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Abstract

[6S]-5-methyltetrahydrofolic acid (MTHF) is a proposed replacement for folic acid (FA) in diets and prenatal supplements. This study compared the effects of these two forms on maternal metabolism and hypothalamic gene expression. Pregnant Wistar rats received an AIN-93G diet with recommended FA (1X, 2 mg/kg, control), 5X-FA or equimolar levels of MTHF. During lactation they received the control diet and then a high fat diet for 19-weeks post-weaning. Body weight, adiposity, food intake, energy expenditure, plasma hormones, folate, and 1-carbon metabolites were measured. RNA-sequencing of the hypothalamus was conducted at parturition. Weight-loss from weaning to 1-week post-weaning was less in dams fed either form of the 5X vs. 1X folate diets, but final weight-gain was higher in 5X-MTHF vs. 5X-FA dams. Both doses of the MTHF diets led to 8% higher food intake and associated with lower plasma leptin at parturition, but higher leptin at 19-weeks and insulin resistance at 1-week post-weaning. RNA-sequencing revealed 279 differentially expressed genes in the hypothalamus in 5X-MTHF vs. 5X-FA dams. These findings indicate that MTHF and FA differ in their programing effects on maternal phenotype, and a potential adverse role of either form when given at the higher doses.

2.1. Animals and Diets

All animal work was approved by the University of Toronto Animal Care Committee (#20011892). First-time pregnant Wistar rats (2–3 days, 170–250 g) were purchased from Charles River Farms (Quebec, Canada) and individually housed upon arrival. Animals were maintained on a reverse 12-h dark-light cycle (lights on at 07:00 a.m. at 22 ± 1 °C) and ad libitum food and water were provided throughout the study. Dams were randomly allocated to one of four dietary interventions that were provided contemporaneously and only during pregnancy until term (3 weeks). Pregnant dams (n = 16–18/group) received an isocaloric AIN-93G diet (energy density 4.0 kcal/g) [] containing either the recommended (1X, 2 mg/kg diet, control) or 5X-quantity of FA for rodents (1X-FA or 5X-FA) or equimolar recommended levels of [6S]-5-methyltetrahydrofolic acid, calcium salt (1X, 2.1 mg/kg diet, Metafolin®, Merck & Cie, Schaffhausen, Switzerland) or 5X-quantity of MTHF (1X-MTHF or 5X-MTHF). FA and MTHF diets at equimolar doses have been used in clinical trials assessing folate status and related-health effects [,,]. The recommended (1X) dose was chosen based on the FA requirements for rodents for adequate growth rate [] and reflects the FDA requirements for non-pregnant women (400 µg/day) []. The 5X dose was chosen to reflect FA intakes commonly consumed in North America above the basal dietary requirements for humans [,]. Diets were custom made by Research Diets Inc. (New Brunswick, NJ, USA) () and stored at 4 °C and in the dark until provided fresh weekly. The additions of the MTHF form of folate into foods has been shown to be stable at both 4 °C and room temperature for up to 24 months []. Analytical testing of the high folate diets following a 36-month storage at −80 °C showed >80% retention of folates (), further confirming their long-term stability.

Introduction

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection and is characterized by a dramatic increase in inducible nitric oxide synthase (NOS) expression and activity with excessive production of nitric oxide (NO) [,]. As a result, sepsis leads to endothelial dysfunction and profound systemic vasodilation with associated hypotension which can have deleterious effects [,]. Therefore, a careful, sustained reduction in NO burden represents a potential therapeutic target. Hydroxocobalamin (HCB), which has traditionally been used in the treatment of cyanide toxicity, affects NO-mediated vasoplegia by (1) directly inhibiting NOS, decreasing NO production, and (2) by binding directly to NO and acting as a scavenger [-]. HCB has been successfully used in the treatment of refractory vasoplegia, particularly in cardiac surgery and liver transplant patients [-], but has not been extensively explored in septic shock. Shapeton et al. comprehensively summarized the pharmacology, toxicology, and clinical evidence surrounding the use of HCB in vasoplegic patients []. In this case report, we describe how a single extended duration infusion of high-dose HCB facilitates the resolution of catecholamine-refractory septic shock.

