Transthyretin (TTR) amyloidosis, also known as transthyretin-related familial amyloidotic polyneuropathy (ATTR-FAP), is a fatal hereditary systemic amyloidosis caused by mutant forms of TTR. Although conventional treatments for ATTR-FAP, such as liver transplantation (LT) and TTR tetramer stabilizer, reportedly halt the progression of clinical manifestation, these therapies have several limitations. Oligonucleotide-based therapy, e.g. small interfering RNA (siRNA)- and antisense oligonucleotides (ASOs)-based therapy, hold enormous potential for the treatment of intractable diseases such as ATTR-FAP, by specifically regulating the gene responsible for the disease. Clinical evidence strongly suggests that LT inhibits mutant TTR production, thus improving the manifestation of ATTR-FAP. Therefore, an oligonucleotide-based therapy for ATTR-FAP, which reduces the production of TTR by the liver, has recently been developed in preclinical and clinical studies. This review focuses on recent advances in oligonucleotide-based therapy and future prospects of next-generation oligonucleotide-based drugs for therapeutic use against ATTR-FAP.

Infliximab shows drastic efficacy for controlling inflammation in rheumatoid arthritis (RA), though the ideal dose of infliximab to keep suppressing inflammation has not yet been identified. Recently, it has been evidenced that the minimum trough serum infliximab levels required for suppressing inflammation are greater than 1 µg/mL. This cross-sectional study was designed to identify the minimum dosage of infliximab for maintaining serum infliximab levels greater than 1 µg/mL. Thirty seven RA patients were enrolled in this study and they were divided into two groups (high-infliximab vs. low-infliximab) in reference to Remi-check Q^®, a kit for examining serum infliximab levels above/below 1 µg/mL by LC. Infliximab dosage (p=0.06) and dosage interval (p=0.05) had trends to have differences between groups. A formula calculated by infliximab dosage divided by dosage interval and body weight (mg/weeks/kg) was shown to have significantly higher levels among high-infliximab group (p=0.04). Based on whether serum infliximab levels above/below 1 µg/mL and values led by the equation, infliximab dosage/infliximab interval/body weight (mg/weeks/kg), a receiver operating characteristic curve (ROC) was depicted with area under the ROC curve 0.750 and the cut-off point for the serum infliximab levels greater than 1 µg/mL was identified as infliximab dosage/infliximab interval/body weight ≧0.750 with the sensitivity 0.393 and the specificity 1.000. In conclusion, we identified that the minimum infliximab dosage to maintain serum infliximab levels greater than 1 µg/mL was infliximab dose/dosage interval/body weight (mg/weeks/kg)≥0.750.

Alternative medicines attract attention because stroke is rarely expected to make a full recovery with the most advanced medical technology. Angelica gigas (AG) is a well-known herbal medicine as a neuroprotective agent. The present study introduced mesenchymal stem cells (MSCs) to identify for the advanced treatment of the cerebrovascular disease. The objective of this research is validation of the enhanced effects of multiple combined treatment of AG extract with MSCs on stroke through angiogenesis. Our results confirmed that AG extract with MSCs improved the neovascularization increasing expression of angiogenesis-regulated molecules. The changes of brain and the behavioral ability showed the increased effects of AG extract with MSCs. As a result, AG extract and MSCs may synergistically increase the therapeutic potential by enhancing neovascularization. This mixed approach provides a new experimental protocol of herbal medicine therapy for the treatment of a variety of diseases including stroke, trauma, and spinal cord injury.

The pentacyclic triterpenoid ursolic acid was previously shown to inhibit the intracellular trafficking of intercellular adhesion molecule-1 (ICAM-1) from the endoplasmic reticulum (ER) to the Golgi apparatus. In the present study, we further investigated the biological activities of three pentacyclic triterpenoids closely related to ursolic acid on the interleukin 1α-induced expression and intracellular trafficking of ICAM-1. In human lung adenocarcinoma A549 cells, asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular transport of ICAM-1 to the cell surface. Endoglycosidase H-sensitive glycans were linked to ICAM-1 in asiatic acid-, corosolic acid-, and maslinic acid-treated cells. Unlike corosolic acid, asiatic acid and maslinic acid increased the amount of the ICAM-1 protein. Moreover, asiatic acid increased the co-localization of ICAM-1 with calnexin (an ER marker), but not GM130 (a cis-Golgi marker). Asiatic acid, corosolic acid, and maslinic acid inhibited yeast α-glucosidase activity, but not Jack bean α-mannosidase activity. These results indicate that asiatic acid, corosolic acid, and maslinic acid interfere with the intracellular transport of ICAM-1 to the cell surface and cause the accumulation of ICAM-1 linked to endoglycosidase H-sensitive glycans.

