Changes in urinary albumin as a surrogate for kidney disease progression in people with type 2 diabetes.

BACKGROUND: It remains unclear whether urinary albumin changes can predict subsequent kidney disease progression in people with diabetes. METHODS: This retrospective cohort study included 4570 Japanese adults with type 2 diabetes (T2D). The exposure was changes in urinary albumin-to-creatinine ratio (UACR) over 3 years, categorized into three categories: ≤ - 30%, minor change, or ≥ 30%. During the exposure period, eGFR decline was also examined and categorized into two categories: < 30% or ≥ 30% decline. The primary outcome was the composite of eGFR halving or initiation of kidney replacement therapy (KRT). The secondary outcome was the initiation of KRT. RESULTS: In the spline model, the hazard ratio for the primary outcome increased linearly on the log2 scale of UACR changes. When classified into six groups based on the categories of UACR changes and eGFR decline, people with a ≤ - 30% UACR change and < 30% eGFR decline had a 38% lower incidence of the outcome compared to those with a minor UACR change and < 30% eGFR decline. Meanwhile, the risk in those with a ≤ - 30% UACR change and ≥ 30% eGFR decline was 2.89 times. People with a ≥ 30% UACR change had the higher risk, regardless of whether a ≥ 30% eGFR decline occurred. Similar results were obtained in the secondary outcome. CONCLUSIONS: UACR changes can be a useful surrogate for kidney disease progression in people with T2D. However, when setting a decrease in UACR as the surrogate, it may be necessary to simultaneously evaluate kidney function decline.

Kidney outcomes and all-cause mortality in people with type 2 diabetes exhibiting non-albuminuric kidney insufficiency.

AIM/HYPOTHESIS: It remains unclear whether people with diabetes exhibiting non-albuminuric kidney insufficiency have higher risk of kidney function decline and mortality compared with those exhibiting preserved kidney function without albuminuria. Furthermore, information regarding the incidence of albuminuria in people with this unique phenotype is sparse. Here, we aimed to elucidate the risk of the kidney outcomes and all-cause mortality in people with diabetes exhibiting non-albuminuric kidney insufficiency. METHODS: In this retrospective cohort study, 8320 Japanese adults with type 2 diabetes were classified into four groups based on the presence of albuminuria and kidney insufficiency at baseline, defined as urinary albumin/creatinine ratio of equal to or above 30 mg/g and eGFR of less than 60 ml min-1 1.73 m-2, respectively. The primary composite kidney endpoint was a 50% decrease in eGFR from baseline or the initiation of kidney replacement therapy. The annual percentage change in eGFR slope and progression of albuminuria category were evaluated as the secondary and tertiary kidney endpoints, respectively. All-cause death was also set as the endpoint. RESULTS: Compared with people exhibiting non-albuminuric preserved kidney function, those with non-albuminuric kidney insufficiency had the higher risk for the primary kidney endpoint (HR 4.1; 95% CI 2.5, 6.7; p < 0.001), steep percentage change in eGFR slope (-1.96%/year vs -1.36%/year, p < 0.001), incidence of albuminuria (HR 2.1; 1.7, 2.6; p < 0.001) and all-cause mortality (HR 1.5; 1.2, 2.0; p = 0.003). In the sensitivity analyses treating the incidence of albuminuria as a competing risk, people with non-albuminuric kidney insufficiency still had higher risk for the primary kidney endpoint and all-cause mortality than those with non-albuminuric preserved kidney function (subdistribution HR 2.8; 1.4, 5.6; p = 0.004; and 1.6; 1.1, 2.2; p = 0.014, respectively). CONCLUSIONS/INTERPRETATION: People with type 2 diabetes exhibiting non-albuminuric kidney insufficiency had poorer kidney outcomes and life prognosis than those exhibiting non-albuminuric preserved kidney function.

Fatty acid-binding protein 5 limits the generation of Foxp3+ regulatory T cells through regulating plasmacytoid dendritic cell function in the tumor microenvironment.