Case presentation

A 73-year-old male with a 30-pack-year tobacco smoking history and poorly differentiated T4N0M0 right upper lobe lung adenocarcinoma status post-neoadjuvant chemotherapy underwent a right upper lobectomy. His comorbidities included type 2 diabetes mellitus, hyperlipidemia, and prostate cancer status post-radiation therapy and radical prostatectomy. He was discharged on postoperative day (POD) 12 but was re-admitted on POD 16 in the setting of a right-sided empyema and septic shock. After administration of empiric antibiotics, the patient was taken to the operating room for right thoracotomy with decortication. His intraoperative course was complicated by poor gas exchange with profound hypoxemia (arterial pO2/fraction inspired O2 or P/F ratio = 50; ref >300) and hypercarbia (partial pressure of carbon dioxide = 100 mmHg). In the context of this new-onset acute respiratory distress syndrome (ARDS) with poor compliance and associated elevated driving pressures (plateau pressure = 37 cmH20, positive end-expiratory pressure = 12 cmH20), the patient suffered an intraoperative, contralateral tension pneumothorax that required emergent needle decompression and subsequent left-sided chest tube placement. Intraoperatively, the patient required escalating doses of epinephrine, norepinephrine, and vasopressin to support hemodynamics. An intraoperative transthoracic echocardiogram (TTE) was performed, which revealed right ventricular (RV) dysfunction with an underfilled and hyperdynamic left ventricle (LV). These compounding factors resulted in a severe respiratory and metabolic acidosis with a pH of 7.1 at the time of arrival to the intensive care unit (ICU).

Case presentation

Despite normalization of his cardiac function (by bedside TTE and calculated CI), the patient continued to require high-dose infusions of norepinephrine (up to 0.09 mcg/kg/min), vasopressin (up to 0.04 units/min), and epinephrine (up to 0.1 mcg/kg/min), suggestive of vasodilatory shock. His vasopressor requirement did not improve after administration of stress dose steroids. Indeed, 23 hours after admission to the ICU, repeat central venous SatO2 was 73.9% for a calculated CI of 6 L/min/m2 by Fick. To address the refractory shock, he was given a one-time sustained infusion of HCB (5 g) administered over four hours. This resulted in a rapid, complete, and definitive weaning from all vasoactive agents over the ensuing 14 hours (Figure ).

Formation of bisretinoid fluorophores by reaction of vitamin a aldehyde with lipid

Incubation of bovine outer segments with all-trans-retinal mimics bisretinoid formation in vivo, and by optimizing the detection of these bisretinoid species, their formation by all-trans-retinal reactivity was confirmed (). Indeed, adjusting the ratio of all-trans-retinal to PE in synthetic mixtures to determine how the two precursors affect bisretinoid production showed that the yield of bisretinoid saturates when the ratio of equivalents of all-trans-retinal to PE (egg-PE as starting material) is 4:2; no further increase occurred with eight equivalents of all-trans-retinal (8:2 ratio) (). Conversely, increasing the concentration of PE from an equivalent ratio of 4:1 through 4:8 (all-trans-retinal to PE) was associated with a steady increase in bisretinoid product. These findings indicated that the concentration of PE in the environment of the outer segment membrane has a marked effect on the rate of bisretinoid synthesis. Formation of NRPE, the first step in the synthetic pathway, is likely the most facile reaction. In the Abca4−/− mouse, a mouse model exhibiting accelerated bisretinoid formation, the level of PE in the outer segment membrane was reported to be increased (). It has been suggested that a higher level of PE in Abca4−/− mice may facilitate sequestering of all-trans-retinal as NRPE, thereby accelerating recovery of the rod photoresponse after a 30–40% bleach ().