We reported a novel transport mechanism of curcumin, independent of improved solubility, which involved direct contact of amorphous solid particles with the cell membrane. This mechanism has potential as a novel systemic delivery system of poorly water-soluble drugs. In this study, the transport mechanism of furosemide (FUR), which is transported by the same novel mechanism, was examined. In vitro cell permeation studies under air-interface conditions (AICs) revealed that the permeation from powders sprayed on cell monolayers was significantly higher than that under liquid-covered conditions (LCCs) from their solutions. The permeation from amorphous solid particles was faster than that from crystals. Similar results were derived from in vitro studies using an artificial membrane, with which the permeation of FUR could be examined without water. These findings clearly indicated that the transport mechanism of FUR is the same as that of curcumin. For the application of this new transport mechanism, the in vivo absorption of FUR was examined after pulmonary insufflation, which allows the solid particles to make direct contact with the epithelial cells. Pulmonary absorption of FUR from the amorphous powder was almost complete and was faster than that after intragastric administration of the solution, suggesting that FUR was absorbed from the lung by the same mechanism as the in vitro study. This new transport mechanism, which is independent of water dissolution, could be exploited to develop a novel delivery system for poorly water-soluble drugs, using pulmonary powder inhalation.

Melatonin has been suggested to play important roles in lipid metabolism as well as circadian rhythm; however, very few studies explored the effects of ramelteon, a selective melatonin receptor agonist, on serum lipid profiles. In this study effects of ramelteon on serum lipid profiles were explored, comparing to those of other sleep-promoting drugs including benzodiazepines and non-benzodiazepines, in patients with insomnia. We retrospectively reviewed medical charts of outpatients who were treated with ramelteon (8 mg/d) or other sleep-promoting drugs for no less than 8 weeks during the period between October 1st, 2011 and September 30th, 2014, and compared the changes in serum lipid profiles between the two groups. Patients with regular dialysis or malignant diseases treated with cytotoxic anti-cancer drugs, or whose lipid-lowering drugs were altered during the study period, were excluded. Among 365 or 855 outpatients treated with ramelteon or other sleep-promoting drugs, 35 or 46 patients, respectively, had complete serum low-density lipoprotein cholesterol (LDL-C) or non-high-density lipoprotein cholesterol (non-HDL-C) data. Serum LDL-C was significantly reduced from 103.1±4.4 to 94.6±4.2 mg/dL (8.2% reduction, p<0.05, n=31) in the ramelteon group, and was not significantly changed (p=0.23, n=40) in the other sleep-promoting drug group. Non-HDL-C was significantly decreased from 138.8±6.0 to 130.6±4.9 mg/dL (5.9% reduction, p<0.05, n=32) in the ramelteon group, and was not significantly altered (p=0.29, n=42) in the other sleep-promoting drug group. Ramelteon, but not other sleep-promoting drugs, specifically lowers serum LDL-C and non-HDL-C levels.

Semaphorin4A (Sema4A) is a family member of semaphorins expressed in immune cells and is also related with disease progression of tumor disease. In this study, we investigate the expression and pathological role of Sema4A in breast cancer (BCa). Our data showed that the expression of Sema4A increased in the tissues and serum of BCa patients when compared with normal controls. The expression of Sema4A in BCa cells could be induced by hypoxic treatment, whereas silencing hypoxia-inducible factor (HIF)-1α could attenuate the above induced. Furthermore, chromatin immunoprecipitation (ChIP) analysis demonstrated that HIF-1α could regulate the expression of Sema4A through directly binding to the promoter of Sema4A gene, whose enrichment could be further enhanced by hypoxic stimulation. In addition, silencing Sema4A could inhibit the proliferation, vascular endothelial growth factor (VEGF) production and the phosphorylation of Akt, extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) and signal transduction and activator of transcription (STAT)3, but induce apoptosis of BCa cells in the presence of hypoxia. In contrast, recombinant human Sema4A treatment showed the opposite effects. Taken together, these results suggest that Sema4A could promote progression of BCa in the presence of hypoxia and it may hold potential for treatment target for BCa.

