Flexible piezoelectric nanogenerators have attracted great attention due to their ability to convert ambient mechanical energy into electrical energy for low-power wearable electronic devices. Controlling the microstructure of the flexible piezoelectric materials is a potential strategy to enhance the electrical outputs of the piezoelectric nanogenerator. Three types of flexible polyvinylidene fluoride (PVDF) piezoelectric nanogenerator were fabricated based on well-aligned nanofibers, random oriented nanofibers and thick films. The electrical output performance of PVDF nanogenerators is systematically investigated by the influence of microstructures. The aligned nanofiber arrays exhibit highly consistent orientation, uniform diameter, and a smooth surface, which possesses the highest fraction of the polar crystalline β phase compared with the random-oriented nanofibers and thick films. The highly aligned structure and the large fraction of the polar β phase enhanced the output performance of the well-aligned nanofiber nanogenerator. The highest output voltage of 14 V and a short-circuit current of 1.22 µA were achieved under tapping mode of 10 N at 2.5 Hz, showing the potential application in flexible electronic devices. These new results shed some light on the design of the flexible piezoelectric polymer-based nanogenerators. Confronting the intemperate consumption of fossil energy and pollutants generated from their use , togethe2–CF2–] monomers is a semicrystalline polymer, while the crystalline PVDF usually exhibits four different phases, i.e., alpha (α), beta (β), gamma (γ), and delta (δ) phases, depending on the chain conformation during crystallization [Compared with inorganic piezoelectric materials, organic piezoelectric materials, such as poly(vinylidene fluoride) (PVDF) and its copolymers, are preferable for wearable nanogenerators because of their naturally flexibility and biocompatibility, chemical stability, and easy synthesis ,7. They lization . The comlization . Furtherlization . Therefolization , mechanilization , epitaxylization , electrolization  and dopilization ,16. Complization ,18. In tResearchers are focusing on the improvement of the electrical outputs of the flexible nanogenerators by optimizing the piezoelectric materials both by designing new flexible piezoelectric materials and forming inorganic-organic composites, designing 3D electrodes, etc. As summarized in the review papers ,4,5,7, tElectrospinning is an efficient way of controlling the microstructure of the nanofibers by tuning the characteristics of the polymer solution, the electrospinning parameters and also the ambient condition. Especially for the aligned polymetric nanofibers, several additional techniques have been employed to fabricate aligned nanofibers such as magnetic, mechanical and electrostatic ways during electrospinning ,23,24. FIn this article, the microstructures of the PVDF nanofiber mats have been controlled by tuning the nanofiber collecting parameters to form the highly aligned nanofibers, random oriented nanofibers and thick films. Accordingly, three types of PVDF-based nanofiber mat of different microstructures have been realized, and the output electrical performances have been compared between nanogenerators based on the highly aligned nanofibers, random oriented nanofibers and thick films. It is shown that the well-aligned nanofiber nanogenerators present the highest output voltage of 14 V, a short-circuit current of 1.22 μA, compared with the nanogenerators fabricated by random oriented nanofibers and thick films. The well-aligned nanofiber nanogenerators also exhibit superior stability and durability. Our research not only provides a valuable, reliable and repeatable but simple and low-cost method without any secondary processes to control the microstructure of the piezoelectric nanofiber of high mechanical energy converting ability, but also sheds some light on the design of the flexible piezoelectric polymer-based nanogenerators and portable/wearable personal electronics.The PVDF nanofibers were synthesized by the advanced electrospinning technique. In the electrospinning process, a high voltage was applied to the polymer solution and the liquid droplets were stretched by the electrical field. At a critical point a stream of liquid erupts from the surface to form a Taylor cone. With the increase of the electric field to a critical point, the surface tension is not large enough to maintain the Taylor cone and the polymer jet is initiated and drawn from the nozzle tip to the collector. While traveling to the collector, the solution jet solidified due to the fast evaporation of the solvent and was deposited on a collector .PVDF (Kynar Flex 2801) was purchased from Arkema Inc. . Dimethylformamide (DMF) was purchased from Sigma-Aldrich . Acetone was acquired from Sinopharm Chemical Reagent Co., Ltd. . DMF together with acetone were used as a solvent to dissolve the PVDF powders. A PVDF solution was prepared by dissolving 1 g PVDF powder into a mixture of 3.5 mL of DMF and 1.5 mL of acetone and then stirring until a homogeneous solution was obtained at room temperature. The solution was transferred into a 5 mL syringe for electrospinning. An electrospinning machine  was used to fabricate the PVDF nanofibers. In the electrospinning process, the air humidity was adjusted to around 40%, the temperature was adjusted to around 30 °C. A nozzle with a nominal inner diameter of 0.4 mm was used and the injection speed was maintained at 0.3 mL/h. A high-voltage generator and a syringe pump were used to control the electrospinning rate. The voltage applied between the syringe needle and the collector was about 15 kV; a distance of 10 cm was maintained between the needle and the collector. The rotation speed of the collector was used to control the alignment of the nanofiber array. For the random nanofibers (r-PVDF), the rotating speed was set to 0, while it was set in the range of 1000~2500 rpm for the aligned nanofiber array. The highly aligned nanofibers with a rotating speed of 1700 rpm were short for a-PVDF. The sample information has been summarized in The nanogenerators were fabricated based on the dry electrospun membranes with 2.5 cm × 2.5 cm in size. Two copper wires were attached to each side of the film while an aluminum foil electrode with ~50 µm thickness was attached to each side of the film. Then, the membrane consisting of two electrodes and connecting wires was packaged in polyimide (PI) tape. The preparation of the PVDF nanofibers and the fabrication of the nanogenerator are summarized in X-ray diffraction  and Fourier transform infrared (FTIR) spectroscopy  were used to characterize the phase structure of the PVDF materials. Field-emission scanning electron microscopy  was used to observe the morphology of the PVDF membranes. The Raman spectra of PVDF membranes were recorded using a Jobin Yvon LabRAM HR800 Raman spectrometer. The piezoelectric output of the nanogenerators was measured on a home-built platform in a repetitive tapping mode of 10 N at 2.5 Hz. A digital force gauge was used to measure the dynamic pressing force applied to the a-PVDF . The open-circuit voltages of the nanogenerators were measured with a digital storage oscilloscope . The short-circuit current was measured by an electrometer .θ~19.9°, and ~20.6°, respectively. These peaks confirm that the α-crystalline and β-crystalline phases exist in pure PVDF [The crystal structure of a-PVDF nanofibers, r-PVDF nanofibers, and f-PVDF thin film were characterized by using XRD, as shown in ure PVDF . The difure PVDF ,27,28. −1, 890 cm−1, and 1430 cm−1. The sharp peak at 841 cm−1 is assigned to the symmetric CF stretching “crystallinity” band of A1 symmetry, corresponding to the β polar phase of PVDF [To further confirm the information of the crystallographic structure of the a-PVDF nanofibers, r-PVDF nanofibers, and f-PVDF thin film, Raman spectra were recorded as shown in  of PVDF . The vib−1, 1072 cm−1, 1276 cm−1, and 1413 cm−1 in FTIR spectra for polar β phase, while the vibrations for the α phase generates absorption bands locate at 614 cm−1, 766 cm−1, 796 cm−1, 875 cm−1, 975 cm−1 and 1144 cm−1 [−1 for β phase presents high intensity, indicating the high proportion of β phase in a-PVDF nanofibers. The relative proportion of the β phase content in PVDF is usually estimated by the following equation:χα and χβ are the degrees of the PVDF α and β phases; Kα (6.1 × 104 cm2·mol−1) and Kβ (7.7 × 104 cm2·mol−1) are the absorption coefficients at 766 and 840 cm−1; Aα and Aβ are the absorption values at ~766 and ~840 cm−1, respectively, corresponding to the α and β phases shown in the FTIR spectra, respectively [The FTIR spectra were further used to confirm the polar phase existing in the PVDF membranes. It was reported that the crystallographic PVDF generates absorption bands at 840 cm144 cm−1 ,31. As sJz is the current density, A is the cross sectional area of the nanogenerator, σz is the surface polarization charge density, and S represents the corresponding surface with charges. The output currents of the piezoelectric nanogenerator rely heavily on the surface polarization charges. The low output currents may due to the low content of the polar β phase in the PVDF film. Non-polar α phase can not generate surface polarization charges, resulting small value of the zσ. Furthermore, the low density of the surface polarization charges may also be attributed to the lack of the high electric field poling process. Thus the f-PVDF nanogenerators lead to low output currents.The functional mechanism of piezoelectric nanogenerators can be generally described as a transient flow of electrons driven by a piezoelectric potential . When anSwitching-polarity measurements are necessary to carry out checks on the output voltage that originated from the piezoelectric behavior of nanogenerators. 2 at 50 MΩ. In order to evaluate the charge storage capabilities from the as-prepared PVDF-based nanogenerators for powering electronic devices, these were demonstrated by charging a commercial capacitor using a bridge rectifier circuit. In summary, we have fabricated three types of flexible PVDF piezoelectric materials of different microstructures by controlling the rotation speed of the collector during the electrospinning process. The well-aligned nanofibers possess the highest content of polar phase compared with that of the random oriented nanofibers and thick films. The high content of the piezoelectric phase in the highly aligned PVDF nanofibers resulted in superior mechanical energy conversion ability. Under an applied pressure, the a-PVDF can generate a maximum output voltage as high as 14 V and a short-circuit over 1.22 µA. These results confirm that the well-aligned piezoelectric PVDF generator has great potential in practical application for wearable electronic equipment. The proposed reliable, simple and cost-effective method without any secondary processes is a promising strategy in fabrication polymer nanofibers for flexible electronic devices. It also sheds some light on the design of the flexible piezoelectric polymer-based nanogenerators and portable/wearable personal electronics.


