Profunda femoris artery aneurysms and pseudoaneurysms are a rare cause of peripheral arterial aneurysms but their risk of rupture is quite high. We have presented a case of a left lower leg pseudoaneurysm. We have shown that endovascular repair with angioplasty and stenting is a suitable treatment method for such a pseudoaneurysm. Due to the limited data on this disease, we suggest multi-institute collaboration to identify and standardize management for the treatment. Profunda femoris artery aneurysms (PFAAs) are a rare cause of peripheral arterial aneurysms; however, the risk of rupture associated with such a finding is quite high. PFAAs are mostly asymptomatic and they usually present as an incidental finding. As for symptomatic patients, a swelling in the groin region is the most common presentation –3. True This is a case of a 38-year-old male who underwent intermedullary nailing of the left hip for avascular necrosis in September 2012. In March 2013, the patient presented with complaints of increasing edema of his left lower extremity associated with a palpable pulsatile mass in the anterolateral aspect of his thigh. At that point, the patient denied calf claudication on ambulation, nocturnal rest pain, or any other symptoms of the lower extremities. On exam, a palpable pulsatile mass in the anterolateral aspect of his thigh was noticed. He had a palpable thrill over it. Nevertheless, all lower extremity pulses were palpable.A computed tomography (CT) angiogram  with 3-dRight common femoral artery (CFA) access was obtained with a micropuncture needle. A crossover catheter was used to gain access to the left common iliac artery and a 7-French Ansel sheath was introduced over a stiff guidewire and parked in the left CFA. Selective angiography was performed again to precisely delineate the area of the pseudoaneurysm and its neck.At this time, a 10 × 38 mm iCAST balloon expandable stent was deployed across the aneurysm and dilated using a 12 × 40 mm balloon. Angiography showed exclusion of the pseudoaneurysm; however, there appeared to be an endoleak from the proximal aspect. Therefore, a second stent was introduced in a similar fashion that overlapped the first one and extended more proximally. A repeat angiogram showed excellent results without any flow into the pseudoaneurysm .The patient tolerated the procedure well. One month followup showed significant decrease in the left lower extremity edema. There existed no associated pain or discomfort. Moreover, on his 6-month followup, the patient was clinically asymptomatic and had no bruit or thrills over the profunda femoris site.PFAA is an uncommon disease with limited data describing the course and optimal management. After reviewing several publications, solitary PFAAs account for roughly 0.5% of peripheral aneurysms , 5. The Most patients are asymptomatic and are diagnosed secondary to other diseases. Symptomatic patients most often present with a swelling of the upper thigh. Other symptoms of PFAAs include local compression of surrounding structures such as veins or nerves and thrombosis leading to ischemia and rupture. Moreover, PFAAs can serve as a distal source of emboli , 3, 8. OCT angiography and ultrasound are the appropriate imaging modalities used in the diagnosis of a PFAA , 6. In aStandard treatment of the PFAA has been open surgery with either ligation of the PFA or reconstruction with a vein or a graft. The decision to undergo either one depends on the condition of the SFA. If the SFA is patent, then ligation of the PFA is permissible since there will be blood flow through the femoralpopliteal tract. However, it is suggested that it is best to repair the PFA rather than ligate, as this will maintain optimal flow to the lower extremity. If there is SFA disease or distal vascular disease, which can be associated with PFAA, it is a must to reconstruct the PFA to prevent ischemia to the lower limb, as the PFA is important for collateral circulation , 9, 10.Data on endovascular repair is limited with reports showing successful repair of a PFAA with endovascular coil embolization and others deploying covered stents to exclude the aneurysm , 10, 11.We reported a rare case of a pseudoaneurysm of the left PFA. On followup with our patient, he is doing well and there have been no complications with the procedure. We have shown that endovascular repair with angioplasty and stent placement is a reasonably safe and effective and a less invasive option for treatment compared to open surgery. Because of the limited data on endovascular repair of PFAAs, we suggest multi-institute collaboration to identify and standardize the management of this disease.


The bioavailability of the exenatide-loaded microspheres, relative to subcutaneous injection of exenatide, reached 10.2%. Collectively, the present study demonstrated the feasibility of orally delivering exenatide with the new cross-linked biomaterial and formulation, and showed therapeutic potential for clinical applications.Exenatide is an FDA-approved glucose-lowering peptide drug for the treatment of type 2 diabetes by subcutaneous injection. To address the issues on the inconvenience for patient use and the difficulty of oral administration of peptide drugs, chemical cross-linking of two pH-responsive biomaterials, alginate and hyaluronate, was carried out to prepare a new material for the encapsulation of exenatide as a form of microspheres. The exenatide-loaded microspheres exhibited spherical structures with excellent loading and release behaviors in the simulated gastrointestinal tract environments. After oral administration of the microspheres in  Helodermatidae venom. This peptide shares 53% sequence homology with glucagon-like peptide-1 (GLP-1) Exendin-4 containing 39 amino acid residues was originally isolated from Generally, protein and peptide drugs are rapidly denatured or degraded by low pH enviornment of the gastric media or the hydrolytic enzymes in the gastrointestinal tract via subcutaneous injection Despite significant developments in oral delivery technologies, successful oral delivery of protein or peptide drugs remains very limited. To address various issues associated with oral administration of macromolecule drugs, different approaches have been attempted in recent years, including chemical modifications by substitution, acylation or PEGlation to alter their physiochemical properties, the addition of other novel functions and the use of more efficient delivery carriers in vitro releases in the conditions mimicking gastrointestinal pH environments, in vivo pharmacokinetics (PK) and glucose-lowering effects in db/db mice were performed and compared. The results indicated that the exenatide-loaded microspheres could effectively deliver the drug to lower plasma glucose level after oral administration.As biodegradable anion polysaccharides, both alginate and hyaluronate have been widely used to encapsulate various drugs for controlled release and targeted drug delivery All experimental animals were cared according to the guidelines of National Institute of Health (NIH), and animal studies were reviewed and approved by the Ethical Committee on Animal Experimentation of China Pharmaceutical University.Sodium alginate  was a gift from Huanghai Biological Pharmaceutical Co. Ltd (Qingdao). Sodium hyaluronate  was purchased from Freda Biotechnology Co. Ltd (Shandong). Exenatide was from GL Biochem Ltd (Shanghai). Blood glucose meter and test strips were purchased from Johnson & Johnson Company (USA). Exendin-4 enzyme immunoassay kit (EK-070-94) was purchased from Phoenix Pharmaceuticals Inc. (USA). Bicinchoninic acid (BCA) protein assay kit was purchased from Generay Biotech Co. Ltd (Shanghai). All other chemicals were pharmaceutical grade and commercially available.db/db mice  were purchased from Cavens Lab Animal Co. Ltd (Changzhou). According to the guidelines of NIH, animals were housed in the specific pathogen free (SPF) laboratory with clean sawdust. Mice were grouped 5 animals per cage and raised under 12 hours light or dark cycle, allowed water and food ad libitum which were replaced frequently to keep fresh. Cages and bottles were cleaned twice a week with changing new sawdust when necessary. After 2-week acclimatization, animal studies were performed and minimization of animal sufferings was undertaken during the experiments. Mice were gently grasped and quickly administered the drugs using smaller syringe or blood sample were extracted by thinner capllary tube. In the end of the experiments, mice were sacrificed between 5-8 weeks after the experiments by removal of cervical spine.C57BL/ksJ The dispersed phase consisting of 75 mg sodium alginate, 25 mg sodium hyaluronate and 3 mg exenatide was dissolved in 5 ml purified water and prepared by magnetic stirring at 400 rpm for 30 min. Then, it was supersonically vibrated for 20 min and held for 1 h to remove the bubbles. Afterward, 10 ml paraffin liquid, the continuous phase, was blended with 2.5% (v/v) Span-80 for 30 min at 200 rpm. Under magnetic agitating at 400 rpm, the dispersed phase was dropped into continuous phase at a speed of 2 drops per second from a needle tube to form droplets. After 30 min, 5 ml 1 N HCl and 5 ml 25% glutaraldehyde (GTA) were added to mediate the cross-linking of alginate and hyaluronate at 40°C and the formation of solid microspheres. Finally, the exenatide-loaded microspheres were washed with ethanol, harvested by filtration under reduced pressure and dried overnight at room temperature.The blank microspheres were characterized with scanning electronic microscope . Residual amount of GTA in the microspheres was analyzed by GC , in which both GTA sample (0.01%) and the microsphere sample (10 mg/ml) were measured with a temperature gradient. Briefly, initial temperature was 100°C and maintained for 1 min, then rose to 180°C at the speed of 10°C per min and held for 30 min. Finally, the column was kept at 280°C for 5 min. Temperatures of sample injector and detectors were 210°C and 250°C respectively.The drug loading efficiency (LE) and loading content (LC) of the microspheres were determined by the amount of free exenatide remained in the supernatant using BCA protein assay kit To simulate the environments of stomach and small intestine, 100 mg exenatide-loaded microspheres were distributed in 10 ml of 0.063 N HCl solutions (pH 1.2) for 2 h followed by transferring to 10 ml of 0.1 M sodium phosphate solution (pH 7.4) at 37°C. A solution of 500 µl was exactly pipeted from the upper layer every 1 h and filtered. At the same time, 500 µl of fresh warmed sodium phosphate buffer was added into the original solution. Exenatide in the supernatant was detected by BCA kit.Swelling ratios (SR) at different time periods were investigated. Free water remained on the surface of swelled microspheres was dried by filtration under reduced pressure and SR was calculated as following T is the weight of swelled microspheres and M0 is the weight of initial dry microspheres.where Mdb/db mice were fasted for 12 h and remained fasting during the experiments, but were allowed water ad libitum. Following experimental groups were designed and divided: blank microspheres via oral administration as a blank group; blank microspheres blended with free exenatide solution (500 µg/kg) via oral administration as a negative control group; exenatide-loaded microspheres (500 µg/kg) via oral administration as an experimental group; and free exenatide solution (50 µg/kg) via subcutaneous injection (SC) as a positive control group. To blend blank microspheres and free exenatide, exenatide was firstly dissolved in physiological saline, and blank microspheres were added to this solution to form a mixture. Each group contained 8 db/db mice. Blood samples were collected from the tail veins of mice prior to the experiments and every 1 h or 2 h during the study, and blood glucose concentrations were measured by glucose meter.All male R) of the microspheres was calculated by the following formula To analyze the concentration of exenatide in plasma at different time points, blood samples were drawn from orbital sinus of mice and centrifuged  prior to experiments and at predeterminate intervals of 1 or 2 h during the experiments. The plasma concentration of exenatide was quantified with exendin-4 ELISA kit. The relative bioavailability (BAwhere AUC represents the area under the curve of plasma exenatide concentration versus time.Data were shown as mean ± standard deviation (SD). Comparison between two groups was analyzed by two-tail t-test with the statistical software (SPSS). A significant difference was defined as P < 0.05.Microspheres were produced by initial emulsion and then suspended in GTA solution for cross-linking To cross-link alginate and hyaluronate, GTA, a widely used and effective cross-linking agent was used to form a hemiacetal between alginate and hyaluronate in the presence of HCl The loading efficiency for exenatide in the microspheres was 71.4±2.57% (n = 3) and loading content was 2.98±0.91% (n = 3). Same as the microspheres prepared by other materials, the present microspheres prepared with cross-linked alginate and hyaluronate also showed a globular structure .. HoweveBecause GTA has the potential to produce the cross-links between DNA and proteins, it could increase the frequency of airway symptoms in humans The swelling pressure inside polymeric network can be enhanced due to electrostatic repulsion between charged moieties. Additionally, charge mobility can also be hindered by covalently cross-linked polymer As shown in R reported by Nguyun et al To determine the pharmacokinetic parameters, the plasma exenatide concentration was measured after orally administrating exenatide-loaded microspheres. As shown in In the present study, the exenatide-loaded microspheres cross-linked for 0.5 h could effectively mimic the effects of subcutaneous injection of exenatide. Compared to reported nanoparticles and microspheres for oral delivery of exenatide After cross-linking of alginate and hyaluronate in the presence of exenatide, the exenatide-loaded microspheres were successfully developed as an oral delivery vehicle for exenatide. Through pH-responsive mechanism, this novel vehicle could keep intact in stomach and then release loaded exenatide in small intestine for absorption. The PD/PK profiles of the present oral vehicle exhibited the comparable effects on lowering blood glucose and kinetic behaviors as subcutaneous injection. Thus, the present study on the microspheres has the potential to substitute subcutaneous injection and provides an alternative therapeutic tool for diabetic patients.


