Molecular mechanism of choline and ethanolamine transport in humans.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

Lack of SPNS1 results in accumulation of lysolipids and lysosomal storage disease in mouse models.

Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1-KO cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global KO of Spns1 caused embryonic lethality between E12.5 and E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1-KO mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K/AKT signaling pathway. Furthermore, we identified 3 human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.

MFSD7c functions as a transporter of choline at the blood-brain barrier.

Mutations in the orphan transporter MFSD7c (also known as Flvcr2), are linked to Fowler syndrome. Here, we used Mfsd7c knockout (Mfsd7c-/-) mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics analysis and detected differential changes of metabolites in the brains and livers of Mfsd7c-/-embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c-/- embryos. Thus, we hypothesized that MFSD7c regulates the level of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch clamp analysis showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c was shown to be conserved in vertebrates, but not in yeasts. We demonstrated that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. We also showed that several missense mutations identified in patients exhibiting Fowler syndrome had abolished or reduced choline transport activity. Mice lacking Mfsd7c in endothelial cells of the central nervous system suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c was required for exporting choline derived from lysophosphatidylcholine in the brain. Collectively, our work identifies MFSD7c as a choline exporter at the BBB and provides a foundation for future work to reveal the disease mechanisms of Fowler syndrome.

Effects of maternal nutrient restriction during gestation on bovine serum microRNA abundance.

The objective was to determine the effects of maternal nutrient restriction during gestation on serum microRNA (miRNA) abundance in cattle. Primiparous Angus-cross cows (n=22) were fed either control (CON; to gain 1 Kg/week) or nutrient restricted (NR; 0.55% NEm) diets based on National Research Council requirements. On day 30 of gestation, cows were blocked by body condition and randomly assigned to one of three diets: CON (n=8) days 30-190; NR (n=7) days 30-110 followed by CON days 110-190 (NR/C); or CON (n=7) days 30-110 followed by NR days 110-190 (C/NR). At 190 days of gestation, maternal serum was collected for RNA isolation and analyzed using a miRNA microarray of known Bos taurus sequences. Data were normalized using LOWESS and analyzed via ANOVA. At 190 days of gestation, 16 miRNAs exhibited differential abundance (P<0.05) between treatments. Cows that underwent NR, irrespective of when the insult occurred, had downregulated bta-miR-126-3p compared to CON cows. Bta-miR-16b was downregulated and three miRNAs upregulated in NR/C compared to C/NR and CON cows. Additionally, seven miRNAs were downregulated and four miRNAs upregulated in C/NR compared to NR/C and CON cows. Comparison of NR/C and C/NR cows revealed three differentially abundant (P<0.04) miRNAs (bta-miR-2487_L-2R-3_1ss15CT, bta-miR-215, and bta-miR-760-5p). Top KEGG pathway enrichment of target genes included: pathways in cancer, PI3K-Akt signaling, focal adhesion, Ras signaling, proteoglycans in cancer, and MAPK signaling. In summary, maternal nutrient restriction altered serum miRNA abundance profiles irrespective of the time at which the nutritional insult was induced.

Synthesis and evaluation of druglike parameters via in silico techniques for a series of heterocyclic monosquarate-amide derivatives as potential carboxylic acid bioisosteres.

Herein, we present a synthetic compound library comprising of 13 structurally diverse heterocyclic monosquarate-amide derivatives. The compounds featured in this library were designed as potential bioisosteric replacements carboxylic acid moiety's. A good selection of the compounds presented exhibit unique molecular architecture and have shown promising results following in silico evaluation of 'druglike properties' using Swiss ADME. The research presented in this work focuses on the preparation of derivatives of 3,4-dihydroxycyclobut-3-ene-1,2-dione, a known carboxylic acid bioisostere.

Mfsd7b facilitates choline transport and missense mutations affect choline transport function.

MFSD7b belongs to the Major Facilitator Superfamily of transporters that transport small molecules. Two isoforms of MFSD7b have been identified and they are reported to be heme exporters that play a crucial role in maintaining the cytosolic and mitochondrial heme levels, respectively. Mutations of MFSD7b (also known as FLVCR1) have been linked to retinitis pigmentosa, posterior column ataxia, and hereditary sensory and autonomic neuropathy. Although MFSD7b functions have been linked to heme detoxification by exporting excess heme from erythroid cells, it is ubiquitously expressed with a high level in the kidney, gastrointestinal tract, lungs, liver, and brain. Here, we showed that MFSD7b functions as a facilitative choline transporter. Expression of MFSD7b slightly but significantly increased choline import, while its knockdown reduced choline influx in mammalian cells. The influx of choline transported by MFSD7b is dependent on the expression of choline metabolizing enzymes such as choline kinase (CHKA) and intracellular choline levels, but it is independent of gradient of cations. Additionally, we showed that choline transport function of Mfsd7b is conserved from fly to man. Employing our transport assays, we showed that missense mutations of MFSD7b caused reduced choline transport functions. Our results show that MFSD7b functions as a facilitative choline transporter in mammalian cells.