Vibrio vulnificus (V. vulnificus) causing fatal septicemia and necrotic wound infections. Our previous work has shown that RpoS regulates the expression and secretion of V. vulnificus RtxA1 toxin. This study was conducted to further investigate the potential mechanisms of RpoS on RtxA1 secretion. First, V. vulnificus TolCV1 and TolCV2 proteins, two Escherichia coli TolC homologs, were measured at various time points by Western blotting. The expression of TolCV1 was increased time-dependently, whereas that of TolCV2 was decreased. Expression of both TolCV1 and TolCV2 was significantly downregulated in an rpoS deletion mutation. Subsequently, we explored the roles of TolCV1 and TolCV2 in V. vulnificus pathogenesis. Western blot analysis showed that RtxA1 toxin was exported by TolCV1, not TolCV2, which was consistent with the cytotoxicity results. Furthermore, the expression of TolCV1 and TolCV2 was increased after treatment of the host signal bile salt and the growth of tolCV1 mutant was totally abolished in the presence of bile salt. A tolCV1 mutation resulted in significant reduction of V. vulnificus induced-virulence in mice. Taken together, TolCV1 plays key roles in RtxA1 secretion, bile salt resistance, and mice lethality of V. vulnificus, suggesting that TolCV1 could be an attractive target for the design of new medicines to treat V. vulnificus infections.RtxA1 is a major cytotoxin of  Vibrio vulnificus (V. vulnificus) is a halophilic Gram-negative bacterium that causes fatal primary septicemia and necrotizing wound infections, and is commonly transmitted by seawater exposure or contaminated seafood consumption  TolC homologs, TolCV1 and TolCV2 (VVM0602608 and VVM0604400), showing 51.3% and 29.6% sequence identity, respectively  at 37°C in a shaking incubator (200 rpm).The bacterial strains and plasmids used in this study are listed in tolCV2 deletion mutant of V. vulnificus MO6-24/O as described previously   and , respectively. These two DNA fragments were used as templates for the second crossover PCR with tolCV2-1 and tolCV2-4 as primers. The resulting PCR products were ligated into suicide plasmid pDM4 and transformed into E. coli SY327λpir and E. coli SM10λpir, generating the pDM4::△tolCV2, which was conjugally transferred into MO6-24/O via triparental mating. The stable transconjugants were selected on TCBS agar plates with chloramphenicol and then heart infusion (HI) agar plates with 10% sucrose. The mutation was verified via PCR and Western blot analysis.The suicide plasmid pDM4 was used to construct an in-frame eviously . The ups14966.1)  using thtolCV2 mutant was constructed using plasmid pLAFR3 with a primer pair . The DNA fragment containing tolCV2 gene and flanking DNA sequence was amplified by PCR and then cloned into pLAFR3, resulting in the pLAFR3::tolCV2, which was introduced into tolCV2 mutant strain by triparental mating. Stable transconjugants were selected and confirmed by PCR and Western blot analysis.The complementation of tolCV2 was amplified from V. vulnificus MO6-24/O chromosomal DNA by PCR with the following primer pair . The resulting PCR products were then inserted into the expression vector PGEX-4T-1  and the GST-TolCV2 fusion protein was purified by GST SpinTrap columns . The rabbit polyclonal anti-TolCV2 antibody was produced using New Zealand white rabbits, and the specificity of the polyclonal antibody against TolCV2 was confirmed by Western blotting.Rabbit polyclonal anti-TolCV2 antibody was produced as described in our previous study . The DNAV. vulnificus strain was inoculated into LB broth and cultured overnight at 37°C in a shaking incubator (200 rpm). The overnight cultures were diluted 200-fold with fresh LB medium and subsequently cultured at 37°C. The pellets were washed twice with DPBS . Bacterial cells (2×108 CFU) resuspended in SDS-PAGE sample buffer were boiled at 100°C for 10 min and separated by 10% SDS-PAGE gels before being transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were then blocked with 5% skim milk in Tris‐buffered saline containing 0.05% Tween 20 (TBS/T) for 2 h at room temperature and incubated with primary antibodies specific to TolCV1  grown overnight in 6-well plates were washed with serum free DMEM medium before being infected with bacteria at an MOI of 20 for 120, 150, or 180 min. The supernatants (300 µL) were precipitated by the addition of 3-fold ice-cold acetone. Bacterial pellets of the supernatants and HeLa cells were lysed with the cell lysis buffer  containing protease inhibitor cocktail  shaking on ice for 30 min and harvested by centrifugation at 13000 rpm for 10 min after scrapping. The protein concentration was quantified using the Bradford’s reagent . Equal amounts of protein were separated by NuPAGE™ 3%-8% Tris-Acetate gels  and subsequently subjected to Western blotting using an anti-RtxA1 antibody specific to amino acid 1492-1970 (RTX-D2 domain). Western blotting was performed as described above.HeLa cells  were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum . 4 cells/well) overnight were washed with serum-free DMEM before being infected with 3 h or 9 h cultured bacterial cells of V. vulnificus strains at an MOI of 20 for 120, 150, or 180 min. A CytoTox96 Non-Radioactive Cytotoxicity Assay kit  was used to measure the amount of lactate dehydrogenase (LDH) released in the supernatants.HeLa cells seeded into 48-well plates  were harvested and resolved in sample buffer for Western blotting, which was performed as described above.V. vulnificus strains in the presence of bile salt, their overnight cultures (3 µL) were dropped on TCBS agar and then incubated at 37°C overnight.Thiosulphate-citrate-bile salt sucrose (TCBS) agar plate  is usually used for the selective isolation of Vibrio species , which cV. vulnificus strains were diluted 200-fold with fresh LB media in the presence or absence of 0.02% bile salt. Bacterial cells were cultured in a 37°C shaking incubator and the growth was measured using a spectrophotometer at 600 nm every 2 h.Overnight cultures of V. vulnificus wild-type, tolCV1 mutant, tolCV2 mutant, or rtxA1 mutant strains (1×107 CFU/mouse) through intraperitoneal (i.p.) injection. Five mice were tested for each group and infected mice were subsequently observed for 72 h. All procedures involving animals were performed in accordance with the guidelines of the Chonnam National University Animal Care and Use Committee (IACUC-YB-2020-81).Eight-week-old female ICR mice  were kept under specific-pathogen-free conditions. Mice were infected with V. vulnificus pathogenesis  has been reported to be mediated by the Type II secretion system, and irrelevant with regard to TolC .As previously reported, the outer membrane protein TolC affects virulence expression in  cloacae . V. vulnA1 toxin Figure 2A1 toxin  stated tV. vulnificus infections affect many people due to the intake of contaminated seafood .YG and RG performed the experiment and analyzed the data. YG wrote the manuscript. JR contributed to the conceptual design. YK conceived and designed the study, wrote and reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a National Research Foundation of Korean (NRF) grant funded by the Korean government (Nos. 2018R1D1A3B07045194 and 2019R1A2C1005884).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.