Plasmacytoid dendritic cells (pDCs) promote viral elimination by producing large amounts of Type I interferon. Recent studies have shown that pDCs regulate the pathogenesis of diverse inflammatory diseases, such as cancer. Fatty acid-binding protein 5 (FABP5) is a cellular chaperone of long-chain fatty acids that induce biological responses. Although the effects of FABP-mediated lipid metabolism are well studied in various immune cells, its role in pDCs remains unclear. This study, which compares wild-type and Fabp5-/- mice, provides the first evidence that FABP5-mediated lipid metabolism regulates the commitment of pDCs to inflammatory vs tolerogenic gene expression patterns in the tumor microenvironment and in response to toll-like receptor stimulation. Additionally, we demonstrated that FABP5 deficiency in pDCs affects the surrounding cellular environment, and that FABP5 expression in pDCs supports the appropriate generation of regulatory T cells (Tregs). Collectively, our findings reveal that pDC FABP5 acts as an important regulator of tumor immunity by controlling lipid metabolism.

Thermal decomposition of spherically granulated malachite: physico-geometrical constraints and overall kinetics.

The thermal decomposition of spherically granulated malachite particles was investigated to unveil the specific kinetic features of the reaction in samples in granular form toward the improvement of the thermal processing of malachite as a precursor of functional CuO. Granular malachite underwent thermal decomposition via a partially overlapping two-step mass loss process upon heating the sample in a stream of dry N2 gas. Morphologically, the process was characterized by swelling of the granular particles and cleavage divisions of the surface layer. The kinetics of the thermal decomposition was investigated through step-by-step kinetic analyses of the systematically recorded thermoanalytical curves. Finally, the kinetics of the component reaction steps was separately characterized by performing a kinetic deconvolution analysis. The first reaction step, which contributed approximately 25% to the overall reaction and followed pseudo-first-order kinetics, was attributed to the thermal decomposition of the granular particle surface. The as-produced surface product layer impeded the diffusional removal of the gaseous products, i.e., CO2 and water vapor, from the interior of the granular particles, which caused swelling of the granular particles owing to an increase in the internal gaseous pressure and the cleavage division of the surface product layer by crack formation. The second mass loss step occurred inside the granular particles under significant variations in the self-generated reaction conditions and geometrical constraints and reached its maximum rate midway through the reaction. Possible causes of the observed specific rate behavior are discussed from the viewpoint of physico-geometrical kinetics in the solid-gas system.

FABP3 in the Anterior Cingulate Cortex Modulates the Methylation Status of the Glutamic Acid Decarboxylase67 Promoter Region.

Polyunsaturated fatty acids (PUFAs) are essential for brain development and function. Increasing evidence has shown that an imbalance of PUFAs is associated with various human psychiatric disorders, including autism and schizophrenia. Fatty acid-binding proteins (FABPs), cellular chaperones of PUFAs, are involved in PUFA intracellular trafficking, signal transduction, and gene transcription. In this study, we show that FABP3 is strongly expressed in the GABAergic inhibitory interneurons of the male mouse anterior cingulate cortex (ACC), which is a component of the limbic cortex and is important for the coordination of cognitive and emotional behaviors. Interestingly, Fabp3 KO male mice show an increase in the expression of the gene encoding the GABA-synthesizing enzyme glutamic acid decarboxylase 67 (Gad67) in the ACC. In the ACC of Fabp3 KO mice, Gad67 promoter methylation and the binding of methyl-CpG binding protein 2 (MeCP2) and histone deacetylase 1 (HDAC1) to the Gad67 promoter are significantly decreased compared with those in WT mice. The abnormal cognitive and emotional behaviors of Fabp3 KO mice are restored by methionine administration. Notably, methionine administration normalizes Gad67 promoter methylation and its mRNA expression in the ACC of Fabp3 KO mice. These findings demonstrate that FABP3 is involved in the control of DNA methylation of the Gad67 promoter and activation of GABAergic neurons in the ACC, thus suggesting the importance of PUFA homeostasis in the ACC for cognitive and emotional behaviors.SIGNIFICANCE STATEMENT The ACC is important for emotional and cognitive processing. However, the mechanisms underlying its involvement in the control of behavioral responses are largely unknown. We show the following new observations: (1) FABP3, a PUFA cellular chaperone, is exclusively expressed in GABAergic interneurons in the ACC; (2) an increase in Gad67 expression is detected in the ACC of Fabp3 KO mice; (3) the Gad67 promoter is hypomethylated and the binding of transcriptional repressor complexes is decreased in the ACC of Fabp3 KO mice; and (4) elevated Gad67 expression and abnormal behaviors seen in Fabp3 KO mice are mostly recovered by methionine treatment. These suggest that FABP3 regulates GABA synthesis through transcriptional regulation of Gad67 in the ACC.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Thermal Decomposition of Maya Blue: Extraction of Indigo Thermal Decomposition Steps from a Multistep Heterogeneous Reaction Using a Kinetic Deconvolution Analysis.

