These results provide new insights into the knowledge of the epidemiology of the disease in Cuban endemic conditions and can contribute to improving the control and the eradication strategy in this situation.Classical Swine Fever (CSF) is an endemic disease in Cuba, and an eradication strategy by zones is planned by the Official Veterinary Service. The aim of this study was to identify high-risk areas of CSF and the risk factors associated with the disease occurrence in the Pinar del Río province, one of the prioritized areas in the eradication strategy. The outbreak occurrence at district level was analyzed through a 7-year period (2009 to 2015). A high-risk cluster  was detected during the last 2 years of the study period in the eastern half of the province, with 38 out of 97 districts included. The rate of CSF-affected holdings had a significant increase during 2014–2015 and seems to have occurred mainly in the high-risk cluster area. Swine population density by district (heads/km Pestivirus, family Flaviviridae  is a highly contagious viral disease caused by an RNA virus belonging to the genus iviridae –3. The diviridae .CSF is a disease notifiable to the World Organization for Animal Health (OIE) and one of the most highly contagious animal diseases, with high morbidity and mortality in susceptible hosts, depending on the virus strain, immune status of the herd, and age of the pigs . Its outThe CSF control strategy recommended in free regionsTaking into account the CSF endemism in Cuba, vaccination with a nationally produced live attenuated vaccine of the lapinized Chinese strain, quarantine of affected premises, and culling of the affected animals and their contacts, are among the most important actions taken to reduce the number of disease outbreaks . Small aCSF has a recognized important economic impact  that in In Cuba, the Veterinary Authority is implementing an eradication plan by zones and Pinar del Río province is one of the starting points. Pinar del Rio is a priority zone for CSF eradication due to both strategic and advantageous aspects. It is an important swine production area in the country, where also herds for swine genetic selection are located . Since iThe key points for the elimination of residual sources of infection in the province are the identification of possible geographical clusters of CSF and the evaluation of potential risk factors associated with the disease occurrence. The present paper describes the results of a study for the identification of spatiotemporal clusters and risk factors for CSF in Pinar del Rio province. This study aims at contributing to a better and deeper understanding of the epidemiology of CSF in this territory and leading to the formulation of better policies for its control via the implementation of more efficient control measures.2. It is divided into 11 municipalities with 97 districts . The mean area of the districts in Pinar del Río is 90.6 km2.Pinar del Río, the province selected for the present study, is the westernmost territory of Cuba with a total area of 8,850 km2); (iii) swine holdings density (holdings/km2); (iv) human population (inhabitants); (v) human population density (inhabitants/km2); (vi) distance (km) from the district centroid to the main road and access to the province; vii) road length (km)—number of km of roads in the district; (viii) road density (km/km2).The provincial department of the National Animal Health Division of the Ministry of Agriculture (DSA) provided data at district level on the swine population by productive sectors. The productive sectors considered were commercial farms and backyard . The available information about swine holdings is recorded in manuscript books . The data were compiled, organized, and carefully revised with an electronic spreadsheet (Microsoft Excel 2010). The reported information on 101 swine holdings affected by CSF (hereinafter “CSF outbreak”) in the province from 2009 to 2015 was also collected. Some potential explanatory variables were analyzed with the aim of identifying those factors associated with the disease occurrence at district level: (i) swine population (heads); (ii) swine population density  were calculated using the Near tool and Tabulate intersection tool was used to calculate the length (km) of roads by district. These variables were calculated in ArcGIS 10.2.The centroid of each district was calculated using Add Geometry Attributes tool (Centroid property); the distances from each district centroid to the main road  around 37 km suggested that at this distance the most pronounced spatial clustering is produced. Thus, the radius of the circular scanning window without geographical overlap in SaTScan was established at 37 km.The Global Moran's The association between the explanatory variables and the outcome variable (number of CSF outbreaks) was assessed using a Generalized Linear Mixed Model (GLMM) with Poisson distribution. The analysis was carried out using the function glmmPQL (package MASS