ADCYAP1/PACAP enhance nerve outgrowth. Brain. 2020;143:2009–2026. doi:10.1093/brain/awaa163Georgios Baskozos, Oliver Sandy-Hindmarch, Alex J. Clark, Katherine Windsor, Pall Karlsson, Greg A. Weir, Lucy A. McDermott, Joanna Burchall, Akira Wiberg, Dominic Furniss, David L. H. Bennett and Annina B. Schmid. Molecular and cellular correlates of human nerve regeneration: The authors apologize for errors in


Among the few existing needs assessment tools for family carers, the 14-item Carer Support Needs Assessment Tool (CSNAT) is the only brief and holistic needs screening tool designed for everyday use in palliative care practices. The aim of this study was to evaluate the reliability, validity, and acceptability of the traditional Chinese version of the CSNAT in palliative care settings in Hong Kong.This adopted a cross-sectional and correlation design with repeated measures. The participants were 125 family carers of palliative cancer patients and 10 healthcare providers (HCPs) that were recruited from two local hospitals. The evaluation of psychometric properties included the following: (1) content validity through HCPs including frontline physicians, nurses, social workers, and clinical psychologists; (2) construct validity between the CSNAT items and those of the validated tools that measured caregiver burden, social support, and caregiving self-efficacy; and (3) one-week test-retest reliability in a sub-sample of 81 caregivers. The acceptability of the tool was assessed by the carers using several closed-ended questions.r = 0.24 to 0.50) and caregiving self-efficacy (r = − 0.21 to − 0.52), but not for social support. All CSNAT items had fair to moderate test-retest reliability (weighted kappa = 0.21 to 0.48), with the exception of two items “managing your relatives’ symptoms, including giving medicines” and “having time for yourself in the day”.The content validity index of the CSNAT at the scale level was 0.98. Each item of the CSNAT was significantly and moderately correlated with caregiver burden (Spearman’s Regarding the acceptability of the CSNAT, almost all HCPs were willing to use the CSNAT for carer assessment and support. 89.6% of the carers demonstrated a comprehensibility of the CSNAT tool and 92.9% felt comfortable answering the questions. Around 90% of the carers agreed to use the tool for screening, discussing needs, and making referrals.The traditional Chinese version of the CSNAT is a tool with high validity and acceptability and adequate reliability that measures family carers’ support needs, which should be considered for wide application in local palliative care practices. The World Health Organization has recognized palliative care as an approach that aims to maximize the quality of life of patients with advanced and incurable diseases and their families . The corThe term “needs” is generally viewed as actions or resources that individuals require from professional help to attain optimal well-being . A recenAmong the few existing needs screening tools for carers, the 14-item Carer Support Needs Assessment Tool (CSNAT) is the only very brief but holistic needs screening tool for practical use in palliative care –14. BuilThough the CSNAT was developed in 2013, it has been translated and validated into different languages, including German, Swedish and Simplified Chinese , 17, 18.The present study adopted a cross-sectional and correlation design with repeated measures. The study setting were palliative care units of the two palliative care hospitals in Hong Kong.The participants were recruited by convenience sampling from December 2019 to December 2020. The participants included HCPs  and carers of palliative cancer patients . A sub-sample of caregivers volunteered to complete the CSNAT again after 1 week . In thisCaregivers were included if they were: (1) adults aged 18+ years old; (2) family members taking care of palliative cancer patients at home; and (3) able to communicate in Cantonese. Caregiver exclusion criteria included: (1) paid caregivers; and (2) mentally incapable of completing the questionnaire  . Eligibir) correlation coefficient was 0.25 based on two published validation reports of the CSNAT, at a significance level of 5% for the two-tailed tests [The sample sizes for the HCPs and carers were estimated based on different psychometric property testing. For content validity testing, according to the international guidelines, at least 10 experts are sufficient to achieve consensus on establishing content validity . Ten HCPed tests , 14. To ed tests .The carers’ support needs were measured using the traditional Chinese version of the CSNAT . The toor = 0.78) [Caregiver burden was assessed using the 13-item traditional Chinese version of the Caregiver Strain Index (C-M-CSI) , which h = 0.78) . In thisThe six-item traditional Chinese version of the Social Support Questionnaire (SSQ-6) was used to measure perceived social support , 28. Eacr = 0.71 to 0.76), internal consistency reliability , convergent validity, and construct validity [The 18-item traditional Chinese version of the Caregiver Inventory (C-CGI-18) was used to assess caregiving self-efficacy in palliative care , 30. It A self-developed questionnaire was used to evaluate the carers’ acceptability of the CSNAT as a routine assessment in palliative care. Five questions are included in the questionnaire, which were aligned with the essential steps of the CSNAT intervention : (1) comAdditionally, socio-demographic data were collected from the cares, including age, gender, marital status, employment status, education level, income, relationship with the patient, duration of caregiving, and living arrangement.The HCP survey was designed to assess the content validity and acceptability of the CSNAT. For content validity testing, the content relevancy of each CSNAT item was measured using a 4-point Likert scale, from 1 (“not relevant”) to 4 (“highly relevant”). For the acceptability of the CSNAT from HCP perspectives, three items are designed based on the key steps of the CSNAT intervention , includiCarers were approached by a research assistant when they were accompanying palliative cancer patients to outpatient units or visiting palliative cancer patients in inpatient units in the two hospitals. If the carers showed an interest in participating in the study, the research assistant conducted screening for their eligibility. Once they were confirmed as eligible, the research assistant provided them with the information sheet and explained the procedures of the study. The participants’ written informed consent forms were collected before data collection. The carers were then asked to fill out the surveys (including the CSNAT) by themselves or as administered by the researcher using a pen-and-paper mode in the hospital settings. A sub-sample of 81 participants volunteered to complete the CSNAT tool again 1 week later through structured telephone interviews following astandardized interview script. The carer survey was pilot-tested on 10 carers and no comprehensibility problems were reported. Regarding the HCPs, one research team member contacted potential participants for participation. Once they agreed to join the study, they were asked to return their signed consent form along with the completed surveys within 2 weeks.p value set at < 0.05.Data were entered and analyzed using SPSS 22.0. Descriptive statistics were used to describe the participants’ profiles and study variables, including acceptability. The mean scores and standard deviations (SD) for the continuous variables and frequency and the percentages for the categorical variables were reported. All inferential tests were two-tailed, with a The percentage of missing data as well as the extent of ceiling and floor effects were calculated to evaluate data quality. A cut-off point of 15% was used as a criterion for determining the presence of ceiling or floor effects based on the proportions of the participants’ highest and lowest scores, respectively .The CSNAT tool is a screening tool, so the total scale was not calculated . Test-rer). It was hypothesized that the CSNAT items would positively correlate with caregiver burden [Each CSNAT item with external constructs, including caregiver burden, social support, and caregiving self-efficacy, was evaluated using Spearman’s correlation analyses  was calculated by the proportion of HCP experts who gave a rating of 3 or 4. Both the item (I-CVI) and scale (S-CVI) levels of the CVI were calculated. If at least nine experts were invited, an acceptable CVI was ≥0.78 .The team validated the traditional Chinese version of the CSNAT with permission from the original developer. Ethical approval was obtained from the researcher’s university and the two hospitals involved in the study. Written consent was collected from each participant. The personal data of the participants were kept strictly confidential and anonymized for data analysis. We ensured the participants’ right to withdraw from research at any time.n = 16), not a primary caregiver (n = 11), no time (n = 9), not in the mood (n = 2), response burden (n = 2), and others, such as language barrier (n = 3). Among the recruited carers who completed the baseline assessments, a subsample of 81 proceeded with the second measurement of the CSNAT within 1 week.Table The mean age of the carers was 54.5 years old SD = 14.3). Most of the carers were female (58.4%) and worked either full or part time (44.8%). The carers were predominantly the children (48.0%) or spouse (31.2%) of the patients. Figure 4.3. MostTen HCPs were recruited for the study, which included frontline physicians, nurses, social workers, and clinical psychologists. Half of them were aged 31 to 40 years old. The majority of them were female (80%) and were master’s degree holders (60%). The mean time of the HCP’s working experience was 14.4 years, ranging from four to 33 years.The data quality of the CSNAT tool is presented in Table p < 0.05 to 0.001), with the exception of two items: “managing your relative’s symptoms, including giving medicines” (Kw = 0.13) and “having time for yourself in the day” (Kw = 0.15).As shown in Table r = 0.24 to 0.50, p < 0.01). Moderate and significant associations were found between the CSNAT items and each domain score of the C-CGI-18 scale, including “managing information” and “self-care” , “care of the care recipient” , and “managing emotional interaction with care recipient” . However, the relationships between the CSNAT item scores and the SSQ-6 scores were not significant.Table The I-CVIs of the CSNAT were between 0.90 and 1.00, and the S-CVI of the CSNAT was 0.98.Table The present study provided evidence of the reliability, validity, and acceptability of the traditional Chinese version of the CSNAT within the Hong Kong palliative care context. The findings indicated that the traditional Chinese version of the CSNAT had high construct validity, content validity, and acceptability as well as acceptable test-retest reliability. This tool should be considered for future use to assess the support needs of carers of palliative cancer patients using traditional Chinese characters.In this study, no missing data from the traditional Chinese version of the CSNAT occurred. Consistent with a previous validation report in Sweden , the stuA validation study in Sweden was the first to examine the CSNAT’s test-retest reliability, and the study reported that all the items had moderate to good agreement at baseline and 1 week later . SimilarAs hypothesized, significant correlations of the CSNAT item scores with those of the C-M-CSI and C-CGI-18 were confirmed in this study, supporting the scale’s construct validity. These findings are also consistent with published studies that measured the relationships of the same constructs among carers of palliative care patients , 18, 33.The study findings are in line with those from prior studies that showed excellent content validity and acceptability of the CSNAT , 14, 17,This study provides further psychometric data in support of the evidence-based CSNAT tool within the Hong Kong palliative care context. Successful validation of the CSNAT tool is an important first step before proceeding with the provision of person-centered care to meet the comprehensive support needs of carers. The CSNAT has been successfully integrated into an intervention with a five-stage process of carer assessment and support during palliative and end of life care . IncreasSeveral limitations should be acknowledged before making its conclusion. First, as the participants were recruited from palliative care hospitals, the findings cannot be generalized to community-based care settings. Second, different mode of administration of the CSNAT at two assessment might introduce the respondent bias due to changes in the social setting (clinic vs. home), possibly influencing the interpretation of the test-retest reliability results . Future The traditional Chinese version of the CSNAT is a holistic screening tool with high validity and acceptability as well as acceptable reliability that measures family carers’ support needs in palliative care settings. Since the CSNAT is brief and easy for practical use, we recommend that this tool should be considered for wide application in local palliative care practices. Future intervention should be developed in the next step in terms of integrating the CSNAT as part of needs-based carer support intervention for family carers.