Formation of bisretinoid fluorophores by reaction of vitamin a aldehyde with lipid

The unsaturated fatty acid 22:6n-3 (DHA) is abundant in the outer segments of photoreceptor cells, and DHA content has been shown to have several effects on retina (), including susceptibility to light damage. When 22:6n-3-containing PE was used as precursor in the synthetic reaction mixture versus egg-PE (a mixture of fatty acid moieties), phosphatidyl-pyridinium bisretinoid (A2PE) production was found to be greater with the latter. Then again, if free DHA was added to the egg-PE/all-trans-retinal reaction mixture, the yield of A2PE was increased. Under deprotonating conditions associated with the addition of base (triethylamine) to the reactants, the generation of A2PE was increased. Altogether, not only is the phospholipid PE the reactant that combines with all-trans-retinal to form bisretinoid, the lipid composition of outer segments may also impact the extent to which the reaction may be favored.

Abstract

Background: to explore the diagnostic accuracy of 18F-Fluciclovine positron-emission tomography (PET) in prostate cancer (PCa), considering both primary staging prior to radical therapy, biochemical recurrence, and advanced setting. Methods: A systematic web search through Embase and Medline was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Studies performed from 2011 to 2020 were evaluated. The terms used were “PET” or “positron emission tomography” or “positron emission tomography/computed tomography” or “PET/CT” or “positron emission tomography-computed tomography” or “PET-CT” and “Fluciclovine” or “FACBC” and “prostatic neoplasms” or “prostate cancer” or “prostate carcinoma”. Only studies reporting about true positive (TP), true negative (TN), false positive (FP) and false negative (FN) findings of 18F-fluciclovine PET were considered eligible. Results: Fifteen out of 283 studies, and 697 patients, were included in the final analysis. The pooled sensitivity for 18F-Fluciclovine PET/CT for diagnosis of primary PCa was 0.83 (95% CI: 0.80–0.86), the specificity of 0.77 (95% CI: 0.74–0.80). The pooled sensitivity for preoperative LN staging was 0.57 (95% CI: 0.39–0.73) and specificity of 0.99 (95% CI: 0.94–1.00). The pooled sensitivity for the overall detection of recurrence in relapsed patients was 0.68 (95% CI: 0.63–0.73), and specificity of 0.68 (95% CI: 0.60–0.75). Conclusion: This meta-analysis showed promising results in term of sensitivity and specificity for 18F-Fluciclovine PET/CT to stage the primary lesion and in the assessment of nodal metastases, and for the detection of PCa locations in the recurrent setting. However, the limited number of studies and the broad heterogeneity in the selected cohorts and in different investigation protocols are limitation affecting the strength of these results.

2. Materials and Methods

A systematic web search through Embase and Medline was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Studies performed from 2011 to 2020 were evaluated. The terms used were “PET” or “positron emission tomography” or “positron emission tomography/computed tomography” or “PET/CT” or “positron emission tomography-computed tomography” or “PET-CT” and “Fluciclovine” or “FACBC” and “prostatic neoplasms” or “prostate cancer” or “prostate carcinoma”. Full-text publications in English were considered. The study population comprised male patients with histologically proven PCa. Studies using 18F-Fluciclovine in pre-surgery setting as well as biochemical recurrence and advanced setting were considered. Two investigators (C.C. and C.R.) independently performed the literature search. The inclusion criteria considered for each study included an assessment of true positive (TP), true negative (TN), false positive (FP), and false negative (FN) results of 18F-Fluciclovine PET or PET / CT for the diagnosis of primary cancer, lymph node staging and the detection of recurrence. Only studies providing these information were finally included in the study. The overall quality of the studies included was independently assessed by two authors, based on the 15 modified items Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2, ) []. Discrepancies between researchers were resolved with consensus.