High-dose vitamin C administration has been reported to exhibit antitumor effect in various mouse models of cancer. However, the underlying mechanism of antitumor effect against colorectal cancer remains to be elucidated. In this study, we investigated the antitumor effect of high-dose vitamin C in a mouse model of chronic inflammation-associated colorectal cancer induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). After cancer induction, the mice were administered vitamin C and/or irinotecan. Because irinotecan is a key drug in colorectal cancer treatment, it was used for comparison in this study. We examined reactive oxygen species (ROS) and interleukin-6 (IL-6) levels in the plasma of mice, as well as collagen type I and caspase-1 expression and neutrophil and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cell counts in the colon tissue. Vitamin C and/or irinotecan administration decreased the plasma level of ROS and IL-6 and increased the expression of collagen type I and caspase-1. Furthermore, it increased neutrophil and TUNEL-positive cell counts. The most significant changes in the parameters analyzed were observed when both vitamin C and irinotecan were administered.

Avian influenza A (H7N9) virus has caused several epidemics and infection in both human and poultry. With mutation, the H7N9 virus gained its fifth endemic in China. Early diagnosis is crucial for the control of viral spread in poultry and prognosis of infected patients. In this study, we developed and evaluated a lateral flow dipstick recombinase polymerase amplification (LFD-RPA) assay for rapid detection of both hemagglutinin and neuraminidase gene of H7N9. Our H7-LFD-RPA and N9-LFD-RPA assay were able to detect 32 fg H7N9 nucleic acid which is more convenient and rapid than previous methods. Through detecting 50 influenza positive samples, cross-reaction was not found with other subtypes of influenza virus. The 100% analytical specificity and sufficient analytical sensitivity results agreed the real time RT-PCR assay. The results data demonstrated that our method performed well and could be applied to the detection of H7N9 virus. This LFD-RPA assay provides a candidate method for rapid point-of-care diagnosis of H7N9.

2,3-Dimethoxy-5-methyl-p-benzoquinone is a common chemical structure of coenzyme Q (CoQ) that conjugates different lengths of an isoprenoid side chain at the 6-position of the p-benzoquinone ring. In a series of studies to explore the cytotoxic mechanism of CoQ homologues with a short isoprenoid side chain, we found that a CoQ analogue without an isoprenoid side chain, CoQ₀, showed marked toxicity against HeLa cells in comparison with cytotoxic homologues. Therefore, we examined the cytotoxic mechanism of CoQ₀. Different from the cytotoxic CoQ homologues that induced apoptosis, 100 µM CoQ₀ induced necrosis of HeLa cells. The CoQ₀-induced cell death was accompanied by a decrease in endogenous non-protein and protein-associated sulfhydryl (SH)-groups, but this improved with the concomitant addition of compounds with SH-groups but not antioxidants without SH-groups. In addition, UV-spectrum analysis suggested that CoQ₀ could rapidly form S-conjugated adducts with compounds with SH-groups by Michael addition. On the other hand, enzyme activities of both glyceraldehyde-3-phosphate dehydrogenase, which has a Cys residue in the active site, and α-ketoglutarate dehydrogenase complex, which requires cofactors with SH-groups, CoA and protein-bound α-lipoic acid, and CoA and ATP contents in the cells were significantly decreased by the addition of CoQ₀ but not CoQ₁. Furthermore, the decrease of an endogenous antioxidant, glutathione (GSH), by CoQ₀ treatment was much greater than the predicted increase of endogenous GSH disulfide. These results suggest that CoQ₀ rapidly forms S-conjugate adducts with these endogenous non-protein and protein-associated SH-groups of HeLa cells, which disrupts carbohydrate metabolism followed by intracellular ATP depletion and necrotic cell death.