The subcutaneous administration of l-menthol significantly increased the number of c-Fos-like immunoreactive nuclei in the dorsal striatum of the mice, and motor activity was promoted. It also increased the extracellular dopamine level in the dorsal striatum of the mice. These observations indicated that after subcutaneous administration, l-menthol enhances dopamine-mediated neurotransmission, and activates neuronal activity in the dorsal striatum, thereby promoting motor activity in mice.Similar to psychostimulants, the peripheral administration of menthol promotes mouse motor activity, and the neurotransmitter dopamine has been suggested to be involved in this effect. The present study aimed to elucidate the effects of  Menthol, which is also known as mint camphor, is a monocyclic terpene alcohol that is found naturally in more than 100 essential oils, and it is a major constituent of the essential oils of spearmint and peppermint. Due to its minty flavor and cool sensation, menthol is among the world’s most widely used flavoring agents; it is used in a variety of commercial products, such as cosmetics, oral hygiene products, including toothpaste and mouthwash, chewing gum, confectionary, foods, beverages, liqueur, cigarettes, and pharmaceuticals. It is estimated that between 30,000 and 32,000 metric tons of menthol is consumed annually -WIN35,428 to the dopamine uptake/binding site of dopamine transporter; this is similar to the action of the dopamine transporter inhibitor GBR12909, and suggests that l-menthol inhibits the binding of dopamine to the dopamine transporter, thereby leading to decreased dopamine uptake [l-menthol on the dopamine transporter would also account for the synergistic interaction with the ambulation-promoting effect of the dopamine transporter inhibitor bupropion, which also enhances the extracellular dopamine level and increases the number of c-Fos-IR nuclei in the striatum of mice [l-menthol may also act on adenosine A2a receptors [l-menthol on c-Fos expression in the striatum and ambulation.Medium-sized spiny neurons that express dopamine D1 or D2 receptors constitute the vast majority (≥95%) of striatal neurons; thus, dopamine is a principal neurotransmitter in the striatum. The binding assay study  suggesteeceptors –75, the e uptake . The act of mice . In addieceptors . Adenosieceptors . Accordil-Menthol may potentially influence the mesolimbic and mesocortical dopaminergic systems through dopamine transporters and adenosine A2a receptors. As l-menthol did not influence c-Fos expression in the brain regions related to the mesolimbic and mesocortical dopaminergic systems, we did not examine the effects of l-menthol on dopamine in those brain regions in the current study. It is noteworthy that l-menthol can act on not only dopamine transporters and adenosine A2a receptors, but also on other kinds of neurotransmitter receptors and channels at the same pharmacologically relevant concentration range [l-menthol on c-Fos expression in different brain regions. Whether l-menthol influences dopamine-mediated neurotransmission in the brain regions related to the mesolimbic and mesocortical dopaminergic systems remains to be examined in future studies.Not only the nigrostriatal system, but also the mesolimbic and mesocortical systems are major dopaminergic systems in the brain. In addition, adenosine A2a receptors are highly expressed in the dopamine-rich regions of the brain. on range , 66. Thul-menthol. Namely, the results showed that after subcutaneous administration, the l-menthol enhances dopamine-mediated neurotransmission, and activates neuronal activity in the dorsal striatum, thereby promoting ambulation in mice.The present study revealed novel information regarding the CNS-activating effects of S1 Data(DOCX)Click here for additional data file.S2 Data(DOCX)Click here for additional data file.S3 Data(DOCX)Click here for additional data file.


Supramolecular chirality plays an indispensable role in living and synthetic systems. However, the generation and control of filament chirality in the supramolecular hydrogel of short peptides remains challenging. In this work, as the first example, we report that the heterodimerization of the enantiomeric mixture controls the alignment, chirality, and stiffness of fibrous hydrogels formed by aromatic building blocks. The properties of the resulting racemic hydrogel could not be achieved by either pure enantiomer. Cryo-EM images indicate that the mixture of L and D enantiomers forms chiral nanofibers, the percentage of which can be readily controlled through stoichiometric co-assembly of heterochiral enantiomers. 2D NOESY NMR and diffusion-ordered NMR spectroscopy reveal that heterodimerization of enantiomers plays a crucial role in the formation of chiral nanofibers. Further mechanistic studies unravel the mechanism of supramolecular chirality formation in this two-component system. Molecular dynamics simulations confirm that the intermolecular hydrogen bond and π–π interaction of heterodimers play important roles in forming a chiral hydrogel. Furthermore, regulation of the adhesion and morphology of mammalian cells is achieved by tuning the relative ratio of L and D enantiomers at the same concentration. This work illustrates a novel strategy to control the supramolecular chirality of aromatic peptide hydrogels for materials science.The online version contains supplementary material available at 10.1186/s12951-022-01285-0. Supramolecular chirality is a critical feature in living and synthetic systems. Remarkable examples are the double-helical DNA which stores genetic information and the triple-helical collagen fibers that constitute the primary structural element of the extracellular matrix (ECM) , 2. The Resembling the properties of native ECM, the supramolecular hydrogel has been attracting more and more attention in different disciplines, including chemistry, materials, and biology. To construct hydrogel with chiral supramolecular structures, self-assembly through non-covalent interactions is an efficient strategy , 12–14. Herein, we report that controlling the intermolecular interaction of enantiomeric tripeptide with aromatic modification affords a chiral nanofibrous hydrogel with tunable alignment, which could not be achieved by either pure enantiomer. We found that L enantiomeric peptide forms an isotropic hollow nanofibrous hydrogel, while its corresponding D isomer forms the anisotropic hydrogel with aligned hollow nanofibers. The mixture of the L and D isomers co-assemble to form chiral nanofibers with controlled alignment and stiffness. Simply tuning the ratio of L and D enantiomers, we can control the ratio of chiral nanofibers in the molecular hydrogel. Detailed mechanistic studies indicated that the chiral nanofibers consist of the heterodimer of L and D enantiomers through non-covalent interactions. Molecular dynamics simulations confirm that intermolecular hydrogen bonding and π–π stacking play a key role in forming heterodimers. Moreover, we also demonstrate the good biocompatibility of the hydrogels by 2D and 3D cell culture. This work illustrates a new and efficient strategy to generate supramolecular chiral nanofibers with tunable alignment and stiffness, which could be used to create programmable niches for directing cell functions.α-Fmoc-protected amino acids with side chains properly protected. We used high performance liquid chromatography (HPLC) to purify all the molecules and characterized them by NMR and LC–MS  and Fourier transform infrared spectroscope (FTIR). The CD spectrum Fig. g of LMW-To further investigate the interaction between LMW-L1 and LMW-D1, we examine the self-assembly performance of these enantiomers at various relative ratios by high-resolution TEM. Gelation test indicates that the mixture of LMW-L1 and LMW-D1 at 0.3 wt% forms stable hydrogels despite the different relative concentrations of LMW-L1 and LMW-D1  experiments were performed. We observe the uniform diffraction rings corresponding to the -L1 Fig. a. The did6-DMSO, 1H NMR signals of LMW-L1 and LMW-D1 in D2O move to the lower field with agminated and broadened peaks, revealing the formation of assemblies  reveals that the diffusion coefficient of hydrogel formed by LMW-L1, LMW-D1, and their equimolar mixture is 2.213 × 10–10 m2 s−1, 2.529 × 10–10 m2 s−1, and 2.275 × 10–10 m2 s−1 . We also find the close contacts between CH in Nap (δ = 7.51 ppm) and F (δ = 6.99 ppm), CH in W  and Y  , indicating the mixtures still hold the co-assembly state, revealing the robust stability of nanostructure co-assembled by LMW-L1 and LMW-D1  simulations to investigate the molecular interactions during the self-assembly processes. The MD simulations were performed by the Gromacs software package  under thThe viscoelasticity and supramolecular chirality of a hydrogel play important roles in controlling cell adhesion and morphologies. Therefore, we investigate the cytocompatibility and cell adhesion of the above hydrogels to evaluate their potential applications in tissue engineering and regenerative medicine. Live/dead assay shows excellent biocompatibility of hydrogels in 2D cell culture Fig. , as evidIn summary, this work describes a novel approach to control the supramolecular chirality and stiffness of molecular hydrogel. Our results highlight the importance of dimerization of aromatic molecules in supramolecular chemistry and represent a general way to create and control supramolecular chirality for various applications in different disciplines . For exaAdditional file 1: All data generated or analyzed during this study are included in this published article and its supplementary information files.