Examining the kinetics of solids' thermal decomposition with multiple overlapping steps is of growing interest in many fields, including materials science and engineering. Despite the difficulty of describing the kinetics for complex reaction processes constrained by physico-geometrical features, the kinetic deconvolution analysis (KDA) based on a cumulative kinetic equation is one practical method of obtaining the fundamental information needed to interpret detailed kinetic features. This article reports the application of KDA to thermal decomposition of clay minerals and indigo-clay mineral hybrid compounds, known as Maya blue, from ancient Mayan civilization. Maya blue samples were prepared by heating solid mixtures of indigo and clay minerals (palygorskite and sepiolite), followed by purification. The multistep thermal decomposition processes of the clay minerals and Maya blue samples were analyzed kinetically in a stepwise manner through preliminary kinetic analyses based on a conventional isoconversional method and mathematical peak deconvolution to finally attain the KDA. By comparing the results of KDA for the thermal decomposition processes of the clay minerals and the Maya blue samples, information about the thermal decomposition steps of the indigo incorporated into the Maya blue samples was extracted. The thermal stability of Maya blue samples was interpreted through the kinetic characterization of the extracted indigo decomposition steps.

Motor Training Promotes Both Synaptic and Intrinsic Plasticity of Layer II/III Pyramidal Neurons in the Primary Motor Cortex.

Motor skill training induces structural plasticity at dendritic spines in the primary motor cortex (M1). To further analyze both synaptic and intrinsic plasticity in the layer II/III area of M1, we subjected rats to a rotor rod test and then prepared acute brain slices. Motor skill consistently improved within 2 days of training. Voltage clamp analysis showed significantly higher α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl-d-aspartate (AMPA/NMDA) ratios and miniature EPSC amplitudes in 1-day trained rats compared with untrained rats, suggesting increased postsynaptic AMPA receptors in the early phase of motor learning. Compared with untrained controls, 2-days trained rats showed significantly higher miniature EPSC amplitude and frequency. Paired-pulse analysis further demonstrated lower rates in 2-days trained rats, suggesting increased presynaptic glutamate release during the late phase of learning. One-day trained rats showed decreased miniature IPSC frequency and increased paired-pulse analysis of evoked IPSC, suggesting a transient decrease in presynaptic γ-aminobutyric acid (GABA) release. Moreover, current clamp analysis revealed lower resting membrane potential, higher spike threshold, and deeper afterhyperpolarization in 1-day trained rats-while 2-days trained rats showed higher membrane potential, suggesting dynamic changes in intrinsic properties. Our present results indicate dynamic changes in glutamatergic, GABAergic, and intrinsic plasticity in M1 layer II/III neurons after the motor training.

Astrocyte-expressed FABP7 regulates dendritic morphology and excitatory synaptic function of cortical neurons.