v.7.3-51.4) in R 3.5.0 .The model used in our study was as follows:The model outcome is the rate of outbreaks by districts. The fixed effects were estimated by the inclusion of the previously mentioned explanatory variables in the model. Relative risk (RR) with 95% confidence intervals (95% CI) of fixed effects was estimated. To adjust by the number swine holdings per district this variable was included in the model as offset and the municipality was included as a random effect to control for the clustering effect.2) were divided by 1000; Road density (km/km2) was not modified, and the rest were divided by 100. Firstly, the association between the dependent and the independent variables was assessed through the univariate regression analysis. Variables with a p-value ≤ 0.2 were included in the multivariate model. The final model was adjusted manually by using the backward elimination procedure to remove the non-significant variables (p < 0.05).In order to present the results on a more interpretable scale, the effect of some independent variables was rescaled. The variables “Distance from the district centroid to the main road in the province (km)” and “Road length (km)” were divided by 10; Swine population (heads), Human population (Inhabitants) and Human population density  in commercial herds and 111 295 (49.15 %) housed in backyard farms. A significant high-risk cluster  of CSF outbreaks at district level  was detep ≤ 0.20) at this step. These variables were included in the multivariate model and two variables were retained. The swine population density was associated with the CSF outbreak. Thus, the RR of outbreak occurrence increases 1.27 (1.09–1.48) times for 100 swine/km2 in the districts. Also, the road length was associated with the disease occurrence, which increases 1.16 (1.04–1.29) times for 10 km of road. The outputs of the models are shown in Seven out of eight variables were associated with CSF occurrence in the univariate model according to the assumed cut-off  province could also be related to the transit of people and trade of live pigs and by-products from Pinar del Río to Artemisa and La Habana provinces, due to the lower prices in Pinar del Rio and the high pork demand of Cuban capital . The strThe association between animal density and increased risk of transmission of infectious diseases has been already identified for CSF , 24 and In addition, the presence of a greater road network can be associated with a higher risk of occurrence of CSF. Roads, railroads, and other transportation ways play a decisive role in infectious disease dissemination. The trade of live animals, by-products and transport of swine production items  are based on vehicles, which can be an important mean of disease spreading , 29. PreAnother related aspect could be the use of undisinfected transport in the movement of the animals . VehicleA significant number of swine in the study territory (~50%) and in Cuba (>50%) are housed in backyard holdings that do not follow the appropriated biosecurity rules such as sanitary barriers, cleaning, disinfection, vaccination, and an appropriated food storage, and sometimes the pigs are fed on swill containing pig by-products . Pigs raThis constant flow could raise the risk of CSF occurrence in Pinar del Río because its sanitary situation was historically favorable. The positive evolution of the CSF until 2002 was key to choose Pinar del Río to start the CSF eradication strategy by zones , in contThis study focuses mainly on the detection of spatiotemporal clusters and the analysis of potential risk factors for CSF outbreaks occurrence considering its spatial distribution and explanatory variables. However, the non-availability of data about other variables made it impossible to include in the analysis many other potentially important factors, such as the number of visits to the farms, variations in total swine population by year and by different animal categories, vaccination coverage at district level, and animal movement, among others, which is an important limitation of this study. Future investigations could consider those factors to provide a more comprehensive assessment of the different factors that may influence both the occurrence and persistence of CSF at sub-district or farm level.The identification of risk factors and the detection of areas where affected holdings are spatially more concentrated can highlight the territories where the sanitary measures must be reinforced to improve the disease control. It will be important to increase the awareness and risk perceptions of pig producers in all sectors using educational activities focused on biosecurity, pig movement control, vehicle disinfection, and vaccination, among others.The methods used allowed to obtain interesting and useful results, which contributed to a better understanding of the CSF distribution in our scenario. These tools have shown the spatiotemporal distribution and risk factors of the disease and can be applied in