The feasibility of this approach is demonstrated by assessing widely described sex differences in dopamine neurotransmission. Reward processing was behaviorally investigated in 36 healthy participants, of whom 16 completed fPET and fMRI during the monetary incentive delay task. A single 50 min fPET acquisition with 6-[18F]FDOPA served to quantify task-specific changes in dopamine synthesis. In men monetary gain induced stronger increases in ventral striatum dopamine synthesis than loss. Interestingly, the opposite effect was discovered in women. These changes were further associated with reward (men) and punishment sensitivity (women). As expected, fMRI showed robust task-specific neuronal activation but no sex difference. Our findings provide a neurobiological basis for known behavioral sex differences in reward and punishment processing, with important implications in psychiatric disorders showing sex-specific prevalence, altered reward processing and dopamine signaling. The high temporal resolution and magnitude of task-specific changes make fPET a promising tool to investigate functional neurotransmitter dynamics during cognitive processing and in brain disorders.The assessment of dopamine release with the PET competition model is thoroughly validated but entails disadvantages for the investigation of cognitive processes. We introduce a novel approach incorporating 6-[ Probing differences between monetary gain and loss consistently shows activation of the ventral striatum (VStr) including the nucleus accumbens, being a pivotal region for reward processing.3 However, blood oxygen level dependent (BOLD) fMRI is directly related to hemodynamic factors and mostly reflects post-synaptic glutamate-mediated signaling instead of mapping specific modulatory neurotransmitter action.The processing of reward and punishment represents an essential aspect of one’s mental health. This is reflected in alterations of the reward system in several psychiatric disorders such as addiction, gambling, eating disorders and depression. The prevailing approach to investigate the neural representation of behavioral effects is functional magnetic resonance imaging (fMRI) with the monetary incentive delay (MID) task being the most widely employed paradigm to study reward and punishment processing. is mediated by different neuronal projections from the ventral tegmental area to the VStr.8 This anatomical separation also implies distinct dopamine signaling that underpin the two motivational signals. In humans endogenous dopamine release can only be assessed indirectly by specific positron emission tomography (PET) radioligands, which compete with the endogenous neurotransmitter to bind at a target receptor. Although the competition model represents a thoroughly validated approach it includes two major disadvantages when investigating human behavior. First, cognitive tasks only yield low signal changes of around 5–15% from baseline, even for a recently introduced advancement that offers high temporal resolution. Second, high specificity of observed task effects implies comparison against a control condition, but this in turn requires separate measurements. As a consequence, among those studies investigating dopamine release during monetary gain1114 only one also evaluated loss, but without observing significant differences between the two conditions.Dopamine plays a crucial role in the processing of reward and punishment by specifically encoding these conditions. Animal research has demonstrated that the behavioral response to rewarding and aversive stimuli17 Furthermore, several studies have reported general sex differences of the dopamine system, including ventral tegmental area functioning, dopamine synthesis rates at baseline and amphetamine-induced release.21 The latter has also been confirmed in rodent studies,23 but in humans this may only be present in young adults. Nevertheless, differences in reward-specific dopamine release between women and men have not yet been investigated, which is potentially attributable to the methodological difficulties mentioned above. Consequently, the neuronal underpinnings of behavioral sex differences in reward and punishment processing remain largely unknown, particularly because fMRI studies of the MID25 or other reward paradigms27 were unable to show any sex differences during reward consumption.An important aspect in the context of reward processing and dopamine neurotransmission is the widely described sex difference thereof. Numerous different testing schemes have shown that women are more sensitive to threats and punishment, thus aiming for risk minimization and harm avoidance. However, men tend to opt for greater rewards in terms of money, status and competitive success irrespective of the associated risks.29 to a neurotransmitter level. Here, task-induced functional dynamics of dopamine synthesis were used as an index of dopamine neurotransmission, focusing on the VStr due to its pivotal role in the processing of reward and punishment. The second aim was to demonstrate the feasibility of this technique by investigating sex differences in the processing of monetary gain and loss on a multimodal level. Thus, we combined task-induced changes in dopamine synthesis with BOLD-derived neuronal activation and modeling of behavioral data to identify the neuronal processes underlying the different behavioral sensitivity to reward and punishment in men and women.Therefore, the primary aim of this work was to introduce a novel approach, which enables the assessment of rapid changes in dopamine signaling during cognitive performance by extending the technique of functional PET (fPET) imagingl-tyrosine to l-3,4-dihydroxyphenylalanine (DOPA) via the enzyme tyrosine hydroxylase, and then to dopamine by aromatic amino acid decarboxylase (AADC). Importantly, these enzymes are subject to fast-acting regulatory mechanisms. Tyrosine hydroxylase and AADC activities increase with neuronal firing in order to refill the synaptic vesicles with de novo synthetized neurotransmitter after stimulus-induced dopamine release and are further regulated by activation or blockade of dopamine receptors.3034 Moreover, the radioligand 6-[18F]FDOPA can be incorporated into this synthesis chain, as it is a substrate for AADC, rapidly forming 6-[18F]F-dopamine. The radioligand is thus specific to the dopaminergic pathway36 and represents an established approximation for dopamine synthesis rates.37 Taken together, the evidence suggests that stimulus-induced activation of dopamine synthesis is also reflected in a proportionally increased radioligand binding , which in turn allows to identify task-specific changes in dopamine synthesis with high temporal resolution.This hypothesis can be directly tested by the application of 6-FDOPA was administered in a bolus + constant infusion protocol (ratio 20:80) similar to previously described procedures29 (see supplement). During the scan the MID task was carried out at 10 (except for the PoC experiments), 20, 30 and 40 min after start of the radioligand application, each lasting for 5 min. Otherwise, a crosshair was presented and subjects were instructed to keep their eyes open and avoid focusing on anything specific (in particular not the task).The radioligand was freshly prepared every day by Iason GmbH or BSM Diagnostica GmbH. One hour before start of the fPET measurement, each participant received 150 mg carbidopa p.o. to block peripheral metabolism of the radioligand by aromatic amino acid decarboxylase.O-methyl-6-[18F]FDOPA (3-OMFD) after carbidopa pretreatment.41 Thus, the 3-OMFD fraction was taken from previous bolus data and modified to match our bolus + infusion protocol . By calculation, the protocol with a bolus:infusion ratio of 20:80 indicated a 51.4% reduction of the 3-OMFD fraction (area under the curve) as compared to a pure bolus. The final arterial input function was then obtained by multiplication of the whole blood curve with the plasma to whole blood ratio and the parent fraction. The 3-OMFD input function was calculated likewise by using the 3-OMFD fraction.Arterial blood samples were drawn from the radial artery (see supplement). Manual samples of plasma to whole blood ratio were fitted with a linear function. Correction for radioactive metabolites was based on previous literature, assuming that the only relevant metabolite is 3- using SPM12 and default parameters unless specified otherwise. fPET images were corrected for head motion  and the resulting mean image was coregistered to the T1-weighted structural MRI. The structural scan was spatially normalized to MNI space and the resulting transformation matrices  were applied to the dynamic fPET data. Images were smoothed with an 8 mm Gaussian kernel, masked to include only gray matter voxels and a low-pass filter was applied with the cutoff frequency set to 2.5 min.Image preprocessing was done as described previously18F]FDOPA time activity curves (TAC) were corrected for the 3-OMFD component using the occipital cortex as reference region. We employed a mathematical correction procedure as this avoids overcorrection compared to simple subtraction of the raw reference TAC.43 Briefly, the 6-[18F]FDOPA reference TAC was extracted with the Harvard-Oxford atlas and adjusted for potential task effects and movement using the same general linear model as described below. The reference TAC was fitted with a one-tissue compartment model in PMOD 3.5. The brain TAC representing the 3-OMFD component was then calculated as convolution of the 3-OMFD arterial input function with the impulse response function given by the fitted values of K1 and k2. The estimated 3-OMFD TAC was then subtracted from every brain voxel. This procedure assumes that the reference region is devoid of specific binding, that the distribution volumes of 3-OMFD and 6-[18F]FDOPA are equal in the reference region and that the distribution volume of 3-OMFD is uniform across the brain. A fixed whole blood component of 5% was used for all calculations.6-FDOPA while performing the MID task. In both subjects the task induced substantial increases in VStr dopamine synthesis of Ki = 0.017 and 0.022/min from baseline  and neurobiological level . The task was extended to four blocks and each of them manipulated to enable the separate assessment of monetary gain and loss.Since the PoC experiment combined monetary gain and loss within a task block, the main study specifically aimed to disentangle these two effects on a behavioral  = 6.9, p = 0.013) and amount  = 4.4, p < 0.001) as well as a trend for an interaction effect sex * amount  = 2.0, p = 0.08). Post-hoc t-test indicated that this seemed to be driven by the –3 € condition with women showing a faster reaction than men .The difference in monetary gain was also reflected in the normalized reaction times, with a main effect of sex , with a faster reaction for higher monetary gains , with faster reaction times for high amounts of loss as compared to men. These distinct relationships for men and women were also obtained for the imaging subsample . Thus, we interpreted the linear (quadratic) term for men (women) as index for reward (punishment) sensitivity, that is, the more negative the parameter, the faster the reaction time for high gain (loss).We further aimed to model the behavioral response in more detail, as the relationship between reaction time and amount for each group. In men, this was best described by a negative linear function . The MID task yielded increased dopamine synthesis rates in the VStr during gain  and loss . Interestingly, the direction of this difference was reversed in women with higher dopamine synthesis during loss vs. gain . However, no such difference was observed for women or the loss condition for both sexes , indicating no