and at 3 days intervals until PN 42. The impact of prenatal exposure to MSG on baseline exploratory, anxiety and depression behaviours as well as spatial and working memory was assessed. In utero exposure to 4 g/kg MSG significantly reduced exploratory drive and increased depression-like behaviours but did not exert any significant impact on anxiety-like behaviours (p < 0.01). Additionally, there was a two-fold increase in KCC2 expression in both 2 and 4 g/kg MSG-exposed offspring. CONCLUSION: This study indicates that, in utero exposure to MSG increases the expression of KCC2 and causes significant effect on locomotion and depression-like behaviours but only marginally affects memory function.

Optimization of a cellular HBV infection model for use in high-throughput drug screening.

Hepatitis B virus (HBV) is a partially double-stranded DNA virus that specifically targets hepatocytes. It is considered a major health issue due to its high prevalence and the life-threatening consequences of chronic infection, including liver cirrhosis and hepatocellular carcinoma. Despite widespread vaccination against HBV, millions of people live with chronic HBV infection. Existing antiviral therapies fail to achieve full HBV elimination, so most patients with the disease require lifelong treatment. The search for new antiviral therapy strategies is hindered by the limited availability of in vitro HBV infection models that are able to support the full HBV life cycle. Therefore, the development and optimization of cellular models are crucial to the search for drugs effective against HBV. In this study, we optimized an in vitro HBV infection model consisting of two cell lines: HepAD38 cells, which are able to produce infectious HBV; and HepG2-NTCP cells, which are susceptible to HBV infection. We showed that prolonged production of HBV in the "donor" cells and HBV inoculation of the "acceptor" cells simultaneously with seeding improves the established procedure. This modified protocol was proven effective in experiments involving compounds with known activity against HBV, suggesting its utility for future high-throughput screening. Keywords: HBV; HBV in vitro models; HepG2-NTCP; HepAD38.

Long-term trans-inhibition of the hepatitis B and D virus receptor NTCP by taurolithocholic acid.

Human hepatic bile acid transporter Na+/taurocholate cotransporting polypeptide (NTCP) represents the liver-specific entry receptor for the hepatitis B and D viruses (HBV/HDV). Chronic hepatitis B and D affect several million people worldwide, but treatment options are limited. Recently, HBV/HDV entry inhibitors targeting NTCP have emerged as promising novel drug candidates. Nevertheless, the exact molecular mechanism that NTCP uses to mediate virus binding and entry into hepatocytes is still not completely understood. It is already known that human NTCP mRNA expression is downregulated under cholestasis. Furthermore, incubation of rat hepatocytes with the secondary bile acid taurolithocholic acid (TLC) triggers internalization of the rat Ntcp protein from the plasma membrane. In the present study, the long-term inhibitory effect of TLC on transport function, HBV/HDV receptor function, and membrane expression of human NTCP were analyzed in HepG2 and human embryonic kidney (HEK293) cells stably overexpressing NTCP. Even after short-pulse preincubation, TLC had a significant long-lasting inhibitory effect on the transport function of NTCP, but the NTCP protein was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were significantly reduced by TLC preincubation. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans-inhibition of the transport and receptor function of NTCP via a particular TLC-binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans-inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides an interesting novel NTCP target site for potential long-acting HBV/HDV entry inhibitors.NEW & NOTEWORTHY The hepatic bile acid transporter NTCP is a high-affinity receptor for hepatitis B and D viruses. This study shows that TLC rapidly accumulates in NTCP-expressing hepatoma cells and mediates long-lasting trans-inhibition of NTCP's transporter and receptor function via an intracellularly accessible domain, without substantially affecting its membrane expression. This domain is a promising novel NTCP target site for pharmacological long-acting HBV/HDV entry inhibitors.