Fatty acid binding protein 7 (FABP7) expressed by astrocytes in developing and mature brains is involved in uptake and transportation of fatty acids, signal transduction, and gene transcription. Fabp7 knockout (Fabp7 KO) mice show behavioral phenotypes reminiscent of human neuropsychiatric disorders such as schizophrenia. However, direct evidence showing how FABP7 deficiency in astrocytes leads to altered brain function is lacking. Here, we examined neuronal dendritic morphology and synaptic plasticity in medial prefrontal cortex (mPFC) of Fabp7 KO mice and in primary cortical neuronal cultures. Golgi staining of cortical pyramidal neurons in Fabp7 KO mice revealed aberrant dendritic morphology and decreased spine density compared with those in wild-type (WT) mice. Aberrant dendritic morphology was also observed in primary cortical neurons co-cultured with FABP7-deficient astrocytes and neurons cultured in Fabp7 KO astrocyte-conditioned medium. Excitatory synapse number was decreased in mPFC of Fabp7 KO mice and in neurons co-cultured with Fabp7 KO astrocytes. Accordingly, whole-cell voltage-clamp recording in brain slices from pyramidal cells in the mPFC showed that both amplitude and frequency of action potential-independent miniature excitatory postsynaptic currents (mEPSCs) were decreased in Fabp7 KO mice. Moreover, transplantation of WT astrocytes into the mPFC of Fabp7 KO mice partially attenuated behavioral impairments. Collectively, these results suggest that astrocytic FABP7 is important for dendritic arbor growth, neuronal excitatory synapse formation, and synaptic transmission, and provide new insights linking FABP7, lipid homeostasis, and neuropsychiatric disorders, leading to novel therapeutic interventions.

Discreet and distinct clustering of five model membrane proteins revealed by single molecule localization microscopy.

Compartmentalization is a functionally important property of the plasma membrane, yet the underlying principles that organize membrane proteins into distinct domains are not well understood. Using single molecule localization microscopy, we assessed the clustering of five model membrane proteins in the plasma membrane of HeLa cells. All five proteins formed discrete and distinct nano-scaled clusters. The extent of clustering of the five proteins, independent of their membrane anchors, increased significantly when the fluorescent protein mEOS2 was employed, suggesting that protein-protein interactions are a key driver for clustering. Further, actin depolymerization or reduction of membrane order had a greater, and in some instances opposing effects on the clustering of membrane proteins fused to mEOS2 compared to PS-CFP2-fusion proteins. The data propose that protein interactions can override the lateral organization imposed by membrane anchors to provide an exquisite regulation of the mosaic-like compartmentalization of the plasma membrane.

Fatty acid-binding protein 7 regulates function of caveolae in astrocytes through expression of caveolin-1.

Fatty acid-binding proteins (FABPs) bind and solubilize long-chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor-mediated signal transduction in response to extracellular stimuli. In FABP7-knockout (KO) astrocytes, the ligand-dependent accumulation of Toll-like receptor 4 (TLR4) and glial cell-line-derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen-activated protein kinases and nuclear factor-κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild-type astrocytes. In addition, the expression of caveolin-1, not cavin-1, 2, 3, caveolin-2, and flotillin-1, was found to be decreased at the protein and transcriptional levels. FABP7 re-expression in FABP7-KO astrocytes rescued the decreased level of caveolin-1. Furthermore, caveolin-1-transfection into FABP7-KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor-α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin-1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780-794.

Improvement of cold injury-induced mouse brain edema by endothelin ETB antagonists is accompanied by decreases in matrixmetalloproteinase 9 and vascular endothelial growth factor-A.