systematic influence of the task timing on dopamine synthesis.n = 9, ρ = –0.67, p = 0.059, n = 7, ρ = 0.79, p = 0.048, Proceeding from the distinct models to characterize the behavioral response of monetary gain and loss , we asse , for percent signal change from baseline  and without weighting by task performance .Finally, VStr dopamine synthesis rates between gain vs. loss were significantly higher for men than women . This was driven by higher dopamine synthesis for loss than gain in women , as men did not show any difference between the two conditions . The caudate indicated no sex difference and also no difference between gain and loss . Furthermore, no significant associations between synthesis rates and behavioral values for putamen and caudate were found . we observed robust neuronal activation in the VStr for gain vs. loss in men and women . It is however unlikely that these baseline differences affect the task-specific estimates (see limitations).Similar to another study,In this work we introduce a novel framework for the assessment of task-specific changes in dopamine neurotransmission, which is based on the dynamic regulation of neurotransmitter synthesis quantified by functional PET imaging. Processing of monetary gain and loss induced robust changes in dopamine signaling in the living human brain even for the direct comparison of these two conditions, demonstrating the high sensitivity and specificity of the approach. Crucially, task-induced changes in dopamine synthesis showed sex-specific differences in the opposite direction with higher synthesis rates in men for gain vs. loss but vice versa in women, directly reflecting behavioral sex differences in reward and punishment sensitivity. Since this sex difference was not present in common BOLD-derived assessment of neuronal activation, our findings have important implications for the interpretation of numerous fMRI studies on reward processing. This is also essential in various clinical populations, where the sex-specific influence on the link between altered reward processing and dopamine signaling is not yet fully understood.17 We hereby extend general sex differences of the dopamine system1921 specifically to the processing of gain and loss and directly link changes in dopamine neurotransmission with the corresponding behavioral response. This is also supported by pharmacological effects observed in animals and humans. For instance, male rats aim for large rewards independent of the risk, whereas females decrease such choices in order to avoid punishment. This sex difference was even more pronounced by the dopamine releasing agent amphetamine, where females abolished the choice for risky rewards to a much larger extent than males. On the other hand, studies in humans have shown that men often opt for selfish rewards, but women take more prosocial choices. However, pharmacological blockade of dopamine D2/D3 receptors shifts these preferences and thereby eliminate the sex difference in prosocial choices, i.e., men and women showed similar preference for selfish rewards. Taken together, these findings suggest that sex differences in reward behavior are substantially driven by dopamine neurotransmission. It is worth to note that pharmacological challenges may represent an unspecific assessment of neurotransmitter action. The systemic manipulation affects the entire brain, possibly eliciting complex downstream effects, and the use of potent challenge agents may overshadow subtle physiological and behavioral differences. Therefore, our results provide novel evidence in this context through the direct and spatially targeted assessment of reward-specific dopamine signaling itself, without manipulation of the neurotransmitter system. This enabled us to disentangle the dopaminergic involvement in monetary gain and loss, which revealed opposing changes in synthesis rates between men and women.The current work provides a biological basis for the well-known behavioral differences in reward and punishment sensitivity between men and women.26 revealed any (and particularly not opposing) sex differences in VStr activation between gain and loss. fMRI based on the BOLD signal is dependent on the link between neuronal activation and changes in hemodynamic factors such as blood flow, volume and oxygenation.48 Blood flow is locally controlled by the major neurotransmitter glutamate, and thus it is widely accepted that the BOLD signal mostly reflects postsynaptic glutamate-mediated signaling.49 Although monoamine neurotransmitters such as dopamine may also modulate blood flow, this does not seem to translate into corresponding fMRI signal changes, at least for the processing of monetary gain and loss using the widely employed MID task. We acknowledge that previous work has indicated a relationship between dopamine release and fMRI,52 but these were again based on potent pharmacological manipulations, which may not be directly comparable to more subtle cognitive effects (see above). Instead, it appears that during cognitive task performance the limited contribution of dopamine to the BOLD signal gets lost in major downstream effects of glutamate action that regulate blood flow. We speculate that the latter two are not sufficiently specific to identify sex differences in neuronal activation during reward processing. This may have substantial implications for the investigation of several brain disorders with dopamine dysfunction such as addiction, schizophrenia or depression, where fMRI represents one of the most widely used methods. Our results suggest that BOLD signal alterations may not primarily reflect the underlying dopaminergic changes, especially when investigating the reward system in men and women. Further work is required to elucidate the exact difference that cognitive and pharmacological stimulation exert on the relationship between BOLD imaging and dopamine signaling and if this extends beyond sex differences of reward processing.In contrast, such an evaluation was not accessible by previous approaches (see introduction for PET findings on the competition model), including reward-specific neuronal activation obtained with fMRI. Again, it needs to be emphasized that neither this nor other fMRI studies The involvement of the raphe nuclei also implies a substantial contribution of the serotonin system in the processing of aversive and rewarding stimuli. This is further supported by changes in reward behavior and neuronal activation after antidepressant treatment that target the serotonin system. Therefore, future work may aim to elucidate the interaction of VStr dopamine signaling with other brain regions and neurotransmitters during reward processing.It also needs to be highlighted that the reward circuit goes beyond the VStr and includes numerous other brain regions such as the frontal cortex and midbrain areas of the substantia nigra, ventral tegmental area and raphe nuclei. In particular, the VStr receives inputs from medial prefrontal, orbitofrontal and dorsal anterior cingulate cortices as well as the amygdala, which mediate reward behavior.55 considering that the MID paradigm also requires a fast motor response. However, the sex difference between gain and loss in the putamen was driven by women, which was less pronounced than in the VStr and no associations with the behavioral response were found.On the other hand, we also observed task-specific dopamine synthesis in the putamen and caudate. This is in line with previous work on motor tasks and cognitive processes,18F]FDOPA signal are related to dopamine release. As mentioned, dopamine synthesis is subject to fast-acting regulatory mechanisms, which is activated by neuronal firing to refill the synaptic vesicles.3032 Moreover, dopamine synthesis is also increased by the dopamine releasing agent amphetamine as demonstrated in rats and monkeys using PET. In a similar manner decreasing dopamine synthesis also decreases amphetamine-induced dopamine release.59 Notably, a previous study reported no relationship between dopamine synthesis and release, but it is important to mention that synthesis was only investigated at baseline . In contrast, we specifically assessed changes in dopamine synthesis during task performance and thus the previous finding is not in contrast to the synthesis model. Hence, the herein proposed approach offers an alternative to the competition model as the crucial factor to identify task-specific changes is the incorporation of radioligands into the dynamic regulation of enzymes responsible for neurotransmitter synthesis (instead of direct competition between radioligand and endogenous neurotransmitter).Although not directly assessed, there are two essential lines of evidence which strongly support the concept that task-specific changes in the 6-FDOPA signal as index of dopamine synthesis and its release, as these processes are tightly coupled.5659Of note, task-specific changes in K Interestingly, recent work indicated generally lower dopamine uptake in women than men in the putamen. However, for the specific age range of subjects included in the current study (third decade) this effect was actually reversed, which concurs with our findings. Irrespective of the direction of this effect, baseline differences  would most likely cause general differences in task-specific dopamine synthesis across all task conditions. However, the observed task-specific changes were higher in men than women for gain, but vice versa for loss, which argues against a dependency of task estimates on baseline synthesis.Another limitation is the use of a literature-based correction for radioactive metabolites instead of an individual one. Although this may indeed change the absolute values of dopamine synthesis to a certain extent, it does not influence the reward-specific effects. Again, in a within-scan design any “global” parameter will affect baseline and task-specific synthesis rates in an equal manner and will thus cancel out when calculating percent signal change or differences between gain and loss. This applies for instance to radioactive metabolites as well as sex differences in dopamine synthesis at baseline.Finally, further work is required to confirm the linear and quadratic relationships of reward-related reaction times in men and women, respectively, and the associations with dopamine synthesis.57 Moreover, our results suggest that reward-specific neuronal activation should not unequivocally be interpreted as corresponding changes in dopamine signaling and that the investigation of sex differences in this context requires further attention. This may be of pivotal relevance for the assessment of numerous psychiatric and neurological patient populations. These include for instance addictive, gambling and eating disorders or depression as well as autism spectrum disorder and Parkinson’s disease, given the different prevalence rates in men and women as well as alterations in reward processing and dopamine signaling.6671 The introduced approach enables to address important future questions of human cognition and to investigate whether the observed reward- and sex-specific differences in dopamine synthesis will translate to clinically relevant characteristics for patient diagnosis or treatment.To summarize, the current work provides a strong motivation for further investigations of functional neurotransmitter dynamics during cognitive processing. The framework of fPET imaging offers important advantages of high temporal resolution, robust effect size of task-induced changes and the possibility to assess multiple task conditions in a single measurement. Future studies should aim for an in-depth evaluation of stimulus-dependent activation of dopamine synthesis, proceeding from previous findings which link neurotransmitter synthesis and release.