3.2.1. Detection of Primary Prostate Cancer Lesion

The per-patient pooled sensitivity for 18F-Fluciclovine PET/CT for the diagnosis of primary PCa was 0.83 (95% CI: 0.80–0.86), with I-square: 89.6% (a) and a pooled of specificity of 0.77 (95% CI: 0.74–0.80) with I –square: 94.8% (b). The likelihood ratio (LR) syntheses gave an overall LR+ of 5.22 (95% CI: 2.15–12.67) (c) and LR– of 0.17 (95% CI: 0.8–0.38) (d). The pooled DOR was 35.43 (95%CI: 6.66–188.55) (e). The SROC curve indicates that the area under the cure was: 0.9224 (f).

Abstract

The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxamine-5′-phosphate and pyridoxine-5′-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6.

Abstract

The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxamine-5′-phosphate and pyridoxine-5′-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6.

Introduction

Since animals and humans are unable to synthesise vitamin B6, these organisms have to take up vitamin B6 with their diet (Kraemer et al. ). Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry (Acevedo-Rocha et al. ). PN hypochloride, the commercial form of vitamin B6, is synthesised from PN and added usually in combination with other vitamins to food products like bakery products, cereals, baby nutrition, multivitamin juices and other beverages (Domke et al. ; Eggersdorfer et al. ). Moreover, PN is added to the food that is used for intensive animal farming to improve the yield (Johnson et al. ; Verbeek ; Eggersdorfer et al. ). Until today, the B6 vitamers are fully chemically synthesised using expensive and toxic chemicals (Eggersdorfer et al. ). Therefore, there is considerable interest in the biotech industry to develop a sustainable fermentation process for the production of vitamin B6.

Abstract

Natamycin (NAT) is the drug of choice for the treatment of fungal keratitis (FK). However, its inherent shortcomings, such as poor solubility, high dosing frequency, and long treatment cycle, need to be urgently addressed by designing a new delivery to widen its clinical utility. Growing research has confirmed that clotrimazole (CLZ) plays a significant role in fungal growth inhibition. Hence, coaxial electrospray (CO-ES) technology is used herein to prepare nano-systems with an average hydrodynamic particle size of 309-406 nm for the co-delivery of NAT and CLZ in chitosan (CTS) and poly(lactic-co-glycolic acid) (PLGA). The resulting NAT/CLZ@CTS/PLGA formulations were characterized by a transmission electron microscope (TEM) and in vitro release test. The results show that the formulations had obvious core-shell structures, uniform particle distribution, and also can sustain the release of drugs over 36 h. Furthermore, in vitro hemolysis, in vivo corneal irritation test, local allergenic test, and antifungal activity analyses are performed to evaluate the safety and efficiency of the formulations. Thus, good biosafety along with a significant anti-candidiasis effect are found in the NAT/CLZ@CTS/PLGA nanoparticles (NPs). Taken together, the results suggest that this design may provide a promising drug delivery system and a new option for the treatment of FK.

Preparation of Electro-Sprayed NPs

Various NP formulations were produced, including the single drug-loaded formulations (NAT@CTS/PLGA and CLZ@CTS/PLGA) and the co-loaded formulation NAT/CLZ@CTS/PLGA.

In vitro Drug Release

The in vitro release of NAT and CLZ from electro-sprayed NPs was performed using a dialysis bag (3,500 molecular weight cutoff, membrane-cell, Chicago, IL, United States) (). First, the samples were centrifuged at 12,000 rpm for 10 min and then the supernatant was discarded and the recovered NPs were rinsed with distilled water to exclude the free drugs. Subsequently, 2.0-ml samples were added into a dialysis bag, which was then immersed in PBS (50.0 ml) and gently swung at 37°C and 100 rpm. The release medium was sampled (1.0-ml) at predetermined time and immediately replaced with 1 ml fresh release medium (phosphate buffered saline, PBS, pH 7.4) after each sampling. The cumulative dose of NAT and CLZ was measured by HPLC method as described above.