Potential photoactivation of certain pharmaceuticals, cosmetic ingredients and natural products by sunlight  has to be considered early on in development in order to avoid serious adverse effects . Current clinical trial registration guidelines  structure-property relationships ((Q)SPR) offers a valuable strategy to reduce experimental testing if an appropriate precision level of the underlying model is guaranteed.insilico analysis  of quantum chemical as well as classical molecular descriptors  has led to predictive photosafety classifiers. Model validation was performed with a proprietary external test set of an in vitro photosafety assay .A diverse data set of known phototoxicants and non-phototoxicants including various molecular chemotypes  was compiled. After geometry optimization the maximum absorption wavelength of each compound was calculated by semi-empirical methods followed by subsequent computation of molecular descriptors. Our Our photosafety models are currently applied in a prospective manner in the prioritization, classification and labeling of newly designed molecules.


Immune perturbation likely affects the development of Kaposi sarcoma (KS) among people infected with the KS-associated herpesvirus (KSHV). We tested whether KSHV-seropositive individuals or cases of classic KS (cKS), which typically originates in the leg, had differing delayed-type hypersensitivity (DTH) in the forearm or leg.Mantoux DTH with three antigens  was performed on the forearm and leg of 15 cKS cases, 14 KSHV-positives without KS, and 15 KSHV-negative controls. The diameters of induration responses were compared by group and body site.P=0.0004), whereas this was not observed in cKS cases  or KSHV-positives . Leg-minus-forearm DTH difference was greater in controls compared with cKS cases (P=0.004) and KSHV-positives (P=0.002). Leg-plus-forearm DTH was similar in controls (mean 28.2 mm) and cKS cases , but it was reduced in KSHV-positives , particularly in the leg (P=0.004) and marginally in the forearm (P=0.07).Leg DTH was greater than forearm DTH among controls (mean difference 5.6 mm, KS cases had weaker DTH only in the leg, whereas both body sites appeared weaker in KSHV-positives without KS. Both systemic and regional immune alterations may influence the development of this malignancy. Kaposi sarcoma (KS) is caused by infection with the KS-associated herpesvirus , but only a fraction of infected people develop the disease. The risk of KS development is markedly increased for people with a severe perturbation in immunity, especially HIV/AIDS. In contrast, the risk for classic KS (cKS) is approximately 100-fold lower than for AIDS KS  responses to dinitrochlorobenzene (DNCB) were noted to be absent in 16 African, non-AIDS KS patients who had very advanced disease but not in 30 who had limited disease  and a smFrom the 2002–2006 population-based cKS case–control study ; PPD (five tuberculin units); and C. albicans ). Each participant was observed for 30 min for possible acute reactions, of which there were none.Following informed consent and an examination for cKS lesions, one dermatologist identified disease-free areas on the volar surface of the forearm and the medial surface of the thigh. Using the conventional Mantoux method, six wheals were raised by intradermal injection of 100 Approximately 48 h after administration, the same dermatologist examined each injection site on the forearm and leg. The perpendicular diameters of palpable induration were determined using the ball-point pen method , measureTotal lymphocyte counts and CD4 and CD8 T-cell subsets were determined by the HIV/AIDS clinical monitoring laboratory using standard cytometric methods.t-test, either paired (within subject) or unpaired (between groups), and with analysis of variance. Categorical data (positive–negative responses) were compared with appropriate tests . They included 35 residents of the province of Palermo, and 9 residents of the neighbouring provinces of Trapani or Agrigento. As we reported previously (P=0.02) but not compared with seronegative controls . Kaposi sarcoma originated on the foot or leg in all 15 cases except 1; a second case developed metastatic KS on the arm. Nine cases currently had no KS lesions, three had 1–2 lesions, and three had approximately 20 lesions. Delayed-type hypersensitivity was performed on skin sites  that were far from KS lesions. The mean diameter of DTH responses in the forearm and leg of each participant is presented in vs K8.1, but the serology was highly concordant. Specifically, nine of the KSHV-seropositives had antibodies against LANA, and all but one had antibodies against K8.1. All cKS cases had antibodies against K8.1, and all but two had antibodies against LANA. As we were interested in cumulative DTH, diameters were summed for each participant, separately in forearm and leg, and then with the two body sites combined.We evaluated 44 participants, of whom 9 were female. The three groups did not differ with respect to age . In contrast, leg and forearm did not differ significantly in the KSHV-positives (P=0.56) or cKS cases (P=0.32) groups.First, we compared mean induration in the forearm ch group . As showP=0.07) and cKS cases , whereas the latter two groups did not differ from each other (P=0.58). In the leg, controls had significantly larger responses in the leg (16.9 mm) compared with KHSV-positives  but not compared with cKS cases . Response sizes in the leg did not differ between cKS cases and KSHV-positives (P=0.17).Second, we performed pair-wise comparisons of the three groups for size of responses in the forearm, then in the leg . As showP=0.002) and cKS cases , and this remained significant after adjustment for diabetes (P=0.003). With Bonferroni adjustment for 15 comparisons (P=0.03–0.06).Within individuals, difference in response sizes by body site was significantly greater in controls  compared with KSHV-positives  and marginally compared with cKS cases , whereas the latter two groups did not differ from each other (P=0.05), and this remained significant after adjustment for diabetes (P=0.05).The sum of responses (leg-plus-forearm) within individuals was significantly lower in KSHV-positives (11.8 mm) compared with controls  than cases with fewer lesions  or no lesions . Cases with >2 lesions also tended to have smaller induration  compared with cases with fewer lesions  or no lesions .There were no differences between or across the groups in total lymphocyte counts, CD4 count or percent, CD8 count or percent, or CD4/CD8 ratio P⩾0.20, . The cKSvs remaining KS free.We sought to identify whether there were differences in DTH, overall or between forearm and leg, which were associated with cKS or with KSHV-seropositivity without KS. We found a large and highly significant difference between the legs and forearms, with stronger responses in the legs of controls. In addition, the KSHV-seropositives had weaker overall responses (arm-plus-leg), including significantly weaker responses in the forearm compared with the cKS cases. These surprising results suggest that cKS cases and seropositives have divergent DTH responses that may relate to the development of KS As our controls have little or no risk of cKS, they represent the basal condition and thereby approximate DTH in this elderly, general population. Compared with these controls, the markedly smaller indurations in the legs of cKS cases support anecdotal reports of fewer and smaller DTH responses in the leg or foot compared with the arm . In contβ2-microglobulin or especially neopterin is more predictive of AIDS KS than is low CD4 lymphocyte count (β2-microglobulin (Stronger DTH in the leg of cKS cases compared with seropositives is the opposite of what we anticipated. It suggests that seropositives may be a special and highly informative group. They have survived with KSHV infection without developing KS, generally for several decades. , includiglobulin , which pDetection of KSHV viremia is the strongest marker of KS risk yet identified . It woulOur study was significantly limited by its small size, which reflects the rarity of cKS, the relatively low prevalence of people without KS who have unambiguous KSHV-seropositivity (In summary, we confirmed that KS cases have diminished DTH responses in the leg, and we also found evidence, albeit weaker, for diminished responses in the arm-plus-leg of elderly KSHV-seropositives who have not developed KS. As we performed several comparisons, these findings might have emerged merely by chance. However, they support the hypothesis that KS, in the absence of HIV infection, is related to intrinsic differences in immune responses, perhaps in the distal extremities even more so than systemically.