Influence of marker weights optimization on scapular kinematics estimated with a multibody kinematic optimization.

Scapular kinematic estimates are altered by soft tissue artefacts, therefore experimental and numerical methods should be developed to improve their accuracy. This study aimed to assess the influence of weights applied to the scapula markers within a closed-loop multibody kinematic optimization on scapular kinematic estimates. Fifteen healthy volunteers performed static postures mimicking analytical, daily living and sport movements. Scapulo-thoracic angles were computed either from a scapula locator as the reference, or from a closed-loop multibody-kinematic optimization (MKO) including a participant-specific point-on-ellipsoid scapulothoracic joint. Weights applied to scapula markers in the MKO were optimized to minimize the difference in scapular orientation from the reference. Optimizing weighting sets significantly (p < 0.0001) improved scapular orientation from 0.9° to 12.1° in comparison to scapular kinematics estimated with non-optimized weighting sets. The mean optimized weighting set contained no neglectable weight for all markers from the acromion to the medial border of the scapular spine but showed no significant difference (p = 0.547) compared to homogeneous weights. Optimized weighting sets were participant- and movement- specific. To conclude, homogenous weights applied on redundant markers located from acromion to scapular medial border spine are recommended when estimating scapular kinematics in upper limb MKO.

Postnatal deletion of Spns2 prevents neuroinflammation without compromising blood vascular functions.

Protein Spinster homolog 2 (Spns2) is a sphingosine-1-phosphate (S1P) transporter that releases S1P to regulate lymphocyte egress and trafficking. Global deletion of Spns2 (Spns2-/-) has been shown to reduce disease severity in several autoimmune disease models. To examine whether Spns2 could be exploited as a drug target, we generated and characterized the mice with postnatal knockout of Spns2 (Spns2-Mx1Cre). Our results showed that Spns2-Mx1Cre mice had significantly low number of lymphocytes in blood and lymphoid organs similar to Spns2-/- mice. Lymph but not plasma S1P levels were significantly reduced in both groups of knockout mice. Our lipidomic results also showed that Spns2 releases different S1P species into lymph. Interestingly, lymphatic vessels in the lymph nodes (LNs) of Spns2-/- and Spns2-Mx1Cre mice exhibited morphological defects. The structures of high endothelial venules (HEV) in the LNs of Spns2-Mx1Cre mice were disorganized. These results indicate that lack of Spns2 affects both S1P secretion and LN vasculatures. Nevertheless, blood vasculature of these Spns2 deficient mice was not different to controls under homeostasis and vascular insults. Importantly, Spns2-Mx1Cre mice were resistant to multiple sclerosis in experimental autoimmune encephalomyelitis (EAE) models with significant reduction of pathogenic Th17 cells in the central nervous system (CNS). This study suggests that pharmacological inhibition of Spns2 may be exploited for therapeutic applications in treatment of neuroinflammation.

Mfsd2b and Spns2 are essential for maintenance of blood vessels during development and in anaphylactic shock.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompanied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our results show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.

Methamphetamine Enhances Cryptococcus neoformans Melanization, Antifungal Resistance, and Pathogenesis in a Murine Model of Drug Administration and Systemic Infection.

Methamphetamine (METH) is a major public health and safety problem in the United States. Chronic METH abuse is associated with a 2-fold-higher risk of HIV infection and, possibly, additional infections, particularly those that enter through the respiratory tract or skin. Cryptococcus neoformans is an encapsulated opportunistic yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients, especially in individuals with AIDS. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. l-3,4-Dihydroxyphenylalanine (l-Dopa) is a natural catecholamine that is frequently used to induce melanization in C. neoformans. l-Dopa-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents, and heavy metals. Using a systemic mouse model of infection and in vitro assays to critically assess the impact of METH on C. neoformans melanization and pathogenesis, we demonstrated that METH-treated mice infected with melanized yeast cells showed increased fungal burdens in the blood and brain, exacerbating mortality. Interestingly, analyses of cultures of METH-exposed cryptococci supplemented with l-Dopa revealed that METH accelerates fungal melanization, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.

Effects of nutrient restriction during early or mid-gestation in bovine on placental development and miRNA expression in the cotyledon.