the epidemiological studies in other provinces of the country to improve the control and eradication strategies at national level. However, this depends on data availability, which is an important limiting factor for this type of study.In conclusion, the disease has space-time clustering and one high-risk area was identified; furthermore, two risk factors of outbreak occurrence were detected. The epidemiological situation showed a worsening over time with the increasing number of CSF outbreaks in the last two years of the study period, being significantly higher in the eastern part of the province. The policymakers should consider these findings in other to improve the CSF control program in Pinar del Río and to reinforce the sanitary measures in this territory. Moreover, these results can contribute to support the strategies in the CSF eradication campaign in Cuba, with stricter regulations of animal movement in districts at high risk, increasing the epidemiological risk-based surveillance and the vaccination prioritization in the high-risk areas.The datasets generated for this study will not be made publicly available. The datasets used in this study belong to the National Animal Health Division of the Ministry of Agriculture (DSA), Cuba. The authors do not have authorization to publish the datasets.The research was performed with secondary and aggregated data. No study (experiment) with human or animal subjects was performed by any of the authors. Also, no private information from owners or farms was used or revealed.OF-R performed data analysis and manuscript drafting. YC, PA, EF-M, DM, and MB critically read the manuscript, suggested corrections, and provided intellectual inputs. MB, YG, EF-M, and OF-R performed data collection. PC, KS, and MP provided supervision and contributed to the epidemiological studies. All authors reviewed and approved the final manuscript.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.


Extra-vascular molecular clearance routes from the brain and cerebrospinal fluid (CSF) remain insufficiently characterized in humans. Animal studies consistently suggest that the cribriform plate and nasal lymphatic vessels are crucial for molecular clearance from CSF. In this study, we aimed to examine human in vivo transport of a CSF tracer from CSF to nasal mucosa. We hypothesised a CSF tracer would enrich in nasal mucosa provided that nasal lymphatic drainage has a significant role in CSF molecular clearance. Consecutive magnetic resonance imaging during 48 h after intrathecal administration of a tracer (gadobutrol) was performed in 24 patients. Despite a strong enrichment of CSF tracer in CSF spaces nearby the cribriform plate, there was no significant enrichment of CSF tracer in nasal mucosa, as measured in superior, medial and inferior turbinates, or in the nasal septum. Therefore, this in vivo study questions the importance of CSF drainage to the human nasal mucosa and emphasizes the need of further human studies. Hence, this route may be hypothesised to be of equal importance in humans. However, only one in vivo human study, utilizing radionuclide tracers, has been able to demonstrate possible clearance of molecules to the nasal turbinates, presumably via the cribriform plate11.There is limited knowledge about molecular efflux routes from cerebrospinal fluid (CSF) to lymphatic pathways in humans. Based on studies of a range of animal species, drainage to nasal mucosa via the cribriform plate is consistently shown to represent a major drainage route13, and the possible connection between meningeal lymphatics and the glymphatic system14, have sparked a renewed interest in lymphatic drainage routes from CSF spaces. Experimentally induced impairment of meningeal lymphatic vessels suggests that lymphatic drainage is crucial for brain molecular clearance15, indicating a role of lymphatic drainage failure in neurodegeneration caused by the accumulation of neurotoxic molecules in brain tissue. Recently, our group used magnetic resonance imaging (MRI) to demonstrate the accumulation of a MRI contrast agent administered intrathecally in the human parasagittal dura, a tissue possibly serving as an intermediate step towards meningeal lymphatics, paralleled by efflux through neuroforamina at the skull base16. The methodology has yet not been used to assess whether this CSF tracer is cleared to nasal lymphatic efflux pathways.The recent reports of lymphatic vessels lining the dural sinusesIn this study, we have therefore examined the efflux of intrathecal gadobutrol to nasal mucosa utilizing multi-phase, long-term MRI. We hypothesised that the MRI contrast agent gadobutrol, when administered intrathecally, enriches in nasal mucosa.The study included 24 patients examined with consecutive MRI scans before and after intrathecal administration of gadobutrol for work-up of tentative CSF circulation disorders. Patient data are given in Table The tracer was detected in CSF close to the cribriform plate in all