In addition, silencing of KSGal6ST attenuated KS expression on the primary cultured microglia and upregulated expression of some activation markers  under the stimulation with lipopolysaccharide and IFN-γ. This study demonstrates for the first time a close association of EAN and disappearance of KS on microglia. KS expression could be a useful marker to evaluate the status of polyneuropathy.Experimental autoimmune neuritis (EAN) is an animal model of Guillain–Barré syndrome, an inflammatory demyelination disease of the peripheral nervous system. Although this disease has been extensively studied on peripheral nerves, the pathology of the central nervous system has not been fully understood. Previous studies demonstrate that expression of keratan sulfate (KS), the sugar chain of proteoglycan, is associated with activated microglia/macrophages accumulated after neuronal injuries. Unexpectedly, we found here that KS is rather diminished in rat EAN. KS was restrictively expressed in microglia in the spinal cord of normal rats. KS was positive in 50% microglia in the ventral horn and 20% in the dorsal horn. In EAN, microglia increased in number and expressed the activation marker CD68, but KS expression was abolished. Concomitantly, pro-inflammatory cytokines, i.e., interferon (IFN)- Microglia transiently increase in number and they express P2X4 receptor involved in allodynia and pain hypersensitivity in the dorsal horn of the spinal cord in EAN.8 Moreover, infiltration of CD4-positive T cells is observed in the spinal cord and triggers apoptosis of motor neurons.9Experimental autoimmune neuritis (EAN) is an animal model of human Guillain–Barré syndrome, and is a helper T-cell-mediated inflammatory demyelination disease in peripheral nerve that is affected by the immunization of P2 peptide.11 KS is a polymer of lactosamine, 3Galβ1–4GlcNAcβ1, sulfated at the C6 of both hexose moieties. GlcNAc6ST-1 mediates sulfation of C6 position of GlcNAc residue, which is an essential step for KS biosynthesis.12 Therefore, GlcNAc6ST-1-deficient (GlcNAc6ST-1−/−) mice show loss of KS in the CNS.13 We and others reported16 that KS expression is enhanced in microglia/macrophages and the extracellular matrix after spinal cord injury (SCI). We found that GlcNAc6ST-1−/− mice show better functional recovery after SCI.15 Ablation of KS with its degrading enzyme keratanase II also promotes functional recovery after SCI.16 Further in vitro studies revealed that KS in extracellular matrix has an inhibitory role in axonal regeneration/sprouting that is relevant to functional recovery with KS ablation after SCI.16 On the other hand, the biological significance of KS expressed in microglia/macrophages remains obscure.Proteoglycans (PGs) have long sugar chains, so-called glycosaminoglycans, i.e., chondroitin sulfate, keratan sulfate (KS), dermatan sulfate, and heparan sulfate.In this study, we attempted to clarify the pathology of the CNS in EAN. We particularly focused on KS expression. Based on an association of KS expression and activated microglia/macrophages accumulated after neuronal injuries, we had speculated that KS expression might be upregulated in the spinal cord to exacerbate EAN. However, we found that KS was rather diminished in rat EAN. We have revealed for the first time the relationship of KS expression and EAN pathogenesis.The clinical scores of control (complete Freund's adjuvant (CFA) alone: open circles) and EAN (closed circles) rats were shown in The expression levels of KS in the spinal cord of control and EAN rats were analyzed by immunoblotting at the peak of EAN. The KS-specific antibody 5D4 was used in this experiment. Tissues were obtained from the cervical, thoracic, thoraco–lumbar and lumbar spinal cord, and SNs . KS was 17 In addition, we used CD11b (OX-42) as an additional marker for microglia/macrophages and found that expressions of Iba1 and CD11b were almost completely merged  than in dorsal horn (20±3.7%)  . KS expr20±3.7%) .19 In general, M1 microglia/macrophages exert pro-inflammatory functions, whereas M2 microglia/macrophages show anti-inflammatory functions. Activation markers detected by antibodies available for rat microglia include CD68 and CD206. CD68 is usually induced upon activation and may be positive for both M1 and M2 microglia, whereas CD206 exclusively represent a subset of M2 microglia. We found that both CD68 and CD206 were strongly expressed during EAN, showing a mirror image of KS expression , and disappeared at onset (12 days), peak (18 days), and remission (35 days) phases -1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, was increased in EAN +IFN-nditions . Importamulation . FurtherWe revealed in this study the reverse relationship between KS expression and progression of EAN. Immunoblotting, immunohistochemistry, and flow cytometric analyses clearly demonstrated that KS expression in microglia/macrophages was lost from onset to remission stages of EAN. Although further studies are required to establish the usefulness of KS expression in human polyneuropathy, our study demonstrates that KS is a good marker for rat EAN.20 A previous report demonstrates that granulocyte/macrophage colony-stimulating factor or TNF-α increases KS expression in cultured microglia.21 If either factor is combined with IFN-γ, KS expression is strikingly suppressed.21 Indeed, we found that IFN-γ and TNF-α are both induced in the spinal cord of EAN  and upregulates anti-inflammatory cytokines and M2 markers .28 Furthermore, it has been recently reported that various microRNAs including miR-27a, -29b, -125a, -146a -155, and -222 are implicated in polarized phenotypes of macrophages.29 Investigations on microRNAs in EAN might provide a clue to reveal the biological role of KS in this disease.Although EAN is a representative model for autoimmune diseases of peripheral neurons, experimental autoimmune encephalomyelitis is a representative model for autoimmune diseases of the CNS and is a model for human multiple sclerosis. It has been reported that KS is downregulated in experimental autoimmune encephalomyelitis.In summary, we found an interesting reverse relationship between KS expression and EAN pathogenesis, as well as microglial activation. Taken together previous reports of KS expression and functional recovery after neuronal injuries, our present study suggest that distinct inflammatory milieu in the CNS may determine expression profiles and biological roles of KS.μl of inoculum containing 100 μg of synthetic P2 peptide (NH2-RTESTFKNTEISFKLGQEFEETTADN-COOH), representing amino acid residues 53–78 of human myelin P2 protein . The peptide was dissolved in phosphate-buffered saline  and then emulsified with an equal volume of CFA  containing 1 mg/ml mycobacterium tuberculosis H37 Ra to get a final concentration of 0.5 mg/ml. Control rats were immunized with 200 μl of inoculum containing PBS and CFA. Neurological symptoms of EAN were evaluated every day as follows: 0, normal; 1, limp tail; 2, abnormal gait; 3, mild paralysis; 4, moderate paralysis; and 5, complete paralysis.30 Control and EAN rats were perfused intracardially with 4% paraformaldehyde (PFA) followed by PBS under anesthesia, and the cervical, thoracic, thoraco–lumbar, lumbar spinal cords, and SNs were quickly isolated at 4, 7, 12, 18, 35, and 90 days after immunization. In order to evaluate the histological findings after the EAN induction, H&E and LFB staining was performed in those SNs. The paraffin-embedded sections were stained by LFB solution (0.1% LFB MBS and 0.05% acetic acid containing in 95% ethanol) overnight at 56 °C. After washing, the sections were briefly rinsed by 0.05% LiCO3 and 70% ethanol. After the rinsing with distilled water, the sections were incubated in Cresyl violet solution to stain the neurons for 6 min. After several washing, the sections were mounted with Eukitt  and examined by an Olympus model BX41 microscope .Male Lewis rats  were housed under a 12-h light–12-h dark cycle with free access to food and water for 1 week. The institutional animal care and use committees of Nagoya University Graduate School of Medicine approved all experimental procedures. Rats were immunized in the hind footpad with 200 g for 15 min, and then the soluble protein concentrations were measured by Bradford protein assay using Protein Quantification Kit . The soluble proteins were treated with chondroitinase ABC  in 50 mM Tris-acetate buffer (pH 8.0) at 37 °C for 1 h and 20 μl of 2 × SDS-PAGE sample buffer  was added. The samples were subjected to SDS-PAGE after the boiling for 10 min. After transferring the proteins to a polyvinylidene difluoride membrane  and blocking it with PBS-T containing 5% skimmed milk, the membrane was blotted by mouse anti-KS antibody , rabbit anti-Iba1 antibody , mouse anti-CD68 anticody , and goat anti-CD206 antibody . Anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG conjugated with horseradish peroxidase  were used as secondary antibodies. Binding antibodies were visualized by ECL plus chemiluminescence system .Spinal cords and SNs were homogenized in the lysis buffer  and 1% Triton X-100 in PBS) at 4 °C for 60 s. The homogenates were centrifuged at 20 000 × Isolated spinal cords were fixed with 4% PFA in phosphate buffer (Wako) overnight, and cryoprotected by immersion in 20% sucrose in PBS during the subsequent night. The tissue samples were embedded in Tissue-Tek O.C.T. compound  and quickly frozen by liquid nitrogen. Frozen tissues were cut into 20 μm sections on a cryostat  and collected on MAS-coated glass slides . The sections were triple rinsed with PBS to wash out the cryoprotectant and fixed with 4% PFA in phosphate buffer, and permeabilized with PBS containing 0.1% Triton X-100. After blocking with 3% bovine serum albumin in PBS containing 0.1% Triton X-100, the sections were treated with chondrotinase ABC (0.5 U/ml) at 37 °C for 1 h, and then incubated with primary antibodies as follows; mouse anti-KS (1 : 200), rabbit anti-Iba1 for microglia/macrophages (1 : 200), mouse anti-CD68 for activated microglia/macrophages (1 : 200), and mouse anti-CD11b for microglia/macrophages . After triple rinsing with PBS, the sections were incubated with secondary antibodies as follows; AlexaFluor 488-conjugated goat anti-mouse IgG, AlexaFluor 488-conjugated donkey anti-mouse IgG, AlexaFluor 594-conjugated goat anti-rabbit IgG, and AlexaFluor 594-conjugated donkey anti-goat IgG . Subsequently, the sections were observed under a confocal microscopy A1Rsi  and a fluorescent microscopy BZ-9000 .34 Briefly, isolated spinal cords and SNs were carefully freed from meninges and mechanically dissociated in HBSS  containing 5 mg/ml collagenase (Wako). The mixture was incubated at 37 °C for 45 min, and the cell suspension was filtered through a 70-μm cell strainer  into a new centrifuge tube. After rinsing twice with PBS, a part of cell pellet was resuspended with PBS and centrifuged through a 40%/70% Percoll gradient (stock isotonic Percoll was diluted with PBS) at 800 × g for 20 min. The cell pellet was rinsed twice by PBS and resuspended with FACS buffer (PBS containing 0.5% (w/v) bovine serum albumin and 2 mM EDTA). FcγR was blocked by the incubation with anti-CD32 antibody  for 15 min at 4 °C. The prepared cells from the spinal cords and peritoneal cavity were stained with the following antibodies for 30 min at 4 °C; FITC-conjugated mouse anti-rat CD11b , AlexaFluor 647-conjugated mouse anti-rat CD45 , and anti-KS labeled using Zenon AlexaFluor 405 mouse IgG1 Labeling Kit (Invitrogen). After adding 300 μl of FACS buffer and centrifuged at 5000 × g for 5 min, cell pellets were resuspended with 700 μl of FACS buffer and were stained using 0.25 μg/ml 7-amino-actinomycin  for 10 min at room temperature. Cells were analyzed by FACS Canto2 3Laser-8color (Becton Dickinson) at the same parameters. The fluorescent compensation was performed using single-stained samples. The data were collected by analyzing 30 000 events using FACSDiva and FlowJo software (Becton Dickinson).The animals were deeply anesthetized and the peripheral blood was completely removed by the perfusion with PBS. Flow cytometory of microglia/macrophages in the spinal cord and SNs was performed as previously described with minor modification.μg/ml LPS and 100 ng/ml IFN-γ  for 24 h. Cells were collected and lysed by 1% Triton X-100 in PBS and lysates were subjected to SDS-PAGE.Mixed glial culture was obtained from the postnatal day 1 rats purchased from Japan SLC. Primary cultured microglia was obtained by the shake-off method. Briefly, the flasks were shaken with 120 r.p.m. for 15 min at 37 °C. Floating microglia was cultured on the PLL-coated dishes in DMEM/F12 media supplemented with 10% FBS and antibiotics. One day later, the media were changed to the serum-free one and siRNAs were transfected using Lipofectamine RNAiMAX reagent (Invitrogen). One day later, microglia was stimulated by 1 μg of total RNA using an oligo(dT) primer and reverse transcriptase  following the standard protocols. Real-time PCR was performed using SYBR Green on the Mx3000P quantitative PCR System . Each PCR mixture contained 0.2 μM of each primer, 10 μl of 2 × SYBR Green Master Mix (Agilent Technologies), 3 μl cDNA, 300 nM ROX reference dye, and PCR grade water  with a total volume of 20 μl. The PCR was carried out with a denaturation step at 95 °C for 10 min and then for 50 cycles at 72 °C for 30 s and 60 °C for 1 min. All PCRs were performed in triplicate and normalized to internal control glyceraldehyde-3-phosphate dehydrogenase mRNA. Relative expression was presented using the 2− method. The sequence of primers was described in RNA isolation was performed using RNeasy Lipid Tissue kit and RNeasy Mini kit  according to the manufacturer's protocols. cDNA was prepared from 2