The objective of this study was to determine effects of maternal nutrient restriction (NR) during early or mid-gestation on uterine composition and miRNA expression in cotyledons. Primiparous Angus-cross cows (n = 38) were synchronized and inseminated using male sexed semen, blocked by body condition score and body weight (BW), and assigned to treatments. Animals were fed either: control (CON; gain 1 kg/week) or NR (55% maintenance energy and crude protein requirements) based on BW. An initial set of animals were fed either NR (n = 8) or CON (n = 8) from day 30-110 of gestation. A second set of animals were fed CON (n = 8) d 30-190 (CON/CON); NR (n = 7) day 30-110 followed by CON day 110-190 (NR/CON); or CON (n = 7) day 30-110 followed by NR day 110-190 (CON/NR). Cows were harvested on day 110 or 190 of gestation to collect placental tissues. RNA was isolated from cotyledon samples (3 animals/group) prior to microarray analysis using known Bos taurus microRNA sequences. Relative microRNA abundance was analyzed via ANOVA. Maternal NR increased (P < 0.05) cotyledon weight and total placentome surface area irrespective of gestational day. At day 110 of gestation, 51 microRNAs were reduced while 91 microRNAs observed greater abundance (P < 0.05) in NR verses CON cotyledons. At day 190 of gestation, 40 microRNAs were reduced and 26 microRNAs were increased (P < 0.05) in both NR/CON and CON/NR verses CON cotyledons. Top KEGG pathway analysis included: axon guidance, endocytosis, neuroactive ligand receptor interaction, and MAPK signaling pathway. Early-gestation maternal NR altered microRNA abundance to a greater extent than mid-gestation NR.

The role of SLC transporters for brain health and disease.

The brain exchanges nutrients and small molecules with blood via the blood-brain barrier (BBB). Approximately 20% energy intake for the body is consumed by the brain. Glucose is known for its critical roles for energy production and provides substrates for biogenesis in neurons. The brain takes up glucose via glucose transporters GLUT1 and 3, which are expressed in several neural cell types. The brain is also equipped with various transport systems for acquiring amino acids, lactate, ketone bodies, lipids, and cofactors for neuronal functions. Unraveling the mechanisms by which the brain takes up and metabolizes these nutrients will be key in understanding the nutritional requirements in the brain. This could also offer opportunities for therapeutic interventions in several neurological disorders. For instance, emerging evidence suggests a critical role of lactate as an alternative energy source for neurons. Neuronal cells express monocarboxylic transporters to acquire lactate. As such, treatment of GLUT1-deficient patients with ketogenic diets to provide the brain with alternative sources of energy has been shown to improve the health of the patients. Many transporters are present in the brain, but only a small number has been characterized. In this review, we will discuss about the roles of solute carrier (SLC) transporters at the blood brain barrier (BBB) and neural cells, in transport of nutrients and metabolites in the brain.

Deletion of Mfsd2b impairs thrombotic functions of platelets.

We recently discovered that Mfsd2b, which is the S1P exporter found in blood cells. Here, we report that Mfsd2b is critical for the release of all S1P species in both resting and activated platelets. We show that resting platelets store S1P in the cytoplasm. After activation, this S1P pool is delivered to the plasma membrane, where Mfsd2b is predominantly localized for export. Employing knockout mice of Mfsd2b, we reveal that platelets contribute a minor amount of plasma S1P. Nevertheless, Mfsd2b deletion in whole body or platelets impairs platelet morphology and functions. In particular, Mfsd2b knockout mice show significantly reduced thrombus formation. We show that loss of Mfsd2b affects intrinsic platelet functions as part of remarkable sphingolipid accumulation. These findings indicate that accumulation of sphingolipids including S1P by deletion of Mfsd2b strongly impairs platelet functions, which suggests that the transporter may be a target for the prevention of thrombotic disorders.

METTL3 Regulates Angiogenesis by Modulating let-7e-5p and miRNA-18a-5p Expression in Endothelial Cells.

[Figure: see text].

The correlation of 18F-FDG PET/CT metabolic parameters, clinicopathological factors, and prognosis in breast cancer

Purpose To study the imaging parameters of 18F-fluorodeoxy glucose (18F-FDG) in breast cancer on positron emission tomography/computed tomography (PET/CT)—the correlation of clinical pathological factors and prognosis among the maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) of lesions for patients. Methods From January 2012 to December 2014, a total of 125 female patients were treated in our hospital for the first time and were diagnosed as breast cancer by histopathology. They were selected as the research subjects. All of them had complete 18F-FDG PET/CT examination data before surgery, the postoperative clinicopathological information, and follow-up data. They were divided into the event group (38 cases) and the event-free group (87 cases) according to whether local recurrence or distant metastasis occurred after the follow-up, with the follow-up time 4–60 months. The correlation on 18F-FDG PET/CT metabolic parameters of breast cancer with clinicopathological factors and prognosis was retrospectively evaluated. Results The primary lesions of 125 cases with breast cancers all had higher 18F-FDG uptake, and the SUVmax, MTV, and TLG of the primary tumors in the event group were significantly higher than those in the event-free group (t = 2.645, 2.782, 15.263, p = 0.011, 0.008, 0.000), p < 0.05; SUVmax, MTV, and TLG of primary breast cancer have no correlation with age and tumor site of patient (p > 0.05); there were statistically significant differences in the SUVmax, MTV, and TLG of primary tumor in the comparison of different tumor size, T stage, N stage, and histological grades (p < 0.05); all of SUVmax, MTV, and TLG in the estrogen receptor (ER) and/or progesterone receptor (PR) positive groups were lower than those in the negative group, with statistically significant difference (p < 0.05) (AU)