patients. There was a highly significant P < 0.001) enrichment of CSF tracer in the subarachnoid space (SAS) adjacent to the straight gyrus with peak enrichment after 3–6 h  enrichment of CSF tracer within the grey matter of the straight gyrus Fig. , as wellNo anaphylactic reactions, other serious adverse reactions or complications were noticed during or after the exams.The main result of this study was that no CSF tracer could be detected in human nasal mucosa in vivo, raising the question of the relative importance of CSF clearance to lymphatic vessels in the nasal mucosa.17. The time from injection until CSF tracer was present in cisterna magna was 20 ± 23 min in humans18. Enrichment of tracer in CSF is a requirement for enhancement in nearby cerebral structures20 as well as in parasagittal dura16. The present observations confirmed entry of tracer into the adjacent straight gyrus and strong enrichment of tracer in the SAS close to the cribriform plate. Tracer was also detected as minute tubular structures inferior to the cribriform plate in 11/24 patients  may also be absorbed by capillaries in the nasal mucosa5 possibly affecting the detectable amount of tracer in the area. Detection of the tracer in both deep cerebral white matter and in the straight gyrus of the frontal lobes confirmed that the applied T1-BB sequence enables for detection of tracer in limited amounts with good sensitivity.The tracer used in this study, gadobutrol, is of low molecular weight (604 Da), distributes freely in the CSF due to its hydrophilic properties and does not cross the blood–brain–barrier. As so, this MRI-tracer should be expected to follow extra-vascular clearance routes for molecules lacking specific transporters4, or even tracer in arachnoid villi34. Perineural spread of tracer through the cribriform plate to nasal mucosa has previously been described in human34. In Johnston et al.10 a tracer injected in the SAS did indeed reach the lymphatic vessels of the nasal mucosa, both in animals and in one human, but no connection to lymphatic vessels was detected by Lowhagen et al.34. These studies provide good anatomical descriptions of the area, but do not necessarily reflect physiological conditions as they are performed post-mortem.In this study, we detected the CSF tracer as minute tubular structures inferior to the cribriform plate in 11/24 patients. This could represent tracer along perineural spaces, in lymphatic vessels or in the interstitial space11. The PET technique used is probably more sensitive compared to MRI, but the image resolution of 4,5 mm is inferior to MRI (1 mm). The superior turbinates in humans are small structures, and the ROI used for superior turbinates in the PET-study may potentially have included adjacent areas. The ROI was also placed 5.2 mm inferior to the frontal lobe to avoid partial-volume effects from the brain, nevertheless, partial volume effects from CSF may have affected the measurements. Our results do confirm tracer inferior to the cribriform plate in a substantial proportion of patients (11/24) .Our study findings contradicts De Leon et al., who utilized dynamic positron emission tomography (PET) to measure CSF clearance in humans and found significant levels of CSF tracer in the superior turbinates35. Substances have been detected in CSF within 30 min after intranasal administration36. One plausible theoretical explanation for such rapid transport is extracellular  convective flow37.Both animal and human studies suggest transport of molecules in the opposite direction, that is, from nasal mucosa to CSF, following anatomical pathways along the olfactory and trigeminal nerves bypassing the blood–brain barrier 39, could have an impact on species-dependent differences in trans-nasal CSF molecular efflux. We, therefore, hypothesise that CSF molecular efflux through other foramina in the skull base and meningeal lymphatics30 could be of more importance in humans compared to many other species. Other postulated molecular efflux routes are via the arachnoid villi to dural venous sinuses40 and across the blood–brain barrier41.From an anatomical point of view, the human bipedal position, and the small olfactory area, which may be considered to be of a rather rudimentary size compared to rodentsThis study included patients with various CSF-disorders Table . 