KAL1 gene that codes for the extracellular protein anosmin-1. Investigation of KAL1 function in mice has been hampered by the fact that the murine ortholog has not been identified. Thus studies performed in other animal models have contributed significantly to an understanding of the function of KAL1. In this review, the main results obtained using the two invertebrate models, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, are illustrated and the contribution provided by them to the elucidation of the molecular pathogenesis of Kallmann Syndrome is discussed in detail. Structure-function dissection studies performed in these two animal models have shown how the different domains of anosmin-1 carry out specific functions, also suggesting a novel intramolecular regulation mechanism among the different domains of the protein. The model that emerges is one in which anosmin-1 plays different roles in different tissues, interacting with different components of the extracellular matrix. We also describe how the genetic approach in C. elegans has allowed the discovery of the genes involved in KAL1-heparan sulfate proteoglycans interactions and the identification of HS6ST1 as a new disease gene.Kallmann Syndrome is a heritable disorder characterized by congenital anosmia, hypogonadotropic hypogonadism and, less frequently, by other symptoms. The X-linked form of this syndrome is caused by mutations affecting the  Kallmann Syndrome (KS) is a congenital disorder in which anosmia or severe hyposmia (deficient sense of smell) is combined with hypogonadotropic hypogonadism (HH) and, less frequently, with synkinesia (mirror movements), mental retardation, cryptorchidism, cleft lip and/or palate, unilateral renal agenesis and others symptoms -5. KS isin vivo neuroimaging techniques in humans [ Post mortem anatomical analysis of a human foetus , studiesn humans , 13 haven humans . HH is in humans , 15, 16.KAL1 [1). The WAP domain shows similarities to small proteins with serine protease inhibitor activity [. KAL1 orthologs were identified in several vertebrates, and in invertebrates like Caenorhabditis elegans and Drosophila melanogaster, but not in mouse [1), indicating that the protein function is also conserved. The main differences among these proteins regard the number of FnIII repeats, which can vary from two to four, the absence of a HR region in invertebrates and the presence at the C-terminus of the C. elegans protein of a glycosyl-phosphatidyl-inositol anchoring site (GPI), which probably mediates the binding of the protein to the cell surface. The gene responsible for the X-linked form of KS was the first to be identified and was named KAL1 , 18. Thiactivity , 20, whiactivity , 22 and in mouse -25. The  KAL1 is responsible for less than 10% of all KS cases [fibroblast growth factor receptor type 1 (FGFR1) [fibroblast growth factor 8 (FGF8) [prokineticin 2 and its receptor (PROK2 and PROKR2) [chromodomain helicase DNA binding protein-7 (CHD7) [heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) [FGFR1 cause autosomal dominant forms of KS [FGF8 gene, which encodes a ligand for FGFR1 [PROK2 and PROKR2 are involved in digenic form of KS, with patients carrying missense mutations both in PROKR2 and KAL1 [PROK2 and PROKR2 [CHD7, the gene responsible for CHARGE syndrome, were found in a heterozygous condition in a minority of KS patients and it has been suggested that KS may be a mild allelic variant of CHARGE syndrome [HS6ST1, which encodes a heparan sulphate (HS) modification enzyme [HS6ST1 mutations were found in KS patients in combination with mutations affecting the FGFR1 gene [KS cases ; howeverKS cases . They ar (FGFR1) , 29, the8 (FGF8) , prokine PROKR2) , chromod7 (CHD7)  and hepa(HS6ST1) . Mutatioms of KS . Heterozor FGFR1 , are resor FGFR1 . PROK2 aand KAL1  or in PRd PROKR2 . Mutatiosyndrome , 36. Then enzyme . HS6ST1 FR1 gene . To date in only about 30% of KS cases a causative gene has been identified. This indicates that large part of KS patients present mutations in unknown genes. In addition, all the studies suggest KS as a disease with a complex and heterogeneous genetics, comprising cases with oligogenic mode of inheritance , 38. The models of molecular pathogenesis of KS have quickly changed over the past ten years. Summarizing the work on humans and vertebrate models it appears that KS is due to alterations of developmental processes occuring during embryogenesis. The most significant defects are in the growth of olfactory axons and the migrations of GnRH-secreting neurons. However also the sporadic symptoms  observed in KS patients could be interpreted as alterations of morphogenesis and development of other tissues/districts.KAL1, by Lutz and coworkers [ The first models of molecular pathogenesis of KS were formulated after the identification of oworkers . At the  Soussi-Yanicostas and coworkers , 40 demo We now know that most of the genes so far implicated in KS code for extracellular proteins. These proteins play functions in the extracellular environment, either as ligands and receptors for signaling or as matrix components acting as substrates for adhesion, growth and migration.in vivo and to identify new disease genes came from studies in C. elegans and Drosophila. The absence of a mouse homolog together with the great advantages offered by invertebrate animal models led different laboratories to focus on: 1) the analysis of the function played in vivo by anosmin-1 homologs in these models, 2) the structure-function relationship of the protein and 3) the identification of genetic interactors of anosmin-1. Research on KS is now focused on the identification of additional genes responsible for KS and on understanding the role of the identified genes in the developmental processes disrupted in KS patients. A significant contribution to understand the role played by anosmin-1  kal-1 gene in C. elegans encodes a protein (CeKAL-1) of 700 amino acids, composed of a signal peptide, a cysteine-rich domain, a WAP-type protease inhibitor domain, three FNIII repeats and a predicted GPI-anchoring site Fig. (1). The protein is 30% identical and 50% similar to human anosmin-1 [kal-1 Fig. (2a): one group is located in the anterior ganglia, a second group at mid body and a third group in the tail region. A deletion mutant was generated by chemical mutagenesis and this null mutant is at the moment the only animal model with a complete loss of function of the kal-1 gene and therefore an unique tool to investigate its function in vivo. Loss of function animals are apparently normal. Using high-resolution microscopy and a variety of GFP markers that help visualize neurons and epithelial cells it was possible to reveal a variety of subtle but specific defects, including: embryonic development retardation and ventral closure defects, alterations in the morphogenesis of the adult male tail, and neurite outgrowth defects [kal-1 expression in neurons influences morphogenesis of nearby epidermal cells by regulating their adhesion and migration, while it controls neuron development in a cell-autonomous fashion, modulating neurite outgrowth and branching [kal-1 mutant animals were successfully rescued by expressing the human KAL1 cDNA under the C. eleganskal-1 promoter [C. elegans as an animal model to study KS in vivo. Thenosmin-1 . The expnosmin-1 , 24, 42. defects , 42, 43.ranching , 24. Notpromoter . These rDrosophila, the kal-1 gene encodes a protein  shorter than vertebrate and C. elegans orthologs, with only two FnIII repeats Fig. (1). DmKal-1 is composed of 525 amino acid and shares 23% identity and 36% similarity with the human protein [kal-1 shows a complex expression pattern during the second half of embryogenesis and its transcripts are detectable in different epithelial cells involved in the morphogenetic processes of germ band retraction, dorsal closure and head involution as well as in cells associated with different sensory organs including the antenno-maxillary organ, which carries out olfactory and gustatory functions in larvae [ In  protein . The pren larvae . The data on nematodes and fruit flies indicate that invertebrate anosmins are involved in axon outgrowth and in epithelial morphogenesis in analogy with what is observed in humans and other vertebrates.C. elegans and Drosophila to better understand the role of the different domains of anosmin-1. Structure-function dissection analysis was used in 2b) [3a, ∆C), did not cause any extra-branching when expressed in the same head neurons. A similar loss of activity was observed after expression of CeKAL-1 carrying a single substitution in the first FnIII motif corresponding to the N267K mutation found in human patients Fig. . These results suggest a fundamental role of the FnIII domains in extra-branches formation. On the other hand, the elimination of two disulfide bonds in the WAP domain Fig.  did not abolish the ability to cause branching alterations but qualitatively changed the phenotypes with a more extensive arborization of the branches. This suggests a possible instructive role of the WAP domain in axon branching. Pan-neuronal expression of CeKAL-1 (not restricted to single head neurons) induced a highly penetrant axon fascicle-misrouting defect in head sensory neurons. The ability to induce this phenotype was maintained by the S241K mutant but was partially lost by the mWAP mutant protein Fig. (3a). This was the first experimental evidence of anosmin-1 as a multifunctional protein, with WAP and FnIII domains exerting distinct roles in different processes. However, these roles are not completely separable as the elimination of the FnIII motifs completely abolishes the ability of the protein to induce the axon branching defect, demonstrating that the WAP domain needs the presence of the other domains to carry out its function.  