Pulmonary emphysema (PE) is a major pathological feature of chronic obstructive pulmonary disease (COPD) and is characterized by proteolytic destruction of the alveolar structure and subsequent inflammation of the respiratory tract. We hypothesized that nitrite attenuates the development of PE via anti-inflammatory actions. PE was induced by intratracheal instillation of porcine pancreas elastase (PPE) in mice. Dietary nitrite dose-dependently (50 and 150 mg/L in drinking water) attenuated emphysematous development and macrophage accumulation in the alveolar parenchyma 21 d after PPE treatment. The present study shows that dietary nitrite might be a possible nutritional strategy in preventing the development of PE in mice.

Mohs paste (MP) is a hospital preparation containing zinc hydrochloride and zinc oxide starch. It is a topical medication used to fixate tissues for the removal of inoperable skin tumors and the management of hemorrhage and exudates, and to prevent foul odor resulting from secondary infections. However, it has problems, such as changes in hardness and viscoelasticity with time and liquefaction by exudate. It has been reported that the modified MP with D-sorbitol (S-MP) and the modified MP using the cellulose instead of starch (C-MP) have excellent physicochemical stability and better handling than original MP (O-MP). In this study, the effect of prescription improvement of MP on the pharmacological effect was examined with reference to water absorbing property, and its tumor tissue invasion fixation depth as an indicator. In the S-MP and C-MP, the amounts of water absorption did not differ significantly from those in the O-MP. The hardness of S-MP was decreased and liquefied like O-MP after absorbing water. In contrast, C-MP retained its form even after water absorption. The subcutaneous tumors in mice treated with modified MP formulations were measured for invasion fixation depth at 6 and 24 h after application. And the tissue status was observed using computed tomography. In all MPs, invasion fixation depth increased depending on application time. S-MP and O-MP depths did not differ significantly. The invasion depths of the C-MP significantly increased compared with those in the O-MP. These results suggest that C-MP had a high tissue fixation rate.

Intravenous immunoglobulin (IVIg) has been shown to be effective in the treatment of a variety of autoimmune diseases. To clarify the role of T regulatory cells (Tregs) in the immunoregulatory effect of IVIg, we focused on human inducible T regulatory cells (iTregs) and investigated the mechanism of action of IVIg. When immunoglobulin G (IgG) was added to a culture system that differentiates iTregs from anti-CD3 antibody activated CD4+CD25− T cells in the presence of syngeneic immature dendritic cells, interleukin (IL)-2 and transforming growth factor-β (TGF-β), the expression of forkhead box P3 (FoxP3), which is the master transcription factor for Tregs in CD4+CD25+ T cells, increased in an IgG concentration-dependent manner. The expression of FoxP3 in iTregs in the 20 mg/mL IgG group was twice as high as that in the saline group. iTregs that highly expressed FoxP3 not only partially suppressed the polyclonal proliferative response of T cells derived from the same individual but also produced significantly more inhibitory cytokines IL-10 and TGF-β. The ability of IgG to enhance iTregs differentiation was also observed in the Fc fragment, but not in the F(ab′)₂ fragment. These results suggest the clinical regulation of immune responses by IVIg administration may contribute at least to enhancing the differentiation of iTregs and partial immunosuppressive functions.

We show that a lectin like protein from the mushroom Agaricus bisporus (LSMT) is capable to permeate the epithelial monolayer barrier of the intestine ex vivo. The protein is not toxic or immunogenic upon prolonged administration and elevated dose in mice. Thus, it could be a candidate as a drug carrier for oral administration. However, its permeability should be tested after the protein has been modified, mimicking the condition in which it is used as a drug carrier. The protein was conjugated to captopril, the selected model of a Biopharmaceutical Classification System (BCS) class III drug, with high solubility but poor permeability. The drug was conjugated to LSMT that had been modified with 4-succinimidyloxycarbonyl-alpha-methyl-2-pyridyldithiotoluene (SMPT) as a linker. The success of LSMT modification was confirmed with TLC and MS; the latter also indicated the amount of captopril molecule linked. The modified LSMT could permeate through the intestinal monolayer barrier, and thus could be absorbed in the intestine after modification. The modified protein appears to remain stable after incubation in simulated gastrointestinal fluids. This pioneering work provides an essential basis for further development of the protein as a drug carrier for oral administration.