13/24 pThe temporal resolution of imaging was low, particularly for late scans. However, MRI scanning was continued throughout a considerable time frame and beyond the time of peak tracer enhancement in CSF. It seems therefore unlikely that mucosal enrichment followed by an expectedly slow molecular escape should have occurred entirely in intervals between MRI scans.4. Visualization of individual lymphatic vessels or perineural spaces in the nasal mucosa using MRI cannot be expected due to the MR image resolution (1 × 1 × 1 mm); the circular ROIs were positioned to measure from representative samples of the mucosa/submucosa covering perineural spaces, lymphatic vessels and the interstitial space. The concentration of the tracer in nasal mucosa would be expected to be lower than in the CSF after dilution from lymph and interstitial fluid in the nasal mucosa. Artefacts from both movement and surrounding areas may have decreased the signal-to-noise ratio and confounded the measurements even if obvious artefacts were avoided and images disturbed by motion were excluded. To this end, our negative result could be explained by low concentrations of tracer undetectable by the methodology utilized in this study.The connections between the SAS, perineural spaces, lymphatic vessels and interstitial spaces in the nasal mucosa are not clarifiedFor some measurements, the signal intensity in the CSF, nasal mucosa and brain parenchyma seemingly declined after contrast agent administration compared to pre-contrast images Figs. , 4 and 542. Therefore, ROI locations had to be manually adapted to local volume changes to avoid misregistration and partial volume effects from adjacent regions, particularly from the airways. The volume differences represent to some extent differences in blood volume. The intrinsic signal in nasal mucosa could thus potentially change for other reasons than the enrichment of tracer. The volume differences in the nasal mucosa also limited the possible regions to include in the measurements. On the other hand, volume changes would be random, and should not produce a systematic effect of the changes in MRI signal units in one direction. Even though manual placement of ROIs may not be optimal, and potentially biased, this was considered the only feasible option due to the inter- and intrapatient physiological fluctuations.Diurnal volume fluctuations of the nasal mucosa derive from dilation and constriction of the venous cavernous tissue in the turbinatesIn conclusion, this study did not confirm clearance of CSF tracer to the nasal mucosa in humans, unlike in many previous animal studies, thus questioning the importance of this molecular clearance route in humans. The method applied in this study has limitations, particularly concerning the use of a small molecular weight CSF tracer. Future studies are needed to address to which extent molecular efflux to nasal mucosa differ between animals and humans.16.The study design was prospective and observational. Patients referred to the Department of neurosurgery, Oslo University Hospital-Rikshospitalet were included for consecutive MRI exams after intrathecal administration of the contrast agent gadobutrol. Inclusion criteria were tentative CSF circulation disorders. Exclusion criteria were age < 18 years or > 80 years, pregnancy and breastfeeding, known adverse reaction to contrast media, history of severe allergic reaction in general, and renal dysfunction. A total number of 34 patients were included for imaging. After imaging, 10 subjects were excluded due to movement artefacts, missing MRI exams, or signs of rhinitis at MRI. Of the remaining 24 patients, 12 were included in a previous study assessing tracer enrichment in the parasagittal duraMRI was obtained approximately 8 a.m. followed by an X-ray guided lumbar puncture performed by an experienced interventional neuroradiologist. Backflow from the puncture needle verified a correct position in the SAS. A radiopaque contrast agent, typically 3 ml of 270 mg I/ml iodixanol , was injected to confirm unrestricted passage in the lumbar SAS. Thereafter, 0.5 ml of 1.0 mmol/ml gadobutrol  was administered. After needle removal, the study subjects were instructed to rotate themselves around the long axis of the body once and remain in a supine position for transportation back to the MRI suite for the first post-contrast image.A 3 T Philips Ingenia MRI scanner  with a 32-channel head coil was used with equal imaging protocol settings at all time points to acquire whole-brain sagittal T1-weighted volume scans with suppression of intraluminal blood signal (“Black Blood”) (T1-BB). The parameters were TR/TE = 700/35 ms, echo train length = 55, flip angle = 80°, 2 averages, 1 × 1 × 1 mm voxel size (isotropic). Acquisition time was 4 min and 54 s.For practical reasons, the consecutive MRI exams could not be obtained at identical time points in every subject. The MRI exams were therefore categorized into the following approximate time points according to an intention-to-scan basis: pre-contrast, < 1 h  after contrast, 3 h  after contrast, 6 h  after contrast, 24 h  after contrast and 48 h  after contrast. The patients were instructed to stay in bed until the 6 h’ time point and were allowed to move freely thereafter.Whole-brain 3D T1-gradient echo (GRE) images were also obtained during each imaging session. The imaging parameters were as follows: TR = shortest , echo time = shortest , echo train length = 232, flip angle = 8°, 1 average, 1 × 1 × 1 mm voxel size and acquisition time 6 min and 29 s.T1-BB scans were deemed superior to T1-GRE with regards to having a better signal-to-noise ratio in the