Overexpression of CeKAL-1 in the AIY interneurons produced highly penetrant and specific AIY axon branching defects Fig. (2b) . This exDrosophila DmKal-1 protein, by analyzing the phenotypes induced by the overexpression of different mutant proteins in the larval cephalopharyngeal skeleton and in adult wing formation [3b, W200-STOP) or only the C-terminal region downstream of the second FnIII motifs Fig. , completely abolished the activity of the protein. These experiments demonstrated that not only the FnIII domains, but also the C-terminal region, containing two consensus sequences for HS binding sites, are essential for the function of the protein. The introduction in the DmKal-1 protein of a mutation analogous to the N267K single substitution found in human patients Fig.  caused, in the larval head skeleton and in adult wings, the same phenotype induced by the wild type protein. However, the N236K mutation also induced strong alterations in the head involution morphogenetic process. This result suggests that N236K does not impair the function of the protein but induces a gain of function effect on a different process. Although a change in the overall adhesive properties of the N236K mutant compared to the wild type protein could not be demonstrated, it is possible that this substitution has produced a change in the affinity towards one or more specific interacting substrates. Interestingly, the same mutated protein did not induce similar gain of function effects in the wing, highlighting the importance of the context in which the protein works. A functional dissection was also performed on the ormation . As repo3b, R159T and R161L) only induces little changes in the protein activity. Proteins carrying point mutations in the CR region Fig.  or in the WAP domain Fig.  were still able to induce the same phenotypes as the wild type in the larval head skeleton and in adult wings. When, however, the C127/128S double substitution in the WAP domain was introduced together with the N236K substitution in the same protein, the gain of function effect produced by the mutation in the FnIII motif was suppressed. Thus, as in C. elegans, the WAP domain of DmKal-1 seems to be dispensable for some of the functions played by the protein, but its role appears essential for others. These results also suggest that the WAP domain may have a regulatory function on the fibronectin domains and on the adhesive properties of the molecule. This is in agreement with experiments performed in cell culture showing that a mutation in one of the cysteines of the WAP domain interferes with the function of the first FnIII motif [ A double point mutation in the consensus sequence of a non-conserved HS binding site in the first FnIII domain Fig. , which encodes an enzyme that catalyzes the transfer of a sulfate moiety to the glucosamine residue in position 6. A second mutation was found in the HS glucuronyl C5-epimerase (hse-5), another HS chain modifier enzyme that epimerizes glucuronic acid units to the isomeric iduronic acid [pst-1), a specific carrier for the transport into the Golgi of PAPS, the universal sulfate donor for all sulfotransferases enzymatic reactions [hse-5 but not in hst-6 suppressed the epidermal defects caused by pan-neuronal expression of kal-1 [kal-1 null mutant were found with C5-epimerized and 6-O-sulfated HS chains attached to proteoglycans during neuroblasts migration [C. elegans mutants are dependent on specific combinations of HS modifications, highlighting again the importance of the extracellular context for the function of anosmin-1 [C. elegans has also opened the way to the discovery that the human ortholog of hst-6 (HS6ST1) is involved in normosmic HH (nHH) and in KS [ In a genetic screening performed in pression . One of nic acid . A thirdeactions . All theof kal-1 . Moreoveigration . A direcigration . In summnosmin-1 . The stund in KS .FGFR1 is responsible for an autosomal dominant form of KS, it was hypothesized that anosmin-1 could interact with FGFR to promote the binding of the FGF ligand [C. elegans the FGF/FGFR signaling is not involved in ventral neuroblasts migration and no genetic interaction with kal-1 was found, the involvement of a different unknown pathway to regulate this particular phenotype has been proposed [KAL1 and Fgfr1 coexpression in different human tissues [in vitro analysis demonstrated that anosmin-1 can act as a co-ligand and modulator of the FGFR1/FGF2/HSPGs complex [C. elegans and Drosophila described above. After the discovery that F ligand . Since iproposed . Neverth tissues , 48-50,  complex , 52. Hu  complex  also dem In vivo confirmation of a functional interaction among anosmin-1/HSPGs/FGF/FGFR was given by genetic studies performed in the C. elegans nervous system, looking at a new phenotype [C. elegans only one FGF receptor and one ligand are present, encoded respectively by the egl-15 and egl-17 genes. These genes are not involved in CeKAL-1 induced axon branching phenotype of the AIY interneuron. However, a similar branching defect was induced also by the misexpression of CeKAL-1 in the AFD sensory neurons which, differently from AIY, require FGFR/ egl-15 for their normal development [egl-15 or of its ligand FGF/egl-17. The suppression of the AFD branching phenotype by loss of the ligand was stronger than by that of the receptor but not as complete as that due to the loss of HS. This result is important because it supports the idea that anosmin-1 can interact with other signaling pathways in addition to the FGF/FGFR one. This work offers the first in vivo demonstration of the role of anosmin-1 as an important modulator of the interaction among HSPGs/FGF/FGFR and also confirms the existence of other pathways, not involving FGFR, in which anosmin-1 and HSPGs may have a similar modulatory role.henotype . In C. eelopment . Also thelopment . In addi4). To carry out its function, anosmin-1 must interact with HSPGs through its HS binding sites mainly localized in the FnIII motifs and, in fact, the deletion of the FnIII motifs completely impairs the function of the protein Fig. . Sulfation of proteoglycans and glycosaminoglycans influences the interactions of the proteins involved in the developmental processes affected in KS and, accordingly, mutations in the gene encoding the modifying enzymes HS6ST1 were found in nHH and KS patients [4a, black arrow). As it has been shown previously, the disruption of disulfide bonds in the WAP domain may have, however, different effects on the function of the protein depending on the context. The evidence emerged from experiments performed in C. elegans, Drosophila and in cell culture and is also confirmed by clinical observations: cases of KS patients with mild sexual phenotypes carrying missense mutations that disrupt disulfide bonds in the WAP domain are well documented [4a, black arrow). In other extracellular contexts however this regulatory function seems to be dispensable Fig. (4b). For example, it is possible that the interaction of anosmin-1-HSPGs with other ligand/receptor complexes may be less dependent on the regulation of the CR region and of the WAP domain Fig. (4b). It is interesting to note that the AFD branching phenotype induced by CeKAL-1 was almost completely suppressed by mutations affecting genes involved in HS modification whereas the loss of function of FGF and FGFR only partially inhibited this branching phenotype. This suggests that alterations of AFD axon branching are produced by the simultaneous involvement of at least two different signaling pathways. The interaction of anosmin-1 with other signaling pathways is confirmed by the observation that both the ventral neuroblast migration in which CeKAL-1 is involved [ It is now clear that KS is a disease mainly involving extracellular interactions. Most of the genes identified so far as involved in KS code for extracellular matrix components or modificators, and for membrane proteins. In the matrix a relatively large number of proteins are present and interact with each other to ensure the correct execution of developmental processes, such as cell and neurite migration. In different districts the combination of proteins present is slightly or significantly different and so are their reciprocal interactions. Although these interactions do not all fall under the ligand/receptor paradigm, the results on anosmin-1 obtained especially with the invertebrate models make it possible to formulate a model for the function of anosmin-1 and of some of the other proteins involved in KS Fig. , Semaphorin and Robo signaling pathways. Since kal-1 enhances the effect of null mutations in all these genes, it has been suggested that CeKAL-1 may act in parallel with these pathways to carry out its function. Consistently, mice lacking Semaphorins pathway genes showed anatomical defects very similar to those associated to KS [SEMA3a to be a gene responsible for the disease [SEMA3a pathway. Work done in le in KS . kal-1 ned to KS -60 and t disease , 62. Sin disease , it is pPROKR2 also carrying a missense mutation in KAL1 [PROK2 and PROKR2 are not present in C. elegans and Drosophila. Another possible involvement of anosmin-1 in a different signal pathway regards the PROK2/PROKR2 pathway. PROK2 have binding affinities for HS-glycosaminoglycans and in addition there is a case of a digenic form of KS with a patient heterozygous for a mutation in  in KAL1 . In thisCaenorhabditis elegans and Drosophila melanogaster.  Kallmann Syndrome is a genetic heterogeneous disorder characterized by two main symptoms, anosmia and hypogonadotropic hypogonadism. Different genes involved in this disease have been identified and their role in the molecular pathogenesis is becoming more clear. In this review we summarized the contribution to the understanding of the molecular pathogenesis of KS provided by the two invertebrate model organisms,  in vivo. These experiments determined that anosmin-1 acts differently in different tissues, with the contribution of each domain varying in the various cellular context. The interactions with other components of the extracellular matrix are strictly dependent on HSPGs and on specific combinations of HS modifications, that are different depending on the tissue. In addition, these studies suggest a novel intramolecular regulation mechanism, which has been confirmed by in vitro studies. The multidomain function and tissue specificity may be the reason why different sets of phenotypes have been observed in different KS patients [ We illustrated how structure-function dissection of anosmin-1 in invertebrates has contributed to better understand the function that the different domains playpatients , 64. Finpatients , 64.C. elegans have been very useful for elucidating, at least partially, the pathways in which anosmin-1 is involved and have contributed to the discovery of a new gene involved in KS [kal-1 expression in C. elegans has already been demonstrated to be transcriptionally activated by the homeobox genes ceh-10/VSX2, ttx-3/LHX2 and lim-4/LHX9 [ Genetic screenings carried out in ed in KS . The facm-4/LHX9 , 67. Intm-4/LHX9 , 69 suggC. elegans and Drosophila will be very useful to understand the still elusive pathogenesis of KS, in particular in elucidating the complex extracellular interactions that are impaired in KS-affected patients. In addition studies in these models can help to understand the function played in vivo by causative genes recently identified in other systems, like SEMA3a and to clarify their eventual interactions with established KS genes. Finally, with their simplicity and easy manipulation they can still play a role in identifying new genetic partners, that can represent new candidate genes to be responsible for the remaining 70% of KS cases, whose causative gene is still not known. The well defined molecular pathways and the availability of powerful genetic techniques in