Dopaminergic neurons originating from the ventral tegmental area (VTA) and the locus coeruleus are innervating the ventral hippocampus and are thought to play an essential role for efficient cognitive function. Moreover, these VTA projections are hypothesized to be part of a functional loop, in which dopamine regulates memory storage. It is hypothesized that when a novel stimulus is encountered and recognized as novel, increased dopamine activity in the hippocampus induces long-term potentiation and long-term storage of memories. We here demonstrate the importance of increased release of dopamine and norepinephrinein the rat ventral hippocampus on recognition memory, using microdialysis combined to a modified novel object recognition test. We found that presenting rats to a novel object significantly increased dopamine and norepinephrine output in the ventral hippocampus. Two hours after introducing the first object, a second object (either novel or familiar) was placed in the same position as the first object. Presenting the animals to a second novel object significantly increased dopamine and norepinephrine release in the ventral hippocampus, compared to a familiar object. In conclusion, this study suggests that dopamine and norepinephrine output in the ventral hippocampus has a crucial role in recognition memory and signals novelty. Through activation of VTA dopamine cells, a novel stimulus would lead to increased dopaminergic activity in the hippocampus, which would activate postsynaptic dopamine receptors resulting in induction of long-term potentiation (LTP) and memory storage of the novel information.The dopaminergic neurons projecting from the ventral tegmental area (VTA) to the ventral hippocampus have been hypothesized to be part of a functional loop through which dopamine signals novelty and strengthens memory consolidation in the hippocampus  expressing neurons projecting from the locus coeruleus (LC) to the dorsal hippocampus improved spatial object recognition. Interestingly, the study by Kempadoo et al.  agonist SKF81297 in the dorsal hippocampus, as well as the prelimbic region of the medial prefrontal cortex (mPFC), potentiates recognition memory  test  and water. The rats were allowed to acclimatize at least 5 days prior to any experiments. All experiments were performed in the light phase between 8 am and 6 pm. The experiments were approved by Karolinska Institutet and the local animal research ethics committee, Stockholm North.Male Wistar rats were obtained from Charles River (Germany) and had an average weight of 250 g upon arrival to the animal facility. The animals were kept under standard laboratory conditions with a 12/12 h light/dark cycle (lights on at 6 am), controlled temperature (21 ± 0.4°C) and relative humidity (55–65%) with ®  and Dormicum®  diluted in purified water . The rats were mounted in a sterotaxic frame  and implanted with a concentric dialysis probe in the ventral hippocampus  according to the atlas of Paxinos and Watson  at a flow rate of 2.5 μl/min and each sample was collected over 30 min . Dopamine and norepineprhine output were continuously measured in the ventral hippocampus. The samples were automatically loaded and injected into a reverse-phased high performance liquid chromatography system with a C-18 column  controlled by a computerized system . The mobile phase for separation of dopamine, norepinephrine and metabolites consisted of 55 mM sodium acetate buffer (pH 4.0) with 12% methanol and 0.55 mM octanesulfonic acid. Dopamine, norepinephrine and their metabolites were quantified online by sequential oxidation and reduction using electrochemical detection in a highly sensitive analytical cell , which was controlled by a potentiostat  with a detection-limit of approximately 0.3 nM/L. A potential of −200 or 400 mV was applied to detect monoamines and their metabolites respectively. All chromatograms were recorded digitally. An external standard of dopamine and norepinephrine was used in order to calculate the dopamine and norepinephrine output. The experiment started when the rats showed a stable dopamine and norepinephrine release (<10% variation). Baseline values (100%) were calculated as the average of the two values prior to the introduction of an object. A separate baseline was calculated for the first object and the second object (novel or familiar).During the experiments the probes were continuously perfused with artificial cerebrospinal fluid . Only animals with correct probe placement were included in the study.The NOR test was first described in a article by Ennaceur and Delacour . In thist-tests. The effect of an object on relative dopamine and norepinephrine output was compared to the last preceding sample using paired t-tests. The comparison between groups (novel vs. familiar) of the relative dopamine and norepinephrine release after the introduction of the second object was evaluated using t-tests. In all statistical evaluations p < 0.05 was considered statistically significant. All p-values were false discovery rate (FDR)-corrected for multiple comparisons.The absolute differences in basal dopamine and norepinephrine output in the ventral hippocampus between groups were analyzed using As the baselines shifted over time, and in order to compare the dopamine and norepinephrine output when the novel or familiar object was present to the preceding sample, separate baseline values were calculated for when the animals were introduced to the first object and a familiar or a novel object. The baselines were calculated from the two values 30 min before the objects were introduced to the animal  was 0.157 ± 0.046 fmol/min for the rats that got a familiar object and 0.110 ± 0.031 fmol/min for the rats that got a novel object (n = 6).The mean dopamine release (presented as mean ± SEM) during the first baseline in the ventral hippocampus was 0.191 ± 0.029 fmol/min. At the second baseline the mean dopamine release in the ventral hippocampus  = 4.320, p = 0.008; After steady baselines of dopamine and norepinephrine were obtained, the animals were presented to the first object. Introduction of the first, novel object significantly increased ventral hippocampal dopamine output (155.8 ± 13.4%) compared with baseline values  = 3.591, p = 0.028; t(11) = 2.775, p = 0.028; Two hours after the introduction of the first object , the second object (either novel or familiar) was introduced to the animal. The introduction of a novel object induced a significant increase of dopamine (163.0 ± 18.4%) output in the ventral hippocampus compared with the second baseline  = 4.269, p = 0.027; The norepinephrine output in the ventral hippocampus (163.9 ± 13.6%) was significantly higher when the first object was presented than it was at baseline  = 3.089, p = 0.036; t(11) = 3.379, p = 0.024; The introduction of a novel object induced an increase of norepinephrine (138.5 ± 11.2%) output in the ventral hippocampus compared with the second baseline (101.0 ± 8.2%; In the present study, we show that dopamine and norepinephrine release in the rat ventral hippocampus increased in response to novelty. Therefore, our data lend support to the hypothesis proposed by Lisman and Grace ; suggest1/5R antagonist SCH23390, indicating that memory persistence is a dopamine dependent process (Vargas et al., The results presented here are also consistent with the findings from a study using object location memory combined to microdialysis in rats, which shows that novelty increased dopamine in the dorsal hippocampus (Moreno-Castilla et al., + neurons projecting from the LC to the hippocampus improved spatial learning in a dopamine receptor dependent manner. Moreover, Kempadoo et al. (Furthermore, recent studies suggest that dopamine in the hippocampus may be co-released from norepinephrine neurons originating from the LC (Kempadoo et al., o et al.  show thao et al. , supportIn the present study, dopamine and norepinephrine release in the ventral hippocampus was increased in a similar way as described by Ihalainen et al. . They fo1/5R agonist SKF81297 in the mPFC or dorsal hippocampus potentiated recognition memory. Similarly, a local injection of D1/5R antagonist SCH23390 in the dorsal hippocampus or mPFC prevented the formation of episodic-like memory. It should be noted that in the present experiments, the objects were presented within 2 h, thus not involving long-term memories. Therefore, comparisons between the present results and previous behavioral data would be speculative.The observed dopamine and norepinephrine output in the ventral hippocampus is in line with a previous study, in which we showed that local administration of the NET inhibitor reboxetine strongly increased hippocampal dopamine release. In contrast, local administration of the DAT inhibitor GBR12909 only caused a negligible increase in dopamine release. Moreover, autoradiography revealed very low levels of DAT expression in the hippocampus. Thus, demonstrating that the dopamine and norepinephrine systems in the hippocampus have a shared uptake mechanism at the NET level (Borgkvist et al., It has previously been shown that other neurotransmitters besides dopamine and norepinephrine play an essential role in recognition memory. Stanley et al.  combinedFinally, by utilizing a version of our previously validated NOR test (Feltmann et al., In conclusion, the present study provides support for the hypothesis that dopamine in the hippocampus signals novelty and suggests that a crucial role for ventral hippocampal dopamine and norepinephrine in recognition memory.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Karolinska Institutet and the local animal research ethics committee, Stockholm North.JT analyzed the data, analyzed the experiments, made figures, and wrote the first draft.CB performed and analyzed the experiments. KF, TM, DM, and CBG performed experiments. BS and ÅK-G led the research, designed experiments and wrote the manuscript. All authors contributed to the article and approved the submitted version.CB is currently employed by Janssen-Cilag AB, but contributed to this study in his prior role at Karolinska Institutet. Janssen had no part in the development, execution or financing of this study. The remaining 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.