Brain edema is a potentially fatal pathological state that often occurs after brain injuries such as ischemia and trauma. However, therapeutic agents that fundamentally treat brain edema have not yet been established. We previously found that endothelin ETB receptor antagonists attenuate the formation and maintenance of vasogenic brain edema after cold injury in mice. In this study, the effects of ETB antagonists on matrixmetalloproteinase (MMP)9 and vascular endothelial growth factor (VEGF)-A expression were examined in the cold injury model. Cold injury was performed in the left brain of male ddY mice (5-6 weeks old) for the induction of vasogenic edema. Expression of MMP9 and VEGF-A mRNA in the mouse cerebrum was increased by cold injury. Immunohistochemical observations showed that the MMP9 and VEGF-A were mainly produced in reactive astrocytes in the damaged cerebrum. Intracerebroventricular administration of BQ788 (10 µg) or IRL-2500 (10 µg) (selective ETB antagonists) attenuated brain edema and disruption of the blood-brain barrier after cold injury. BQ788 and IRL-2500 reversed the cold injury-induced increases in MMP9 and VEGF-A expression. The induction of reactive astrocytes producing MMP9 and VEGF-A in the damaged cerebrum was attenuated by BQ788 and IRL-2500. These results suggest that attenuations of astrocytic MMP9 and VEGF-A expression by ETB antagonists may be involved in the amelioration of vasogenic brain edema.

Fatty acid binding protein 7 regulates phagocytosis and cytokine production in Kupffer cells during liver injury.

Kupffer cells (KCs) are involved in the progression of liver diseases such as hepatitis and liver cancer. Several members of the fatty acid binding proteins (FABPs) are expressed by tissue macrophages, and FABP7 is localized only in KCs. To clarify the role of FABP7 in the regulation of KC function, we evaluated pathological changes of Fabp7 knockout mice during carbon tetrachloride-induced liver injury. During liver injury in Fabp7 knockout mice, serum liver enzymes were increased, cytokine expression (tumor necrosis factor-α, monocyte chemoattractant protein-1, and transforming growth factor-ß) was decreased in the liver, and the number of KCs in the liver necrotic area was significantly decreased. Interestingly, in the FABP7-deficient KCs, phagocytosis of apoptotic cells was impaired, and expression of the scavenger receptor CD36 was markedly decreased. In chronic liver injury, Fabp7 knockout mice showed less fibrogenic response to carbon tetrachloride compared with wild-type mice. Taken together, FABP7 is involved in the liver injury process through its regulation of KC phagocytic activity and cytokine production. Such modulation of KC function by FABP7 may provide a novel therapeutic approach to the treatment of liver diseases.

Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice. This study demonstrates that FABP5 binds to DHA and is involved in the brain endothelial cell uptake and subsequent BBB transport of DHA, confirming the importance of this cytoplasmic carrier protein in the CNS exposure of this PUFA essential for neuronal function.

Fatty Acid Binding Protein 3 Is Involved in n-3 and n-6 PUFA transport in mouse trophoblasts.

BACKGROUND: Low placental fatty acid (FA) transport during the embryonic period has been suggested to result in fetal developmental disorders and various adult metabolic diseases, but the molecular mechanism by which FAs are transported through the placental unit remains largely unknown. OBJECTIVE: The aim of this study was to examine the distribution and functional relevance of FA binding protein (FABP), a cellular chaperone of FAs, in the mouse placenta. METHODS: We clarified the localization of FABPs and sought to examine their function in placental FA transport through the phenotypic analysis of Fabp3-knockout mice. RESULTS: Four FABPs (FABP3, FABP4, FABP5, and FABP7) were expressed with spatial heterogeneity in the placenta, and FABP3 was dominantly localized to the trophoblast cells. In placentas from the Fabp3-knockout mice (both sexes), the transport coefficients for linoleic acid (LA) were significantly reduced compared with those from wild-type mice by 25% and 44% at embryonic day (E) 15.5 and E18.5, respectively, whereas those for α-linolenic acid (ALA) were reduced by 19% and 17%, respectively. The accumulation of LA (18% and 27% at E15.5 and E18.5) and ALA (16% at E15.5) was also significantly less in the Fabp3-knockout fetuses than in wild-type fetuses. In contrast, transport and accumulation of palmitic acid (PA) were unaffected and glucose uptake significantly increased by 23% in the gene-ablated mice compared with wild-type mice at E18.5. Incorporation of LA (51% and 52% at 1 and 60 min, respectively) and ALA (23% at 60 min), but not PA, was significantly less in FABP3-knockdown BeWo cells than in controls, whereas glucose uptake was significantly upregulated by 51%, 50%, 31%, and 33% at 1, 20, 40, and 60 min, respectively. CONCLUSIONS: Collectively FABP3 regulates n-3 (ω-3) and n-6 (ω-6) polyunsaturated FA transport in trophoblasts and plays a pivotal role in fetal development.