Aqueous solutions of isovaline and its precursor molecule, 5-ethyl-5-methylhydantoin, were irradiated with ultraviolet and γ-ray photons, to evaluate their structural stability against space radiation. The degree of photolysis was measured and irradiation products were identified using chiral, reversed-phase and ion-exchange high-performance liquid chromatography. The experimental results show that the degree of photolysis of 5-ethyl-5-methylhydantoin is more significant than that of isovaline under ultraviolet light irradiation, while the results under γ-ray irradiation are the opposite. As the products of isovaline photolysis, aspartic acid, serine, glutamic acid and alanine were dominantly detected. Therno acids –17. For no acids –20.etc. [The radiation field in the low Earth orbit (LEO), up to an altitude of 450 km, contains three types of radiation: galactic cosmic radiation (GCR), solar cosmic radiation (SCR), and radiation belts generated by the interaction of trapped GCR and SCR with Earth’s magnetosphere (van Allen belts). GCR, SCR, and the radiation belts are composed of protons, electrons, α-particles, heavy ions, etc. . Additioetc. . Althougetc. .On the other hand, the polarization of light is an important property with respect to the asymmetric photochemistry of bioorganic molecules. Neutron stars near presolar nebula are the possible astronomical sources of circularly polarized light (CPL) which can trigger enantiomeric excess (EE) of interstellar organic materials through asymmetric photochemical reactions ,16,24–26l-enantiomeric excess up to 18.5% due to aqueous alteration of meteorite parent bodies [−1) and Murchison (47 pmol·g−1) meteorites [Isovaline , a structural isomer of valine and a non-proteinogenic α-methyl amino acid , was fout bodies ,34. Furtteorites . But theThe recovery amount (fraction of unphotolyzed molecules in percent) in Φ (the ratio between the number of photolyzed molecules and the number of absorbed photons), is shown in I/I0 = 10−ɛlc, where I and I0 are the intensity of the incident light and transmitted light, respectively, ɛ is the molar extinction coefficient, l is the length of the solution through which light passes, and c is the molar concentration of the solution). As the deuterium lamp  produces continuous spectra (190–400 nm), we calculated the average absorbed photon number for every 10 nm of wavelength and obtained the total average absorbed photon number . The average Φ-value obtained in the continuous UV (190–400 nm) irradiation experiment suggests that approximately 89% of an Ival and 80% of an EM-Hyd molecule can be decomposed by one photon. Although the Φ value for UV-photolysis of EM-Hyd is about 9% less than that of Ival, photodecomposition of EM-Hyd is approximately 2~2.5 times larger than that of Ival as EM-Hyd can absorb more UV-photons, which can be clearly understood from Ival’s and EM-Hyd’s molar extinction coefficients, as shown in The quantum yield of photolysis, With γ-ray irradiation, Ival was more significantly photodecomposed than EM-Hyd in the same conditions, as shown in Φ) under γ-ray irradiation for Ival and EM-Hyd is enormously high, as shown in 2O) molecules (mainly Compton scattering) during irradiation. The secondary electrons and scattered photons then lose energy through a sequence of interactions with the reactant and solvent molecules again. Thus, the amount of decomposition of Ival and EM-Hyd depends on the secondary chain reactions in the radioactive process. As a consequence, the Φ value during γ-ray irradiation is four orders of magnitude higher than that during UV irradiation. Because the photon-energy ratio between γ-rays (1.25 MeV) and UV  is 2.0 × 105, the Φ value ratio between γ-ray and UV irradiation in this study  and ethyl (−CH2CH3) groups from Ival in the transition period, which would lead to the formation of Ser and Ala  besides Ser and Ala were formed from Ival by γ-ray irradiation, whereas Ser and Ala were the only common photo-products in the case of UV- and γ-alteration of Ival. This difference might be due to the γ-ray-induced reactions as we mentioned above (see section 2.2). Additionally, two other peaks after the peak of Ival  and alanine (Ala) were formed . The pho and Ala . For the of Ival , trace fThe analysis of the photolysis products of irradiated EM-Hyd will be addressed in a future study. As mentioned in the introduction, EM-Hyd is one of the precursors of Ival and it is well known that hydrolysis of EM-Hyd produces Ival . Therefod- and l-EM-Hyd) and Ival  were abiotically synthesized in the Mita Laboratory at the Fukuoka Institute of Technology, Fukuoka, Japan. Ethylmethylketone was condensed with ammonium carbonate in the presence of potassium cyanide to produce EM-Hyd according to the Strecker synthesis. Then, Ival was synthesized by hydrolyzing EM-Hyd with barium hydroxide. Purity of the synthesized Ival and EM-Hyd was over 99%.As starting material for the radiation experiment, EM-Hyd . The wavelength range of the measurement was 190 to 400 nm. 2 for 8 and 16 h at a 30-cm distance from the photon source of the lamp. The total power density emitted from the deuterium lamp was calculated by integrating the spectral irradiance  using a deuterium lamp , as shown in radiance  over the60Co source (1.25-MeV-photon energy) at the Japan Atomic Energy Agency, Takasaki, Japan. The total irradiation doses were 10 and 20 KGy (20 and 40 J) as absorbed by the solvent.Ival and EM-Hyd were sealed in borosilicate glass tubes in air. Every sample  was irradiated with γ-rays from a 4·5H2O, 1 mM) was used as the mobile phase and the flow rate was 1 mL/min.After the UV and γ-ray irradiation, an aliquot of EM-Hyd was hydrolyzed with 6-M HCl at 110 °C for 24 h to convert it into Ival. Following the acid hydrolysis and evaporation to dryness, the hydrolyzed fraction was dissolved in milli-Q water. Finally, a portion of the irradiated sample  was injected into a chiral chromatographic system to measure the amount of decomposition. The chromatographic system was equipped with a pump (TOSOH DP-8020), a column , and a detector . Copper (II) sulfate pentahydrate , and a detector . The temperature of the column was maintained at 37 °C. Phosphate buffer  and acetonitrile  were used as the mobile phase under isocratic conditions.In addition, the decomposition of irradiated EM-Hyd was analyzed (without hydrolysis) by reversed-phase high-performance liquid chromatography (RP-HPLC). The RP-HPLC system was composed of two pumps (SHIMADZU LC-20AD), a column , a cation-exchange column , a post column derivatization system, and a Shimadzu RF-535 fluorometric detector . The temperature of the column was maintained at 55 °C. Gradient elution was performed using eluents A  and B . The flow rate of the carrier was 0.3 mL/min. A Wako amino acid mixture (type AN-II and type B) was used as the amino acid standard for IE-HPLC analysis.Furthermore, the photolysis products of irradiated Ival were analyzed by ion-exchange high-performance liquid chromatography (IE-HPLC) using post-column derivatization with 3) aerogel and analyze micro-organisms and organic compounds in it. The mission will also involve irradiation of Gly, Ival and their precursors at LEO in a real space environment by using the exposed facility of the Japanese Experimental Module (JEM) on the International Space Station.The present results of laboratory simulation suggest that EM-Hyd  is photochemically much more stable under γ-rays and less stable under UV environment than Ival. Moreover, the photo-alteration process of Ival produces Ser and Ala suggesting that Ival and its precursors might play an important role for the formation of Ser and Ala in the prebiotic chemistry. Our second phase simulation experiments will focus on the photochemistry of solid-state samples including glycine (Gly), hydantoin (a precursor of Gly), Ival, EM-Hyd and complex amino acid precursors synthesized from possible interstellar molecules. We are also planning to irradiate them in a real space environment. Recently, the “Tanpopo Mission” , a proje


Accumulating evidence indicates integration of dopamine function with metabolic signals, highlighting a potential role for dopamine in energy balance, frequently construed as modulating reward in response to homeostatic state. Though its precise role remains controversial, the reward perspective of dopamine has dominated investigation of motivational disorders, including obesity. In the hypothesis outlined here, we suggest instead that the primary role of dopamine in behavior is to modulate activity to adapt behavioral energy expenditure to the prevailing environmental energy conditions, with the role of dopamine in reward and motivated behaviors derived from its primary role in energy balance. Dopamine has long been known to modulate activity, exemplified by psychostimulants that act via dopamine. More recently, there has been nascent investigation into the role of dopamine in modulating voluntary activity, with some investigators suggesting that dopamine may serve as a final common pathway that couples energy sensing to regulated voluntary energy expenditure. We suggest that interposed between input from both the internal and external world, dopamine modulates behavioral energy expenditure along two axes: a conserve-expend axis that regulates generalized activity and an explore-exploit axes that regulates the degree to which reward value biases the distribution of activity. In this view, increased dopamine does not promote consumption of tasty food. Instead increased dopamine promotes energy expenditure and exploration while decreased dopamine favors energy conservation and exploitation. This hypothesis provides a mechanistic interpretation to an apparent paradox: the well-established role of dopamine in food seeking and the findings that low dopaminergic functions are associated with obesity. Our hypothesis provides an alternative perspective on the role of dopamine in obesity and reinterprets the “reward deficiency hypothesis” as a perceived energy deficit. We propose that dopamine, by facilitating energy expenditure, should be protective against obesity. We suggest the apparent failure of this protective mechanism in Western societies with high prevalence of obesity arises as a consequence of sedentary lifestyles that thwart energy expenditure. In this view, dopamine's reward related effects arise secondary to and in the service of adaptively managing energy expenditure. We are profoundly indebted to Salamone's elegant work and dogged focus on the role of dopamine in regulating effort and his persistent criticism of the reward hypothesis of dopamine. The present hypothesis represents an integration and expansion of his fundamental insights into a broader hypothesis in which dopamine adaptively regulates both effort and reward—scaling the impact of prior reward history on current behavior—with respect to the availability of energy in the environment.To date, no compelling framework has integrated these two distinct domains of dopamine effects and putative function, the widely recognized reward function and the less prominent but equally demonstrable effects of dopamine on activity and energy expenditure. Apparent dopaminergic effects on activity are often framed as a consequence of reward processes. For example, the role of dopamine in modulating voluntary wheel running in rodents has been proposed to arise from dopaminergic modulation of the reward and reinforcement associated with wheel running  pressed significantly more on the high cost lever than wild-type mice, consistent with prior literature that shows dopamine enhances effort toward reward. However, in this instance the increased effort did not increase reward, merely the amount of effort expended toward that reward. Detailed analysis of the data show that the DATkd were not insensitive to or unaware of switches between the levers as their peri-switch behavior was essentially identical. The difference arose during the stable periods between lever switches where the DATkd mice distributed their effort equally to both levers while the wild-type mice preferentially pressed the cheap lever. To better understand the strategy underlying the DATkd behavior, we fit the data to a temporal difference learning (TD) model  indicates a large genotype effect where the DATkd mice ate overall hyperdopaminergic mice adapt their consumption to response costs similarly to wild-type. On the surface this appears contradictory to many studies that suggest that stimulating or impeding dopamine transmission can enhance or diminish effort-based responding, respectively   Rather than over-riding homeostatic mechanisms and promoting excess consumption, dopamine appears to work within the