Vibrio vulnificus cases in coastal regions of Korea. All V. vulnificus cases in coastal regions notified to the Korea Disease Control and Prevention Agency between 2003 and 2016 were included in this work. Data for seawater temperature on the south, west, and east coast during the study period were provided by the Korea Oceanographic Data Center of the National Institute of Fisheries Science. We used a generalized additive model and performed a negative binomial regression analysis. In total, 383 notified cases were analyzed . The maximum seawater temperature was the most significant predictor of V. vulnificus cases on the south and east coasts (relative risk according to the 1 °C increase in seawater temperature (RR) = 1.35 : 1.19–1.53) and 1.30 (95% CI: 1.06–1.59), respectively). However, the mean seawater temperature was the most significant predictor for the west coast (RR = 1.34 (95% CI: 1.20–1.51)). These results indicate that continuously monitoring seawater temperature increase in each coastal area is crucial to prevent V. vulnificus infections and protect high-risk groups, such as persons with liver disease.The purpose of this study was to assess the association between seawater temperature and  Vibrio vulnificus infection is an acute and fatal infectious disease caused by V. vulnificus. V. vulnificus is a halophilic Gram-negative bacterium that belongs to the Vibrio genus of the Vibrionaceae family. This pathogen can cause primary septicemia in high-risk populations, such as individuals with chronic liver disease, diabetes, immunodeficiency, iron storage impairment, and end-stage kidney disease, and it has a fatality rate of 50–60% [V. vulnificus-contaminated warm seawater during recreational and leisure activities. Moreover, one can be infected with this bacterium through the consumption of uncooked or undercooked seafood [V. vulnificus cause severe disease in human; biotype 1 is the most common and accounts for the entire illness, including primary sepsis associated with a fatality rate exceeding 50% [V. vulnificus infection requires antibiotic treatment and surgical removal of necrotic tissue [V. vulnificus infections are commonly observed in several coastal cities in Japan [f 50–60% ,2. Besid seafood . The fat seafood  to 25% [ seafood . Three bin Japan , and thein Japan ,9,10,11.V. vulnificus can be found in warm coastal regions worldwide and in estuarine environments where water temperatures range from 9 to 31 °C [V. vulnificus was isolated only at water temperatures between 15 and 27 °C. Furthermore, its presence was associated with water temperature, dissolved oxygen levels, and levels of other Vibrio species [V. vulnificus proliferation and its ability to infect humans. Therefore, water temperature is a reliable predictor of pathogenic V. vulnificus infection cases [V. vulnificus is known to be a seawater temperature of 13 °C, and below 13 °C, V. vulnificus cells enter the viable but nonculturable (VBNC) state [V. vulnificus infection [V. vulnificus infection has been poorly assessed quantitatively. Thus, this study was conducted to assess the risk of V. vulnificus infection depending on coastal water temperature. species . It is kon cases ,17,18. Ton cases ,13,14. Hnfection  and geognfection  and devenfection . HoweverV. vulnificus infection was declared as a notifiable infectious disease on August 1 2000, and population surveillance programs were implemented afterward. A total of 761 V. vulnificus infection cases were reported to the Korea Disease Control and Prevention Agency (KDCA) by National Infectious Disease Surveillance System (NIDSS) from 2003 to 2016. Epidemiological investigations were conducted in all 761 of these cases. The data for this study were derived from epidemiological investigations conducted during that period and complete epidemiological datasets. Among these cases, 383 were linked to the consumption of raw seafood or exposure to seawater in coastal cities, counties, and districts.In South Korea, V. vulnificus infection (hereinafter referred to as cases). Cases met the case definition criteria of nationally notifiable infectious diseases put forth by the KDCA in 2020. Confirmed cases included people with clinical symptoms of a V. vulnificus infection and a confirmatory infection diagnosis according to the examination criteria. Suspected cases included people with clinical symptoms indicative of V. vulnificus infection and epidemiological association  who tested negative for the pathogen.The present study included confirmed and suspected cases of The subjects of the study were 383 cases residing in the inland or coastal areas of Korea during the study period. They were selected from the 761 cases for which epidemiological investigations were conducted. In the study, the selection criterion for the coastal area was where the 383 cases were infected in cities, counties, and districts adjacent to the sea. V. vulnificus cases reported between 2003 and 2016, seawater temperature data were obtained from the Korea Oceanographic Data Center. Seawater temperatures were analyzed for each day and coastal region  to calculate the minimum, average, and maximum monthly temperatures.To assess the association between seawater temperatures and V. vulnificus can proliferate in July (21.2 °C), August (24.1 °C), and September (23.1 °C), as well as at the maximum number of identified cases and the maximum mean temperature in August (24.1 °C) were used in this study.Considering that it is more important to examine the duration of high-water temperatures than temperature alone, the number of days within a month on which the seawater temperature exceeded the expected temperature range (18–24 °C) was calculated. The seawater temperature indices were determined through a literature review. The mean temperature at which V. vulnificus cases per coastal region and the seawater temperature indices was investigated.We performed a descriptive analysis to examine the epidemiological characteristics of confirmed cases at city, county, and district levels. In addition, the absolute water temperature  and the number of days on which the water temperature exceeded the expected temperature range (18–24 °C) were calculated. The link between the number of monthly V. vulnificus infections between 2003 and 2016 and each seawater temperature indicator. A generalized additive model (GAM) was applied to predict the nonlinear relationship between the two variables. The monthly number of cases in coastal regions (N) was the dependent variable and the seawater temperature indicators were the independent variables; a smoothing spline function was used. After each coastal model fit, the variable with the lowest Akaike information criterion (AIC) value among the water temperature indicators was selected as a predictor (Equation (1)).Pearson’s correlation analysis was performed to determine the linear relationship between the monthly number of V. vulnificus cases in coastal regions, and a cubic spline smoothing function was used. The analysis was adjusted for the effector variables time trends  and seasonality. The analysis was also adjusted for the population as an offset variable throughout the study period (2003–2016).In this equation, E(Yi) refers to the monthly number of V. vulnificus infection cases). Therefore, negative binomial regression was used to calculate the risk of infection depending on seawater temperature increase. The relative risk (RR) of V. vulnificus cases incidence per 1 °C increase in seawater temperature along with 95% confidence intervals (CIs) and the relationship between V. vulnificus cases and seawater temperature were analyzed. SAS software version 9.4  and MGCV package in R 3.1.4 software were used for the analysis. The level of significance was set at p < 0.05.Overdispersion (variance greater than the mean) was observed for the dependent variables .V. vulnificus cases were reported between 2003 and 2016. Five cases of infections that occurred abroad and 248 with unknown locations at which infection occurred were excluded. Of the 508 remaining cases, 117 registered in the inland area and 8 lacking necessary information  were excluded from the study. Thus, 383 cases in coastal cities, counties, and districts were included in the descriptive epidemiology study and for the analysis of the association between water temperature and V. vulnificus infections , and the maximum seawater temperature was the highest in 2005 (21.0 °C).V. vulnificus cases mostly occurred between July and September of each year  and 1.30 (95% CI: 1.06–1.59), respectively. The mean seawater temperature was the most reliable predictor on the west coast and showed a relative risk of 1.34 (95% CI: 1.20–1.51).After adjusting for time and seasonality, the maximum seawater temperature was the most reliable predictor of V. vulnificus cases. V. vulnificus cases were most frequently observed in August and September. In August, the mean and maximum seawater temperatures were highest at 24.1 and 28.9 °C, respectively. A positive correlation was observed between each seawater temperature indicator and the number of infection cases. After adjusting for time trend and seasonality, the risk of V. vulnificus infection was significantly higher when seawater temperatures were high.The purpose of this study was to assess the association between domestic seawater temperature and V. vulnificus cases associated with the consumption of contaminated seafood or exposure to contaminated seawater have been reported in coastal regions worldwide. A total of 4754 cases of Vibrio infections were reported by the Centers for Disease Control’s Cholera and Other Vibrio Illness Surveillance (COVIS) system in the United States between 1997 and 2006. Of these, 1210 (25%) cases had developed non-foodborne Vibrio infections after being in contact with seawater, and 72% of the V. vulnificus infection cases involved residents of Gulf Coast states [V. vulnificus infection occurred between 1999 and 2000, and 50 (53%) occurred in Kyushu. Interestingly, 43 cases occurred in the coastal regions of the Ariake and Yatsushiro seas, which have several tidal flats [Several V. vulnificus cases were frequently observed between July and September in Korea. The number of cases was highest on the west coast, followed by the south and east coasts. This study also shows that the risk of V. vulnificus cases increases with increasing seawater temperatures, consistent with findings from previous studies [In the present study,  studies ,27,28,29V. vulnificus case in our study was observed in May, when the maximum seawater temperature at the coast was 20.2 °C. In contrast, the maximum seawater temperature from January to April, when cases were not reported, was 12.4–16.3 °C. The cases of V. vulnificus infection from 1988 to 2010 reported to the COVIS system in the United States had a distinct seasonal distribution. Infections rarely occurred when the seawater temperature was below 13 °C [The first ow 13 °C . It is kow 13 °C .p < 0.001) were observed between the number of V. vulnificus infection cases in coastal regions and the nine water temperature indicators. The adjusted-R2 of water temperature for the incidence of total V. vulnificus infections was 70%. A previous study reported a linear increase in the presence of V. vulnificus at seawater temperatures between 11 and 30 °C [V. vulnificus infection on the south, west, and east coasts according to the 1 °C increase in seawater temperature were 1.35 (95% CI: 1.19–1.53), 1.34 (95% CI: 1.20–1.51), and 1.30 (95% CI: 1.06–1.59), respectively. The estimated exposure–response relationship between Vibrio infections and the threshold of 16 °C demonstrated an RR of 1.14 (95% CI: 1.02–1.27) for a two-week lag. The estimated risk was in line with the findings of previous studies [V. vulnificus cases increased tenfold [In this study, positive correlations  increases when seawater temperature is high, posing greater health risks to people who eat raw oysters [The maximum seawater temperature was the most sensitive index to predict the number of cases on the south and east coasts, while the mean seawater temperature was the most reliable one on the west coast in this study. The highest incidence was observed in August on the south and east coasts when the maximum and mean seawater temperatures were the highest. However, on the west coast, which had the highest infection prevalence among the three coasts, the highest incidence was registered in September, although the seawater temperature was highest in August. Given that the same surveillance and reporting system is used nationwide, delayed reporting cannot explain this discrepancy. However, it has been reported that the abundance of perature . In addi oysters . FurtherV. vulnificus infection cases in coastal regions. Second, marine environmental factors, such as salinity, turbidity, dissolved oxygen, and suspended particulate matter, which may affect V. vulnificus infections, were not considered in this study. Lastly, our work did not consider the number of beach visitors, and behavioral patterns, which may affect the risk of infection in the population during the summer.The primary limitation of this study is that it was an ecological study that assessed the relationship between seawater temperatures and V. vulnificus incidence depending on the increase in seawater temperature in a quantitative manner using national incidence data of over 14 years. Our work also assessed the risk of infection in each of the coastal regions of Korea. Unexpectedly, our findings show that temperature predictors differ between coastal areas and that the sea temperature and the peak case incidence do not coincide on the west coast.The significance of this study is that it is the first to estimate the risk of V. vulnificus cases appears different, so it is necessary to further investigate whether liver diseases , which are the representative risk diseases for V. vulnificus infection, differ by coast.Considering population by coast, the incidence of V. vulnificus infection between 2003 and 2016 in coastal regions of Korea. We calculated the risk of V. vulnificus infection for every 1 °C increase in seawater temperature on the three coasts. The most reliable infection predictor based on seawater temperature indicators differs between coastal areas. On the west coast, the highest seawater temperature and the peak incidence of cases do not coincide, suggesting that factors other than seawater temperature are also relevant to predict infections. Therefore, it is necessary to consider people’s water activity and eating habits of raw seafood in hot seasons as well as seawater temperature to prevent V. vulnificus infections in coastal areas.This study included 383 registered cases of