region of the nasal mucosa and were chosen for the ROI-analysis in all areas of interest. The T1-GRE images were used for visually detection of tracer inferior to the cribriform plate and in elaboration of the image showing the tracer immediately below the cribriform plate Fig. .43. Recently published data showed the same safety profile for intrathecal administration of gadobutrol as for intrathecal administration of the CT contrast agent iodixanol18, and no signs of deposition in brain tissue have been detected after 4 weeks19.Gadobutrol is approved for intravenous use only by the United States’ Food and Drug Administration (FDA). Intrathecal administration of gadobutrol has so far been used off-label in a controlled study setting at our institution in patients with specific clinical indications for work-up of CSF circulation disorders. Gadobutrol is a macrocyclic gadolinium contrast agent. Concerns about gadolinium brain deposition have primarily been associated with linear contrast agentsAll T1-BB volume scans were reconstructed to 1 mm thick coronal sections perpendicular to a line between the anterior and posterior commissure. A board-certified neuroradiologist (EM) with 7 years’ experience in neuroradiology manually placed 24 circular, pre-defined regions of interest (ROIs) at all time points using the hospital’s Picture Archiving and Communication System . The placements of the ROIs in the nasal mucosa and the SAS in one patient are illustrated in Fig. 2, a study of lymphatic vessels in humans44, post-mortem studies of humans10 and the PET-study by de Leon et al.11 to be as anatomically correct as possible for detection of tracer in lymphatic vessels. The lymphatic vessels were expected to be of size far below the image resolution of 1 mm, and therefore not directly detectable.The ROIs in the nasal turbinates, including the posterior parts, were based on anatomical considerations in rodentsThe exact placement and size of each set of ROIs were chosen with the intention to avoid artefacts and to cover as much relevant tissue as possible and at the same time striving to be anatomically stringent between different time points. 16 of the ROIs were placed in the mucosa/submucosa of the nasal turbinates and nasal septum as follows: 3 ROIs were placed bilaterally on each middle and inferior turbinate including the posterior parts of the turbinates; 1 ROI bilaterally on the superior turbinates; 1 ROI on both sides in the superior part of the nasal septum. 8 ROIs outside of the nasal cavity was measured: bilaterally in the deep white matter of the frontal lobes; bilaterally in straight gyrus of the frontal lobe and the adjacent SAS, respectively.In addition, ROIs were placed bilaterally in the vitreous body of the ocular bulb for reference  with 13 years’ experience in neuroradiology verified the placement of the ROIs and the overall quality of the images.www.slicer.org)45. Figure The 3D T1-GRE images were visually analysed in reconstructed 1 mm thick coronal and sagittal images for tracer inferior to the cribriform plate in all 24 patients for all time points. The 3D T1-GRE images were also used as source data for Fig. SPSS software version 24  was used for statistical analyses. Linear mixed model analysis was used to assess whether changes in signal unit ratios over time were statistically significant. Pearson Chi-square test was used to determine differences between categorical data. Statistical significance at the 0.05 level (two-tailed) was accepted.The study was approved by the Institutional Board of Oslo University Hospital (2015/1868), the Regional Ethical Committee of South-East Norway (2015/96) and the National Medicines Agency (15/04932-7). The protocol was conducted in accordance with the relevant guidelines and regulations. Oral and written informed consent was obtained from all participants.Supplementary file1


The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point toward allosteric mechanisms in regulating GDAP1 oligomeric state and function.Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive toward classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca GDAP1 gene, coding for the ganglioside-induced differentiation-associated protein 1 (GDAP1), are associated with several forms of Charcot-Marie-Tooth disease (CMT), which is one of the most common inherited neurological disorders, affecting one in 2,500 people  . Endogenonomeric . In ligh2+ uptake and release to modulate cytosolic Ca2+ signaling (2+ inflow through store-operated Ca2+ entry (SOCE) activity and endoplasmic reticulum (ER) Ca2+ levels , can induce cyclosporin A-insensitive permeability of the inner membrane of liver mitochondria  shares a 55% sequence identity with GDAP1, and the HD and TMD are conserved . The HD Here, we describe the crystal structure of the complete dimeric GDAP1 core domain, and based on its unique mode of dimerization, we propose a model for full-length GDAP1 on the MOM. We also provide a low-resolution model for monomeric GDAP1L1 based on small-angle X-ray scattering (SAXS) data. As no GST activity was detected for GDAP1, a metabolite library was screened for GDAP1 binding partners. We find that HA binds to GDAP1 and affects its stability, conformation, and oligomerization. The HA binding site in the crystal structure of GDAP1 is located close to the CMT-linked residue cluster and the membrane-binding surface.Chemicals were from Sigma-Aldrich unless otherwise stated. Crystallization screens were from Molecular Dimensions. The Human Endogenous Metabolite Compound Library was from MedChemExpress.