Erythrocytes efficiently utilize exogenous sphingosines for S1P synthesis and export via Mfsd2b.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that exerts its activity via activation of five different G protein-coupled receptors, designated as S1P1-5. This potent lipid mediator is synthesized from the sphingosine precursor by two sphingosine kinases (SphK1 and 2) and must be exported to exert extracellular signaling functions. We recently identified Mfsd2b as the S1P transporter in the hematopoietic system. However, the sources of sphingosine for S1P synthesis and the transport mechanism of Mfsd2b in erythrocytes remain to be determined. Here, we show that erythrocytes efficiently take up exogenous sphingosine and that a de novo synthesis pathway in part provides sphingosines to erythrocytes. The uptake of sphingosine in erythrocytes is facilitated by the activity of SphK1. By converting sphingosine into S1P, SphK1 indirectly increases the influx of sphingosine, a process that is irreversible in erythrocytes. Our results explain for the abnormally high amount of sphingosine accumulation in Mfsd2b knockout erythrocytes. Furthermore, we show that Mfsd2b utilizes a proton gradient to facilitate the release of S1P. The negatively charged residues D95 and T157 are essential for Mfsd2b transport activity. Of interest, we also discovered an S1P analog that inhibits S1P export from erythrocytes, providing evidence that sphingosine analogs can be used to inhibit S1P export by Mfsd2b. Collectively, our results highlight that erythrocytes are efficient in sphingosine uptake for S1P production and the release of S1P is dependent on Mfsd2b functions.

The correlation of 18F-FDG PET/CT metabolic parameters, clinicopathological factors, and prognosis in breast cancer.

PURPOSE: To study the imaging parameters of 18F-fluorodeoxy glucose (18F-FDG) in breast cancer on positron emission tomography/computed tomography (PET/CT)-the correlation of clinical pathological factors and prognosis among the maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) of lesions for patients. METHODS: From January 2012 to December 2014, a total of 125 female patients were treated in our hospital for the first time and were diagnosed as breast cancer by histopathology. They were selected as the research subjects. All of them had complete 18F-FDG PET/CT examination data before surgery, the postoperative clinicopathological information, and follow-up data. They were divided into the event group (38 cases) and the event-free group (87 cases) according to whether local recurrence or distant metastasis occurred after the follow-up, with the follow-up time 4-60 months. The correlation on 18F-FDG PET/CT metabolic parameters of breast cancer with clinicopathological factors and prognosis was retrospectively evaluated. RESULTS: The primary lesions of 125 cases with breast cancers all had higher 18F-FDG uptake, and the SUVmax, MTV, and TLG of the primary tumors in the event group were significantly higher than those in the event-free group (t = 2.645, 2.782, 15.263, p = 0.011, 0.008, 0.000), p < 0.05; SUVmax, MTV, and TLG of primary breast cancer have no correlation with age and tumor site of patient (p > 0.05); there were statistically significant differences in the SUVmax, MTV, and TLG of primary tumor in the comparison of different tumor size, T stage, N stage, and histological grades (p < 0.05); all of SUVmax, MTV, and TLG in the estrogen receptor (ER) and/or progesterone receptor (PR) positive groups were lower than those in the negative group, with statistically significant difference (p < 0.05); the SUVmax, MTV, and TLG of human epidermal growth factor receptor 2 (HER2) positive group, proliferating cell nuclear antigen (Ki-67) high expression group were higher than those in the negative group and low expression group, with statistically significant difference (p < 0.05). There were 38 recurrence and metastasis cases within 125 cases with breast cancer in 5 years after operation, with the total recurrence and metastasis rate as 30.40% (38/125). The event-free survival rate in the SUVmax ≥ 8.64 group was significantly lower than that in the SUVmax < 8.64 group (p < 0.01). CONCLUSIONS: The metabolic parameters of 18F-FDG PET/CT in breast cancer can reflect the biological behavior of the tumor indirectly; therefore, it was studied on the related correlation to provide the guidance of clinical individualized comprehensive treatment and prognostic judgment.