Ulcerative colitis is characterized by colonic mucosal bleeding and ulceration, often with repeated active and remission stages. One factor in ulcerative colitis development is increased susceptibility to commensal bacteria and lipopolysaccharide (LPS). LPS activates macrophages to release nitric oxide (NO) through Toll-like receptor 4 (TLR4) signaling. However, whether NO is beneficial or detrimental to colitis remains controversial. In this study, we investigated whether NO enhances the development of colitis in mice treated with dextran sulfate sodium (DSS) and inflammation in cells treated with low-dose LPS. An NO donor, NOC18, induced colitis and increased CD14 protein and nitrotyrosine levels in colonic macrophages from mice treated with DSS for 7 d (molecular weight: 5000). In the mouse peritoneal macrophage cell line RAW264.7 stimulated with 3 ng/mL LPS, NO activated the CD14-TLR4-nuclear factor kappa B (NF-κB) axis. Low-dose LPS stimulation did not change the levels of signal transducer and activator of transcription (STAT) 3 phosphorylation, CD14, inducible NO synthase, interleukin (IL)-6, or NF-κB. In addition, low-dose LPS increased phosphorylation of src homology protein tyrosine phosphatase 2 (SHP2), a negative regulator of STAT3 phosphorylation. However, NO decreased SHP2 phosphorylation and significantly activated the downstream signaling molecules. NO increased SHP2 nitration in LPS-stimulated RAW264.7 cells and DSS-treated mice. These results indicate that SHP2 nitration in macrophages might be involved in activation of the CD14-TLR4-NF-κB axis through STAT3 signaling in mice with DSS-induced colitis.

Contact hypersensitivity (CHS) to preservatives is receiving increased attention. Parabens are widely used in foods, pharmaceutics and cosmetics as preservatives. The skin sensitizing activity of parabens remains controversial but a few investigations have been made as to whether parabens could facilitate sensitization to other chemicals. We have shown that di-n-butyl phthalate (DBP), a phthalate ester, has an adjuvant effect in a fluorescein isothiocyanate (FITC)-induced CHS mouse model. We have also demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. Comparative studies of phthalate esters revealed that TRPA1 agonistic activity and the adjuvant effect on FITC-CHS coincide. Here we focused on two commonly used parabens, butyl paraben (BP) and ethyl paraben (EP), as to their adjuvant effects. BALB/c mice were epicutneously sensitized with FITC in acetone in the presence or absence of a paraben. Sensitization to FITC was evaluated as the ear-swelling response after FITC challenge. BP but not EP enhanced skin sensitization to FITC, but the effect of BP was much weaker than that of DBP. Mechanistically, BP enhanced the trafficking of FITC-presenting CD11c⁺ dendritic cells (DCs) from the skin to draining lymph nodes as well as cytokine production by draining lymph nodes. When the TRPA1 agonistic activity was measured with a cell line expressing TRPA1, BP exhibited higher activity than EP. The present study provides direct in vivo evidence that BP causes sensitization to other chemicals by means of a mouse FITC-CHS model.

The lateral hypothalamic area contains neurons expressing neuronal nitric oxide synthase (nNOS), in addition to orexin neurons. Here we examined whether the activity of orexin neurons was regulated by endogenous nitric oxide (NO) in male C57BL/6 mice. Caffeine (30 mg/kg, intraperitoneally (i.p.)) increased the number of orexin neurons positive for c-Fos, a marker of neuronal activity, and also increased the number of NOS/c-Fos-positive cells as identified by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and c-Fos immunohistochemistry. Diphenhydramine hydrochloride (10 mg/kg. i.p.) decreased c-Fos-positive orexin neurons but had no significant effect on the number of c-Fos-positive NOS neurons. nNOS inhibitor 7-nitroindazole (25 mg/kg, i.p.) alone increased c-Fos-positive orexin neurons, and combined treatment with caffeine and 7-nitroindazole did not show additive effect in the number of c-Fos-positive orexin neurons. In contrast, 7-nitroindazole decreased c-Fos-positive NOS neurons and attenuated caffeine-induced increase in c-Fos-positive NOS neurons. Sleep deprivation increased c-Fos-positive cells in both orexin neurons and NOS neurons, and 7-nitroindazole did not show additive effect with sleep deprivation in the activation of orexin neurons. Together, these results suggest that endogenous NO negatively regulates the activity of a subset of orexin neurons, and this subset of orexin neurons overlaps with that activated by awakening stimuli.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive myeloid cells found in patients with cancer and in mouse tumor models. They suppress anti-tumor immunity, resulting in the promotion of tumor growth. The relationship between nutrition and cancer has recently been reported by several research groups. Tumor cells rely on glutaminolysis, in which glutamine is metabolized into glutamate for energy production, and hence, glutamate levels are elevated in tumor-bearing hosts. However, the mechanism of regulation of tumor progression by glutamate still remains unclear. In this study, we found that the metabotropic glutamate receptor (mGluR) 2/3 was expressed on MDSCs, and an mGluR2/3 antagonist LY341495 attenuated the immunosuppressive activity of MDSCs. Furthermore, we observed that LY341495 treatment inhibited B16-F10 melanoma growth in vivo. Taken together, our data suggest that glutamate signaling promotes tumor growth by increasing the potency of immune suppression.