[Elucidation of the pathology of mental disorders focusing on polyunsaturated fatty acids and FABPs].

Polyunsaturated fatty acids (PUFAs) are essential for brain development and function, and an imbalance of brain PUFAs is linked to mental disorders like autism and schizophrenia. However, the cellular and molecular mechanisms underlying the effects of PUFAs on the brain remain largely unknown. Since they are insoluble in water, specific transporters like fatty acid binding proteins (FABPs), are required for transport and function of PUFAs within cells. We focused on the relationship between FABP-mediated homeostasis of brain PUFAs and neural plasticity. We found that FABP3, with a high affinity for n-6 PUFAs, is predominantly expressed in the GABAergic inhibitory interneurons of the anterior cingulate cortex (ACC) in the adult mouse brain. FABP3 knockout (KO) mice show increased GABA synthesis and inhibitory synaptic transmission in the ACC. We also found that FABP7 controls lipid raft function in astrocytes, and astrocytes lacking FABP7 exhibit changes in response to external stimuli. Furthermore, in FABP7 KO mice, dendritic protrusion formation in pyramidal neurons becomes abnormal, and we have reported a decrease in spine density and excitatory synaptic transmission. Here, we introduced recent advances in the understanding of the functions of PUFAs and FABPs in the brain, focusing especially on FABP3 and FABP7, in relation to human mental disorders.

Catenulopyrizomicins, new anti-Hepatitis B virus compounds, from the rare actinomycete Catenuloplanes sp. MM782L-181F7.

Hepatitis B virus (HBV) causes chronic hepatitis in humans, and current antiviral therapies rarely treat viral infections. To improve the treatment efficacy, novel therapeutic agents, especially those with different mechanisms of action, need to be developed for use in combination with the current antivirals. Here, we isolated new anti-HBV compounds, named catenulopyrizomicins A-C, from the fermentation broth of rare actinomycete Catenuloplanes sp. MM782L-181F7. Structural analysis revealed that these compounds contained a structure that is composed of thiazolyl pyridine moiety. The catenulopyrizomicins reduced the amount of intracellular viral DNA in HepG2.2.15 cells with EC50 values ranging from 1.94 to 2.63 µM with small but notable selectivity. Mechanistic studies indicated that catenulopyrizomicin promotes the release of immature virion particles that fail to be enveloped through alterations in membrane permeability.

Polyunsaturated fatty acids-induced ferroptosis suppresses pancreatic cancer growth.

Despite recent advances in science and medical technology, pancreatic cancer remains associated with high mortality rates due to aggressive growth and no early clinical sign as well as the unique resistance to anti-cancer chemotherapy. Current numerous investigations have suggested that ferroptosis, which is a programed cell death driven by lipid oxidation, is an attractive therapeutic in different tumor types including pancreatic cancer. Here, we first demonstrated that linoleic acid (LA) and α-linolenic acid (αLA) induced cell death with necroptotic morphological change in MIA-Paca2 and Suit 2 cell lines. LA and αLA increased lipid peroxidation and phosphorylation of RIP3 and MLKL in pancreatic cancers, which were negated by ferroptosis inhibitor, ferrostatin-1, restoring back to BSA control levels. Similarly, intraperitoneal administration of LA and αLA suppresses the growth of subcutaneously transplanted Suit-2 cells and ameliorated the decreased survival rate of tumor bearing mice, while co-administration of ferrostatin-1 with LA and αLA negated the anti-cancer effect. We also demonstrated that LA and αLA partially showed ferroptotic effects on the gemcitabine-resistant-PK cells, although its effect was exerted late compared to treatment on normal-PK cells. In addition, the trial to validate the importance of double bonds in PUFAs in ferroptosis revealed that AA and EPA had a marked effect of ferroptosis on pancreatic cancer cells, but DHA showed mild suppression of cancer proliferation. Furthermore, treatment in other tumor cell lines revealed different sensitivity of PUFA-induced ferroptosis; e.g., EPA induced a ferroptotic effect on colorectal adenocarcinoma, but LA or αLA did not. Collectively, these data suggest that PUFAs can have a potential to exert an anti-cancer effect via ferroptosis in both normal and gemcitabine-resistant pancreatic cancer.