Abstract

Natamycin (NAT) is the drug of choice for the treatment of fungal keratitis (FK). However, its inherent shortcomings, such as poor solubility, high dosing frequency, and long treatment cycle, need to be urgently addressed by designing a new delivery to widen its clinical utility. Growing research has confirmed that clotrimazole (CLZ) plays a significant role in fungal growth inhibition. Hence, coaxial electrospray (CO-ES) technology is used herein to prepare nano-systems with an average hydrodynamic particle size of 309-406 nm for the co-delivery of NAT and CLZ in chitosan (CTS) and poly(lactic-co-glycolic acid) (PLGA). The resulting NAT/CLZ@CTS/PLGA formulations were characterized by a transmission electron microscope (TEM) and in vitro release test. The results show that the formulations had obvious core-shell structures, uniform particle distribution, and also can sustain the release of drugs over 36 h. Furthermore, in vitro hemolysis, in vivo corneal irritation test, local allergenic test, and antifungal activity analyses are performed to evaluate the safety and efficiency of the formulations. Thus, good biosafety along with a significant anti-candidiasis effect are found in the NAT/CLZ@CTS/PLGA nanoparticles (NPs). Taken together, the results suggest that this design may provide a promising drug delivery system and a new option for the treatment of FK.

Preparation of Electro-Sprayed NPs

Various NP formulations were produced, including the single drug-loaded formulations (NAT@CTS/PLGA and CLZ@CTS/PLGA) and the co-loaded formulation NAT/CLZ@CTS/PLGA.

Zone of Inhibition Tests

The antifungal activity of electro-sprayed NPs was assessed against C. albicans by the zone of inhibition (ZOI) test (), which determined the level of antifungal activity that inhibits the growth of C. albicans. The NAT/CLZ@CTS/PLGA NPs were tested at a concentration of drugs equivalent to that of the NAT@CTS/PLGA NPs and CLZ@CTS/PLGA NPs. First, the C. albicans were co-cultured with sterilized SDB slants at 37°C and diluted with fresh medium to a concentration of 106 CFU/ml after 24 h of co-culture. Meanwhile, the sterilized SGA medium was poured into a disposable Nest culture plate and allowed to coagulate at room temperature (25 ± 1°C). The C. albicans (0.5 ml) were then evenly swabbed onto each plate and a 6-mm-diameter sterilized filter paper was then placed in the center of each culture plate. Then, an 8-μl sample of the NAT/CLZ@CTS/PLGA NPs was added to a fresh, sterilized 6-mm-diameter filter paper in a separate plate, and PBS (0.02 M, pH 7.4) was added as the negative control. To allow sufficient time for the fungi to grow and the formulations to diffuse, all the plates were placed in a low-temperature incubator (Shanghai Jinghong Experimental Equipment Co. LTD, SHP-150, China) at 37°C for 48 h. The potential antifungal activity was then presented as the diameter (mm) of ZOI.

Abstract

Treating neuropathic pain remains challenging, and therefore new pharmacological strategies are urgently required. Here, the enhancement of glycinergic neurotransmission by either facilitating glycine receptors (GlyR) or inhibiting glycine transporter (GlyT) function to increase extracellular glycine concentration appears promising. Propacetamol is a N,N-diethylester of acetaminophen, a non-opioid analgesic used to treat mild pain conditions. In vivo, it is hydrolysed into N,N-diethylglycine (DEG) and acetaminophen. DEG has structural similarities to known alternative GlyT1 substrates. In this study, we analyzed possible effects of propacetamol, or its metabolite N,N-diethylglycine (DEG), on GlyRs or GlyTs function by using a two-electrode voltage clamp approach in Xenopus laevis oocytes. Our data demonstrate that, although propacetamol or acetaminophen had no effect on the function of the analysed glycine-responsive proteins, the propacetamol metabolite DEG acted as a low-affine substrate for both GlyT1 (EC50 > 7.6 mM) and GlyT2 (EC50 > 5.2 mM). It also acted as a mild positive allosteric modulator of GlyRα1 function at intermediate concentrations. Taken together, our data show that DEG influences both glycine transporter and receptor function, and therefore could facilitate glycinergic neurotransmission in a multimodal manner.