In traditional Tibetan medicinal system, Berberis herbs mainly originate from the dried barks of Berberis kansuensis, Berberis dictyophylla, Berberis diaphana, and Berberis vernae. In this study, molecular phylogenetic method based on four markers (i.e., rbcL, internal transcribed spacer (ITS), ITS2, and psbA-trnH) and HPLC chemical analysis were used to evaluate the chemical and genetic differences between the four Berberis species. The results showed that the discriminatory power of ITS, ITS2 and psbA-trnH was low, but the rbcL marker was highly effective and reliable for the species differentiation. The four Berberis species can be successfully classified based on phylogenetic analysis of the rbcL sequences. Moreover, the results of chemical analysis showed that four main alkaloids (i.e., berberine, palmatine, magnoflorine, and jatrorrhizine) cannot be used as chemical markers for discrimination of the four Berberis species. These findings provide valuable information for distinguishing the four Berberis Tibetan herbs.

The aims of this study were to determine the effects of gamma-aminobutyric acid (GABA) on immunoglobulin A (IgA) secretion from Peyer’s patch (PP) cells; to assess rat alpha-defensin-5 (RD-5) expression in the rat small intestine; and to determine the effect of GABA on intestinal ischemia reperfusion (I/R) injury-induced intestinal innate immunity. We found that GABA caused an increase in IgA secretion in the presence and absence of lipopolysaccharide (LPS). Moreover, GABA also significantly increased the mRNA levels of RD-5 and superoxide dismutase (Sod) 1, 3. Intestinal I/R was induced by a 30-min occlusion of the superior mesenteric artery followed by a reperfusion for 60-min. This led to a significant decrease in IgA secretion, and mRNA levels of RD-5 and Sod 1-3 in the ileum. On the other hand, administration of GABA before I/R induction had a significant protective effect against oxidative injury and attenuated the effects on intestinal immunity.

Nicotine-withdrawal symptoms have been indicated as a possible risk factor for neuropsychiatric events, such as depression and suicide, during use of smoking-cessation drugs. We aimed to investigate whether the results of the metabolomic analysis of the rat brain reflect nicotine-withdrawal symptoms. We also aimed to investigate the relative changes in each metabolite in the brains of rats with nicotine-withdrawal symptoms. We created rats experiencing nicotine-withdrawal symptoms through repeat administration of nicotine followed by a 12-h withdrawal period, and rats recovered from nicotine-withdrawal symptoms followed by an 18-h withdrawal period. We then implemented brain metabolic profiling by combining high-resolution magic-angle spinning ¹H-NMR spectroscopy with partial least square discriminant analysis (PLS-DA). We found that metabolic profiling of the brain reflects the state during nicotine-withdrawal symptoms and the state after recovery from nicotine-withdrawal symptoms. Additionally, N-acetylaspartate and glutamate increased and aspartate, γ-aminobutyric acid (GABA), and creatine decreased in the hippocampus of rats experiencing nicotine-withdrawal symptoms. Therefore, it is suggested that neurogenesis and neuronal differentiation could be changed and abnormal energy metabolism could occur in the hippocampus during nicotine-withdrawal symptoms.