constraints of homeostatic regulation, altering the distribution of effort in pursuit of food without changing overall consumption; that is, dopamine induces greater vigor but not greater “wanting.” (3) Rather than shifting effort, consumption, and behavioral choice to more preferred foods, dopamine again increases vigor without altering consumption, preference or choice; that is, the apparent decreased sensitivity to costs does not shift appetitive goals. The studies described all point to a common theme: that dopamine is modulating behavioral energy expenditure. Dopamine modulation of effort and expenditure has been viewed as either a non-specific effect—“generalized activity”—and/or as a reallocation of effort that overcomes response-costs associated with pursuing goals  Rather than increasing the impact of reward on behavior, we observe a reduced coupling between reward history and choice, suggesting dopamine induces greater exploration; that is, Energy and its use is the final bottom line for adaptation. All organismal needs and functions, from thermoregulation to reproduction to procurement of energy itself, require energy. Maintaining an adequate supply might be thought of as an evolutionary prime directive. Much focus has been given to systems that regulate the pursuit, consumption and storage of energy, but much less attention to systems that control its expenditure. Yet, aside from being an “equal partner” in determining energy balance , achieving an optimal distribution of energy to different activities is critical to adaptation. That is, what an animal does with its available energy is arguably as important as acquiring energy. How to best direct energy expenditure, however, is contingent upon environmental conditions. In an energy rich environment, exploration, exercise, and energy expenditure is adaptive. In an energy poor environment, exploitation of prior experience and energy conservation—getting the most bang for one's proverbial energy buck—is essential. In this view, effective energy management entails deciding 1) how much energy do I have to expend and (2) how carefully, or selectively, do I need to deploy it. We characterize these two questions as two axes of energy management: expend vs. conserve and explore vs. exploit, respectively  learning about reward—that is, assigning value to stimuli that directs energy expenditure and (2) the degree to which that reward information biases behavioral choice, construing the second axis, explore-exploit, as energetic thrift: how carefully do I need to exploit my available resources?In the current hypothesis, we associate these two axes of energy expenditure with two well-documented dopamine functions. First, we link to dopamine's role in regulating generalized activity to the expend-conserve axes. Second, we suggest that dopamine's role in reward is not to modulate reward incentive and appetitive pursuit By emphasizing the management of energy expenditure rather than reward modulation as a primary function of dopamine, many otherwise hard to reconcile observations can cohere into a central organizing metaphor for understanding the role of dopamine in behavior. In the following sections, we briefly detail the core elements of this hypothesis in the context of current ideas and literature about dopamine.skeletal alternative framework and do not attempt to assimilate everything known about dopamine nor detail every mechanism potentially involved in dopamine signaling, an intractable challenge for a single paper.It should be noted that the dopamine system is complex and multi-faceted. Aside from extracellular dopamine concentrations and rates of tonic and phasic dopamine cell firing, “dopamine function” can include modulation of release at its targets, modulation of synthesis, vesicular packaging and readily releasable pools, changes in receptor expression and function and alterations in dopamine transporter and reuptake. As a necessary simplification for the purposes of exposition, we will speak of “high” and “low” dopamine function, a not uncommon practice in the literature. The complexities this obscures represent potential mechanisms by which the dopamine system can exert more nuanced, flexible and sophisticated regulation of its function(s). In Figure The dopamine system is interposed between two worlds of stimuli: the external and internal. On one hand, dopamine modulates an organism's response to environmental stimuli. In the reinforcement learning perspective of dopamine and reward , toward exploration such as in the open field or merely moving around doing more mouse-like stuff day to day in the home cage. As psychostimulant users for decades attest: dopamine is energizing. Thus, the effect of dopamine on generalized activity represents a fundamental effector in the regulation of energy expenditure by up- or down-regulating how much energy is expended in behavioral activity independent of how that activity may be directed: The literature is replete with evidence that increased dopamine increases effort in pursuit of goals, canonically illustrated by increased performance in progressive ratio paradigms where after each reward earned, the cost of each subsequent reward increases Hodos, . Historibenefit  actually bias and shape behavioral choices. We integrate into this axis reinforcement learning and incentive-salience perspectives arguing that phasic dopamine mediates learning about and updating reward values, as widely believed, while tonic dopamine, as incentive views suggest, scales the impact of these values on behavioral choice, the expression of learned values. We reconceptualize this incentive scaling formally as a function within reinforcement learning that regulates the degree to which prior reward learning biases choice; that is, how much established learning is exploited. We assign this latter function to tonic dopamine and construe it as regulating thrift; that is, regulating the frugality of energy expenditure.In the following sections we argue that reward serves a central function in the dopamine mediated management of energy: controlling the s Figure . In thisThe frugality of one's expenditures depends upon the resources available. A rich person need not quibble about thousands of dollars while a poor person needs to count pennies. Similarly, animals living in energetic environments of plenty need not worry about energy conservation while those living under conditions of scarcity must expend energy judiciously. Thus, energy management entails not only determining the overall magnitude of energy expenditure, as discussed above, but also its allocation to specific activities.We start from the premise that when energy is readily available, energy expenditure is adaptively advantageous, for two primary reasons. First, physical activity has been shown in countless studies to contribute significantly to health and longevity . The current hypothesis encompasses both within a TD learning framework.As noted in the introduction, the term “reward” can be ambiguous. Here we adopt an information perspective and define reward as positively valenced outcome information. The primary question to be addressed is what role dopamine plays in linking reward history to future choices. Reinforcement learning perspectives argue that dopamine modulates corticostriatal plasticity in response to reward, i.e., positive outcome information, thus mediating learning t − 1) is compared to what was actually received at time t plus remaining expected rewards in the future, that is, the value estimate at t. If there is a discrepancy, called a prediction error, the prior value at t − 1 is adjusted so that when that state is encountered again, it will be more accurate. When the animal moves forward in time to t + 1, the same process will occur, this time adjusting the predicted value of t by comparing it to actual reward at t + 1 plus the future expected value of t + 1, and so on. The name TD arises because an entire succession of states, each with its own value prediction of “remaining” future reward, is adjusted one step at a time. It is this collection of values associated with different states—stimuli and actions—that enables accurate prediction of future reward. In short, it is an algorithm that facilitates trial and error learning where the animal is always, as it were, in medias res, and an accurate estimate of the value of particular stimuli and actions accrues gradually over time through experience.Temporal difference models are a class of reinforcement learning algorithms that have been successfully applied to understanding how neural substrates, such as dopamine and the basal ganglia, mediate behaviorally observed reinforcement learning. Within these models, stimuli and actions are assigned a “value” that incorporates all expected future rewards associated with those stimuli or actions. As time unfolds and the animal moves incrementally forward in time, advancing through successive states , at each step forward the prior prediction  biases action selection, frequently referred to as the “explore-exploit” parameter.in order to better allocate and utilize its available energy.Substantial evidence supports a role for dopamine in mediating reinforcement learning and corticostriatal plasticity, which will not be reviewed here  to minimal influence (explore). This could be construed as a frugality axis where greater exploitation maximizes reward for energy expended while reduced exploitation facilitates exploration and greater energy expenditure.Although, through its role in reinforcement learning, (phasic) dopamine contributes to learning about value, the range of the vertical axes in Figure external environment needs to be taken into account as well, specifically the availability of reward, particularly energy. As discussed above, the richness of the environment determines how frugal an animal needs to be with its energy expenditure and how much it needs to maximally exploit prior learning. Niv et al. .How and to what extent learned values direct behavioral choices clearly depends upon many factors, most obviously the motivational state of the organism, the “salience” in “incentive-salience,” taking into account the organism's internal environment. The v et al.  have sugFinally, it is widely believed that learned values can be part of a goal-directed or a habit system  contribute to energy management in potentially different ways. The development of a dopamine and energy management hypothesis demands asking how dopamine modulation in different targets may contribute to coordinated energy management. At this point, we cannot reasonably speculate on this question.is an overall, grand function of dopamine—most frequently, “reward” processing, in some form or another. It seems not unreasonable to think that as the function of a neurotransmitter elaborates and transforms over evolution, those elaborations arise as variations on a theme, a further enhancement of some function that has already conferred adaptive fitness. Even if such a notion ultimately proves false, seeking a “common thread” among seemingly diverse, unrelated functions may provide deeper insight into how these apparently disparate functions interact to contribute to behavioral adaptation. “Mediating reward,” as broad and ambiguous as it is, has served as this common denominator in much of the literature and has yielded rich results. Here we suggest an alternative, overarching function, energy management.Reflecting on this question, however, provokes the question of whether there really is some overriding “function” of dopamine at all? Or has dopamine acquired various, unrelated functions so that the notion of a finding “the” function of dopamine represents a fool's errand: perhaps the dopamine functions we observe today evolved as patchwork, evolutionary bric-a-brac, an opportunistic collection of fortuitous adaptations. From the literature, one gets the sense that many believe there coordinated activity across multiple substrates in response to external conditions, in a way that reward, an event