1. Introduction

To foster a fast translation to clinical application, we searched for substances that might have potential effects on glycine sensitive proteins, but that are already used in clinics. We focused on substances that display, by themselves or by one of their metabolites, structural similarities to known GlyT1 substrates (i.e., glycine, sarcosine, or N-ethylglycine). Here, we identified propacetamol, a N,N-diethylglycine ester of acetaminophen that was developed initially to increase its solubility for a possible intravenous application and to reduce hepatotoxicity by prolonged release of acetaminophen [,]. After systemic application of propacetamol, it is cleaved by esterase to acetaminophen and N’N’-Diethyl-glycine (). In this study we assessed the effect of N,N-diethylglycine (DEG) on both glycine transporter (GlyT) and glycine receptor (GlyR) function in a Xenopus laevis oocyte expression system by a two-electrode voltage clamp approach. We demonstrate that DEG, but not acetaminophen or propacetamol, functions as a low affinity alternative substrate for both GlyT1 and GlyT2. Moreover, DEG shows partial agonistic activity on homomeric GlyRα1, but not on GlyRα2 or GlyRα3. Taken together, our data raise the possibility that propacetamol, in addition to its well-characterized function as a COX inhibitor via acetaminophen, might influence glycine-dependent neurotransmission via a multimodal mechanism.

2.4. Electrophysiology

All two-electrode-voltage-clamp (TEVC) recordings were performed using a TurboTec-03X amplifier (npi elctronics, Tamm, Germany), and a custom-made recording chamber with the membrane potential set to −50 mV. Only oocytes showing a stable leak current <300 nA at the beginning of the experiment were used. Oocytes were superfused with ND96-Ca2+. Solutions containing substances at the indicated concentration for 20 s followed by a washout for at least 30 s or until stable baseline currents were observed. All measurements were performed at room temperature (21–23 °C). Current traces were evaluated using the software Cell Works and Cell Works Reader (npi electronics, Tamm, Germany). Excel (Microsoft, Redmond, WA, USA) and Graphpad Prism 7 (Graphpad Software, Inc., San Diego, CA, USA) were used for statistical analysis. Data are presented as mean ± SEM in dose-response curves or boxplots with whiskers from minimum to maximum with median and quartiles.

3.2. GlyT Responses to Propacetamol and Paracetamol and DEG

To test if propacetamol or acetaminophen had any effect on the activity of GlyT1 or GlyT2, GlyT-expressing oocytes were superfused with each of these substances. Neither propacetamol nor acetaminophen alone produced any current response. Alkaline pretreatment, however, which is predicted to cause hydrolysis of the propacetamol (10 mM) to acetaminophen and DEG, resulted in significant inward currents in both GlyT1 and GlyT2-expressing oocytes, reaching 20–40% of the maximum current amplitude. Currents of slightly larger amplitudes (50–70%) were observed after the application of 10 mM DEG alone. Adding 20 µM glycine to 10 mM acetaminophen and 10 mM propacetamol solution each did not significantly alter the current response of any GlyT compared to 20 µM glycine solution alone (A,B).