Differential expression and regulatory roles of FABP5 and FABP7 in oligodendrocyte lineage cells.

Fatty-acid-binding proteins (FABPs) are key intracellular molecules involved in the uptake, transportation and storage of fatty acids and in the mediation of signal transduction and gene transcription. However, little is known regarding their expression and function in the oligodendrocyte lineage. We evaluate the in vivo and in vitro expression of FABP5 and FABP7 in oligodendrocyte lineage cells in the cortex and corpus callosum of adult mice, mixed cortical culture and oligosphere culture by immunofluorescent counter-staining with major oligodendrocyte lineage markers. In all settings, FABP7 expression was detected in NG2(+)/PDGFRα(+) oligodendrocyte progenitor cells (OPCs) that did not express FABP5. FABP5 was detected in mature CC1(+)/MBP(+) oligodendrocytes that did not express FABP7. Analysis of cultured OPCs showed a significant decrease in the population of FABP7-knockout (KO) OPCs and their BrdU uptake compared with wild-type (WT) OPCs. Upon incubation of OPCs in oligodendrocyte differentiation medium, a significantly lower percentage of FABP7-KO OPCs differentiated into O4(+) oligodendrocytes. The percentage of mature MBP(+) oligodendrocytes relative to whole O4(+)/MBP(+) oligodendrocytes was significantly lower in FABP7-KO and FABP5-KO than in WT cell populations. The percentage of terminally mature oligodendrocytes with membrane sheet morphology was significantly lower in FABP5-KO compared with WT cell populations. Thus, FABP7 and FABP5 are differentially expressed in oligodendrocyte lineage cells and regulate their proliferation and/or differentiation. Our findings suggest the involvement of FABP7 and FABP5 in the pathophysiology of demyelinating disorders, neuropsychiatric disorder and glioma, conditions in which OPCs/oligodendrocytes play central roles.

Novel cognitive enhancer ST101 enhances acetylcholine release in mouse dorsal hippocampus through T-type voltage-gated calcium channel stimulation.

We recently developed a novel cognitive enhancer, ST101 (spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one), that activates T-type voltage-gated calcium channels (VGCCs). Here, we address whether T-type VGCC activation with ST101 mediates its cognitive effects in vivo and the relevance of T-type VGCC activation to acetylcholine (ACh) release in the hippocampus. Acute intraperitoneal administration of ST101 (1 mg/kg, i.p.) improved memory-related behaviors in both olfactory bulbectomized (OBX) and scopolamine-treated mice. Effects of ST101 administration were abolished by both intraperitoneal and intracerebroventricular pre-administration of the T-type VGCC inhibitor mibefradil. Acute administration of ST101 enhanced basal and nicotine-induced ACh release in the dorsal hippocampus in both OBX and sham-treated mice. Enhanced ACh release was abolished by infusion with mibefradil (10 µM) but not with the L-type VGCC inhibitor nifedipine (10 µM). As expected, significantly reduced CaMKIIα, PKCα, and ERK phosphorylation was restored by acute ST101 administration in the OBX mouse hippocampal CA1 region. Enhancement of CaMKIIα and PKCα but not ERK phosphorylation was inhibited by mibefradil (20 mg/kg, i.p.) preadministration. Increased CaMKIIα and PKCα phosphorylation was confirmed by increased phosphorylation of GluR1, synapsin I, and NR1. Taken together, stimulation of T-type VGCCs is critical for the enhanced hippocampal ACh release and improved cognitive function seen following ST101 administration.