in the external world, perhaps does not. This coordinated orchestration of multiple substrates toward a single goal or purpose is characteristic of hormones, such as reproductive or stress hormones. Given dopamine's diffuse projections and actions on multiple targets, it is tempting to entertain the notion that dopamine may have evolved a hormone-like function to coordinate multiple neural substrates and orchestrate behavior optimally adapted to the prevailing energy environment in which the organism finds itself. Though admittedly broad and speculative  adaptation as well, as frequently proposed in theories likening obesity to addiction  in rodents fed high fat, high calorie diets is a prevalent model of environmentally induced obesity. Though widely construed as a model of obesogenic environments, rodents on such diets do not universally become obese, showing degrees of resistance that vary between strains (Brownlow et al., This hypothesis would predict that a ready supply of energy, as is generally the case in modern western societies, would enhance dopamine and facilitate energy expenditure, providing and expenditure but in the absence of voluntary exercise opportunities, the increased energy expenditure is blocked, resulting in energy imbalance, obesity, and metabolic disorder.Although wheel running has been shown to protect against DIO in several models of obesity (Zachwieja et al., increase in D2 (South and Huang, initial response to high fat and increased caloric availability is to increase dopamine and increase expression of the disinhibitory D2 receptor, inducing greater activity. In order to answer the question of how the dopamine system responds to an abundance of available calories, it is critical to disambiguate initial from chronic response and assess the degree to which pathological adaptations, such as leptin insensitivity, are present.Some studies have demonstrated that high fat/calorie diets diminish dopamine function, including reduced TH, reduced stimulated dopamine release and reduced D2 receptor expression (Geiger et al., contribute to this pathology? Evidence suggest that wheel running can alter functional characteristics of the dopamine system (MacRae et al., To what degree do the changes observed in the dopamine system under DIO arise not from high calorie diet itself but from a lack of exercise opportunity? That is, in observations of diminished dopamine function associated with DIO, we can ask not only to what extent does this reflect a pathological adaptation, but also to what degree does a lack of opportunity for voluntary energy expenditure thwarting energy expenditure under conditions of energetic abundance. Many have suggested that sedentary lifestyles characteristic of modern western cultures may contribute to obesity as much or more than diet (Powell and Blair, In the hypothesis proposed here, increased availability of energy would, via up-regulation of dopamine mediated behavioral activity, result in increased exploration and energy expenditure, allowing the animal to take advantage of a plentiful energy supply and protect against obesity. Insofar as the DIO paradigm does not provide an opportunity for exploration and energy expenditure by confining rodents to small cages with little to no novelty, stimulation or exercise opportunities, it may reflect the consequences of The reward hypothesis of dopamine has dominated investigation and thinking about dopamine; experiments are typically designed within that conceptual framework. As a consequence, the information necessary to evaluate the proposed hypothesis is more often than not missing. Frequently, activity levels are simply ignored. This ranges from broad, overall investigation in which there is a profound bias toward studying appetitive behavior and consumption with only recently a nascent literature on mechanisms controlling voluntary energy expenditure to specific experiments, such as many DIO studies in which activity is not measured or taken into account at all [e.g., Geiger et al., range of conditions to which an animal must adapt, but it does not reflect the primary condition believed to underlie the rise in obesity, which is an environment of plenty, not scarcity. A semi-naturalistic home cage approach, as illustrated by our work as well as others (Hursh et al., Second, adaptation is environment-dependent. Currently, the vast majority of animal studies effectively investigate one energy economy: periodic, temporary food scarcity arising from food restriction employed to motivate animals. Not only does this fail to reflect the Third, virtually all work focuses on the impact of altered dopamine on behavior with little examination of how behavioral interactions with the environment and subsequent outcomes alter the dopamine system itself. These types of studies are admittedly challenging, as evidenced in the literature on how stress alters dopamine function. Nonetheless, they seem critical to fully understanding the adaptive function of dopamine. Does a sustained environment of plenty or scarcity up- or down-regulate dopamine function? Though the question may well be critical, there is no clear or compelling answer to date and the question is rarely asked.Here we develop a broad hypothesis of dopamine function which suggests that the myriad apparent functions of dopamine might be understood collectively as mechanisms by which energy expenditure is adapted to the energy economy in which the animal finds itself: a substrate for reconciling the pursuit of reward with resources. We first elaborated this hypothesis in theoretical terms attempting to integrate different views of dopamine function into a broader energy management framework. We then applied this framework to re-interpreting ideas and data in the growing field of dopamine and obesity. We propose the novel hypothesis that dopamine, by favoring energy expenditure, would normally be protective against obesity but that the sedentary lifestyles of modern society impede this protective process and induce pathological adaptations that contribute to rather than protect against obesity. Though beyond the scope of the current discussion, we believe the framework sketched here in broad strokes may be fruitfully applied to other areas of research in dopamine, including attention-deficit hyperactivity disorder and addiction, potentially yielding novel insights and testable hypotheses.Reward—the linkage of external events and stimuli to internal needs—clearly represents a critical function from an evolutionary, adaptive point of view. However, reward and value are fundamentally relative. What is the yardstick by which reward is modulated? Here we suggest that the dopamine system, at its root, arose to address an even more fundamental function than reward: optimally utilizing energy resources, the very heart of adaptive survival and, literally, a matter of life or death.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Secretion of the anterior pituitary hormone prolactin can be significantly increased by antipsychotic drugs, leading to a range of adverse effects in patients with schizophrenia. However, there is evidence from a variety of studies that prolactin may also be related to symptom profile and treatment response in these patients, and recent work has identified variations in prolactin secretion even in drug-free patients. In this paper, a selective review of all relevant studies pertaining to prolactin and schizophrenia, including challenge and provocation studies, is presented. The implications of this work are discussed critically. A tentative model, which synthesizes these findings and argues for a significant role for prolactin in the development of schizophrenia, is outlined. Prolactin is a polypeptide hormone secreted by the anterior pituitary gland. Prolactin has multiple functions, including lactation and maternal-infant bonding, in mammals. Recent work has found it to be relevant to parental and sexual behaviour in humans , 2 as we2) dopamine receptors. Most available antipsychotic drugs can therefore cause elevations in prolactin secretion. This increase is associated with a variety of adverse effects: lack of libido and erectile dysfunction in men [The production of prolactin is inhibited by dopamine release in the hypothalamo-pituitary circuit and can be increased by blocking type 2  is linked to dopamine receptor supersensitivity , alteratBecause of the small sample sizes involved, the results of these studies must be interpreted with caution. They may indicate dopamine receptor supersensitivity  or GABA Alterations in genes regulating circadian rhythm have been linked to both schizophrenia and the affective disorders . Like otThese studies suggest that there are clear alterations in the circadian rhythm of prolactin in schizophrenia, particularly with regard to sleep , 49. TheSuicide attempts and completed suicide are not uncommon in schizophrenia . A link Two recent studies have examined genetic variants in prolactin in patients with schizophrenia. Rybakowski et al.  examinedThe literature reviewed above, though complex, suggests fruitful lines for further enquiry. First, the association between raised prolactin and acute episodes of schizophrenia needs to be replicated in larger samples. Riecher-Rössler et al.  provide Second, the association of lower prolactin levels with positive symptoms and “paranoid” symptoms in particular are in line with the revised dopamine hypothesis of schizophrenia. This association can certainly be interpreted as suggesting increased dopamine transmission in this subgroup, with the same caveat as above: we do not know how well prolactin correlates with dopamine activity in the mesolimbic pathway. If this finding can be replicated, it provides some support for the notion of subtypes within the broad category of schizophrenia. Given that the most recent edition of the DSM has consciously omitted the traditional subtypes , a biomaThird, provocation studies provide further support for the idea of blunted or lowered prolactin secretion in patients with “positive” schizophrenia. Whether this relationship holds good for other conditions in which “positive” symptoms are prominent—such as psychotic mood disorders or acute and transient psychosis—needs to be decided by further research. Though alterations in dopamine, serotonin, and GABA activity are all hinted at by individual provocation studies, the true nature of these abnormalities is likely to be slight and subtle, and their significance is unclear. A similar conclusion would apply to the results of studies of circadian rhythm.Fourth, the association between prolactin and treatment response to antipsychotics, as well as its relationship to tardive dyskinesia, is inconsistent and, in the latter case, unlikely to be of much clinical significance. Given the numerous confounders that can affect prolactin levels, the inconsistency of positive findings and the negative results of recent genetic research, it is unlikely that a consistent association can be made. Changes in prolactin level may be a more sensitive indicator of response in some cases, though these probably reach a plateau at higher doses and may depend crucially on the drug being studied.Finally, the association of prolactin gene variants with schizophrenia—though preliminary—suggests that there is more to the schizophrenia-prolactin association than simple dopamine-based models. Though Rybakowski et al.  invoked A review of the literature on prolactin and schizophrenia suggests that the relationship between them is complex and not confined to the adverse effects of antipsychotics. Though the above interpretations must be regarded as imperfect and tentative, they do call for a reappraisal of the role of prolactin in the various stages of schizophrenia, particularly with regard to its onset and to the development of positive symptoms. Research in this area may lead to an improved understanding of schizophrenia, as well as a better delineation of the effects of prolactin on social behaviour and cognition in humans.