Abstract

Supplementation with [6S]-5-methyltetrahydrofolic acid (MTHF) is recommended as an alternative to folic acid (FA) in prenatal supplements. This study compared equimolar gestational FA and MTHF diets on energy regulation of female offspring. Wistar rats were fed an AIN-93G diet with recommended (2 mg/kg diet) or 5-fold (5X) intakes of MTHF or FA. At weaning, female offspring were fed a 45% fat diet until 19 weeks. The 5X-MTHF offspring had higher body weight (>15%), food intake (8%), light-cycle energy expenditure, and lower activity compared to 5X-FA offspring (p < 0.05). Both the 5X offspring had higher plasma levels of the anorectic hormone leptin at birth (60%) and at 19 weeks (40%), and lower liver weight and total liver lipids compared to the 1X offspring (p < 0.05). Hypothalamic mRNA expression of leptin receptor (ObRb) was lower, and of suppressor of cytokine signaling-3 (Socs3) was higher in the 5X-MTHF offspring (p < 0.05), suggesting central leptin dysregulation. In contrast, the 5X-FA offspring had higher expression of genes encoding for dopamine and GABA- neurotransmitter receptors (p < 0.01), consistent with their phenotype and reduced food intake. When fed folate diets at the requirement level, no differences were found due to form in the offspring. We conclude that MTHF compared to FA consumed at high levels in the gestational diets program central and peripheral mechanisms to favour increased weight gain in the offspring. These pre-clinical findings caution against high gestational intakes of folates of either form and encourage clinical trials examining their long-term health effects when consumed during pregnancy.

2.1. Animals and Diets

First-time pregnant (2–3 days) Wistar rats weighing 170–250 g (Charles River Farms, QC, Canada) were singly housed in a 12 h dark-light cycle (lights on at 0700 hrs at 22 ± 1 °C) and provided ad libitum food and water throughout the study. The protocol (# 20011892) was approved by the University of Toronto Animal Care Committee. Upon arrival, dams (n = 16–18/group) were randomly allocated to one of four custom-made dietary interventions (Research Diets Inc., New Brunswick, NJ, USA) that were provided only during their 3-week pregnancy: an AIN-93G diet (energy density 4.0 kcal/g) containing FA at either recommended (1X-FA, 2 mg/kg diet, control) or high (5X-FA, 10 mg/kg diet) quantities for rodents or [6S]-5-methyltetrahydrofolic acid calcium salt (Metafolin®, Merck & Cie, Schaffhausen, Switzerland) at equimolar recommended (1X-MTHF, 2.1 mg/kg diet) or high quantities (5X-MTHF, 10.4 mg/kg diet). Diets were kept in the dark at 4 °C until freshly provided to the animals weekly. Diets were analytically tested for long-term stability []. FA and MTHF were provided at equimolar levels in the diet due to a suggested comparable bioavailability of MTHF at equimolar levels to FA. Equimolar levels are used in clinical trials comparing folate status and homocysteine level maintenance [,]. The FA form was used as a reference to calculate the MTHF equimolar dose equivalent as only the synthetic folate form contains a tolerable upper level (1000 µg/day). The recommended (1X) FA dose provides 2 mg/kg of FA (4000 kcal) and represents the basal requirement for rodents for adequate growth rate []. This dose reflects recommendations of 0.4 mg of FA/day for women at low risk of neural-tube defects planning a pregnancy []. The supplemented FA diet contains 5X the basal requirement for rodents or 10 mg/kg of FA (4000 kcal). This dose reflects FA intakes commonly consumed by pregnant women in North America during the post-fortification era at >2-fold above basal dietary requirements for humans [,]. As MTHF is incorporated at the equivalent dose as FA in supplements, intakes may soon increase beyond recommendations [].

3.1. X-MTHF Offspring Gained more Weight and Ate more Food Than 5X-FA Offspring

A Dose×Form interaction effect was also found on measures of cumulative food intake up to 19 weeks post-weaning (Dose p = 0.7, Form p = 0.05, Dose×Form p = 0.02, B). The 5X-MTHF female offspring consumed ~8% more food compared to the 5X-FA offspring, but food intakes of the 1X folate offspring were similar to both 5X groups. Cumulative food intake and total weight gain of the offspring were also strongly and positively correlated within each group (1X-FA r = 0.89, p < 0.001, 1X-MTHF r = 0.90, p < 0.001, 5X-FA r = 0.71, p < 0.01; 5X-MTHF r = 0.72, p < 0.01).

5. Conclusions

MTHF compared to FA consumed at high levels in the gestational diet program central and peripheral mechanisms to favour increased weight gain in the female offspring. These pre-clinical findings caution against high gestational intakes of folates of either form, and encourage clinical trials examining their long-term health effects when consumed during pregnancy.

Abstract

The probiotic Bacillus subtilis strain 29784 (Bs29784) has been shown to improve performance in broilers. In this study, we used a metabolomic and 16S rRNA gene sequencing approach to evaluate effects of Bs29874 in the broiler intestine. Nicotinic acid and hypoxanthine were key metabolites that were produced by the strain in vitro and were also found in vivo to be increased in small intestinal content of broilers fed Bs29784 as dietary additive. Both metabolites have well-described anti-inflammatory effects in the intestine. Furthermore, Bs29784 supplementation to the feed significantly altered the ileal microbiome of 13-day-old broilers, thereby increasing the abundance of genus Bacillus, while decreasing genera and OTUs belonging to the Lactobacillaceae and Enterobacteriacae families. Moreover, Bs29784 did not change the cecal microbial community structure, but specifically enriched members of the family Clostridiales VadinBB60, as well as the butyrate-producing families Ruminococcaceae and Lachnospiraceae. The abundance of various OTUs and genera belonging to these families was significantly associated with nicotinic acid levels in the cecum, suggesting a possible cross-feeding between B. subtilis strain 29784 and these beneficial microbes. Taken together, the data indicate that Bs29784 exerts its described probiotic effects through a combined action of its metabolites on both the host and its microbiome.

2.1. Bacterial Strain and Growth Conditions

Bs29784 is a commercially available probiotic for broilers (Alterion® NE, Adisseo, Commentry, France). The commercial product contains 1010 CFU/g spores of B. subtilis strain 29784 and is mixed at 1 g/kg in the feed that is supplied to broilers. For in vitro experiments, a pure culture of Bs29784 was obtained by inoculating the commercial probiotic in Luria–Bertani (LB) broth (Sigma-Aldrich, St. Louis, MO, USA). The bacteria were grown overnight at 37 °C under aerobic conditions. Bacteria were plated on LB plates and their identity was confirmed via matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) [] and Sanger sequencing of the 16S region []. Bacterial growth was determined in LB broth over a 24-hour time span (grown in triplicate). Bacterial supernatant was obtained by centrifugation (5 min, 13,300 rpm) and filtered using a Polyvinylidene difluoride (PVDF) membrane filter (0.22 µm × 13 mm diameter, Kynar 500®, Arkema, Amsterdam, The Netherlands). Blank samples (medium without bacteria) were incubated simultaneously with the bacterial samples and processed in the same way to serve as controls. Samples were stored at −80 °C until metabolomic analysis.

2.2. Animal Trial

The study was undertaken following the guidelines of the ethics committee of the Faculty of Veterinary Medicine, Ghent University, in accordance with the EU Directive 2010/63/EU. One-day-old Ross 308 broiler chicks were obtained from a local hatchery and divided into 2 groups of 5 birds consisting of (1) a control group that received a standard commercial diet and (2) a group that received a standard commercial diet supplemented with the commercial Bs29784 probiotic at a dose of 1010 CFU/kg feed (FARM 1&2 mash, Versele-laga, Deinze, Belgium). Animals were housed on a solid floor covered with wood shavings at a density of 5 birds/m2. Animals were subjected to a light schedule of 12 h light and 12 h dark. All broilers were given water and feed ad libitum. At 13 days of age, all birds were weighed, the birds were euthanized, and digestive content from the jejunum, ileum and cecum was collected. These samples were frozen in liquid nitrogen directly after sampling and stored at −20 °C until further processing. The material from the 3 sections was used for metabolomic analysis and Bacillus quantification, while the ileal and cecal content was used for 16S sequencing. At 13 days of age, no differences in bodyweight could be observed, with an average bodyweight of 273.4 g ± 19.54 g (mean ± SD) for the control group and 254.3 g ± 38.37 g for the Bs29784-supplemented group (p = 0.358).