® technology-based site-specific recombination (Invitrogen). An N-terminal Tobacco Etch Virus (TEV) protease digestion site was included in each construct.The open reading frame (ORF) of full-length human GDAP1 isoform 1 (UniProt ID: Q8TB36) was ordered from DNA2.0 as a synthetic codon-optimized gene for bacterial cytosolic expression in the pJ201 vector. The C-terminally truncated GDAP1∆295-358, GDAP1∆319-358, and GDAP1∆303-358 constructs were generated by PCR and transferred into the pDONR221 entry vector using Gateway6 and thioredoxin tags, respectively. Point mutations were generated using site-directed mutagenesis PCR (6 tag (Genscript). All constructs were verified with DNA sequencing of both strands.For structural and biochemical characterization, GDAP1 constructs were transferred into pTH27  and pDESesis PCR . The ORFE. coli BL21 (DE3) in ZYM-5052 auto-induction medium (E. coli B834 (DE3) in SelenoMet™ -media (Molecular Dimensions) (Recombinant protein expression was done using n medium . Selenomensions) .2+-NTA affinity resin by gravity flow (Thermo Fisher Scientific). Unbound proteins were washed with 25 mM HEPES, 300 mM NaCl, 2% (v/v) glycerol, and 25 mM imidazole (pH 7.5). The protein was eluted with an identical buffer, with imidazole at 250 mM. The affinity tag was cleaved with a 1:20 molar ratio of TEV protease . The His6 tag and TEV protease were then removed by another Ni2+-NTA affinity step. SEC was performed on a Superdex 200 or Superdex 75 10/300 GL increase column  using 25 mM HEPES (pH 7.5), 300 mM NaCl (SEC buffer) as eluent. An anion exchange chromatography (IEX) step was added for GDAP1L1, using a HiTrap HP Q XL column . GDAP1L1 was eluted using a linearly increasing gradient up to 1 M NaCl in 30 mM Tris (pH 7.9). Peak fractions were analyzed with SDS-PAGE, and Coomassie-stained bands were analyzed using a Bruker UltrafleXtreme matrix-assisted laser desorption/time-of-flight mass spectrometer (MALDI TOF-MS). Tryptic peptides extracted from the gel were identified by searching NCBI and SwissProt databases using BioTools2.2 (Bruker).The soluble recombinant protein was captured on a Ni2. Data collection was conducted on the synchrotron beamlines P11 , I24, and I04 , at 100 K  nano-dispenser. The protein concentration was between 5–25 mg/ml in SEC buffer. Apo GDAP1∆303-358 was crystallized in 0.2 M magnesium formate, 20% (w/v) PEG 3350. GDAP1∆303-358 crystals with HA were obtained by co-crystallization with 1 mM HA (2% (v/v) EtOH as solvent), using 0.1 M succinic acid, 15% (w/v) PEG3350 as mother liquor. SeMet-GDAP1 crystals were grown in 0.2 M ammonium formate, 20% (w/v) PEG3350. Crystals were briefly soaked in 30% (v/v) glycerol before flash-freezing in liquid Nat 100 K .http://doi.org/10.5281/zenodo.3988189).Data were processed and scaled with XDS  and AIMLhttp://www.pymol.org) and Chimera /λ, where 2θ is the scattering angle) at a fixed temperature (+15 °C). 50 µL of a protein sample at 8.5–10 mg/ml were injected into a BioSEC3-300 (Agilent) or Superdex 75 10/300 GL increase column  and eluted at a flow rate of 0.2 ml/ml or 0.5 mg/ml. Data reduction to absolute units, frame averaging, and subtraction were performed using Foxtrot  using a miniDAWN TREOS II detector (Wyatt Technologies), coupled to a Shimadzu Prominence HPLC system with RID-20A (RI) and SPD-M30A (diode array) detectors. SEC to separate oligomeric species was performed using Superdex 75 10/300 GL or Superdex 200 15/150 GL increase columns  in SEC buffer. The protein concentration was 1–10 mg/ml, and the injected protein amount 15–150 µg. Data processing, baseline reduction, and molecular weight calculation were done in ASTRA 7 (Wyatt Technologies).GDAP1∆319-358 and GDAP1∆295-358 in SEC buffer were titrated with HA ) in a 96-well PCR plate. After adding SYPRO Orange fluorescent dye, the plate was sealed with an optical PCR plate sheet, and thermal denaturation was analyzed by differential scanning fluorimetry (DSF) in an Applied Biosystems 7500 device. Melting curves were analyzed with GraphPad Prism.350/F330) were monitored to determine apparent melting temperatures (Tm).Thermal unfolding of wild-type and C88A GDAP1∆303-358 in SEC buffer was studied by nanoDSF using a Prometheus NT.48 instrument (NanoTemper). The fluorescence of tryptophan was excited at 280 nm and recorded at 330 and 350 nm. The samples were heated from +20 to +90 °C with a heating rate of 1 °C/min, and changes in the fluorescence ratio  in SEC buffer with and without 2% (v/v) DMSO for HA and GSH, respectively. The sample cell and injection syringe were filled with 50 μM GDAP1 and 500 μM HA or 8 mM GSH, respectively. The system was equilibrated to a stable baseline before initiating an automated titration. The injection volume was 2.5 μL with 15 injections for HA and 3 μL with 12 injections for GSH. Injections were repeated at 180-s intervals at +25 °C. The sample was stirred at 750 rpm. The data were analyzed with the one-site binding model in Origin  to obtain thermodynamic parameters.BLI measurements were performed in SEC buffer containing 0.005% Tween 20 and 2% DMSO, using an Octet RED instrument (FortéBio) at +25 °C. Biotinylated GDAP1∆319-358 was loaded onto Super Streptavidin (SSA) biosensors (FortéBio) and quenched with 250 µL of 10 μg/ml biocytin. The association of GDAP1∆319-358 with HA at a series of concentrations was measured for 180 s. The dissociation was performed by washing the biosensors with binding buffer for 180 s. A reference measurement without biotinylated protein was subtracted from all curves. Data were analyzed using Data Analysis 11.0 (FortéBio).p-nitrophenoxy)propane (EPNP) together with every GDAP1 construct. Absorbance was followed at 360 nm for a 500 µL reaction at +25 °C for 5 min, with a Jasco V-730 UV-VIS spectrophotometer  in a 1-mm quartz cuvette . Substrate concentrations in the assays were 1 mM (CDNB), 0.25 mM (pNBC), and 0.3 mM (EPNP) in 100 mM potassium phosphate buffer, pH 6.5. The GSH concentration was between 1–5 mM, and GDAP1 amount was 50 µg. As a positive control, 0.5 µg of recombinant S. japonicum GST (SjGST)-TEV fusion protein was used. Data were analyzed using Jasco Spectral Analysis software. All measurements were done in triplicate.Spectrophotometric activity measurements were done using the generic GST substrate analogs 1-chloro-2,4-dinitrobenzene (CDNB), 4-nitrobenzyl chloride (pNBC), and 1,2-epoxy-3- were sampled for SDS-PAGE. The matrices were washed three times with 250 µL of 20 mM HEPES, 150 mM NaCl, pH 7.5, and after each wash, the matrices were collected as above and the supernatants sampled for SDS-PAGE. Bound proteins were eluted with 40 µL of 12 mM HEPES, 90 mM NaCl, 20 mM GSH, pH 7.5 for 15 min at +4°C, collected as above, and supernatants were sampled for SDS-PAGE.100 µL aliquots of GSH-sepharose 4B  matrix slurry were washed twice with deionized water. The matrices were collected in between washes by centrifuging at 300 We used the constructs GDAP1∆295-358, GDAP1∆303-358, and GDAP1∆319-358 to get detailed insights into human GDAP1 structure and potential functions . Notablym by ∼3 °C. Due to its limited solubility, HA was titrated up to 250 μM, and a concentration-dependent Tm shift was observed  and −14.8 kcal/mol , respectively. The results suggest that the GDAP1 interface is part of the biological assembly rather than an artifact in vitro, despite having a small interface area. The above observations are consistent with the lack of GST activity in GDAP1, as the canonical dimerization mode generates the active site with sites for substrate binding. We could not detect any activity toward the conventional GST substrates CDNB, NBC, and EPNP, even at high protein concentration  from one monomer into a hydrophobic pocket on the second one, formed by five conserved residues on helices α4 and α5 . A CMT-related mutation cluster of GDAP1 mainly localizes on helices α3 and α6, and less on helices α7, α8, and their connecting loops , and it may play a role at the interface between mitochondria and the ER . GDAP1-l2+ homeostasis, GDAP1 may regulate this metabolism through its binding to fatty acids.Fatty acids are a source of metabolic energy and function as building blocks for complex lipids. GDAP1 could be a fatty acid transport protein due to its localization on MAM and MOM and its fatty acid binding shown here. Moreover, since both GDAP1 and HA are linked to Cain vivo. As shown by earlier studies from neuronal cell line protein extracts (via an unknown mechanism (Another aspect arising from these findings is the oligomeric state of GDAP1 extracts , GDAP1 sechanism . Thus, tThe crystal structure of the dimeric GDAP1 core domain lacks the HD and TMD, but full-length GDAP1 does form dimers in cells. Co-immunoprecipitation of full-length GDAP1 from HEK-293T cells confirmed that the protein formed homodimers . We builvia the incorporation of acidic lipid headgroups in the binding site. These questions can be answered when the structure of full-length GDAP1 on a MOM-like lipid membrane eventually becomes available.The fatty acid-binding site observed in the GDAP1 crystal structure faces the membrane-binding surface in the context of the modeled full-length dimer, suggesting that the observed ligand could mimic the lipid membrane surface. This, in turn, suggests that membrane binding could affect the conformation of the GDAP1 core domain, for example, GDAP1 is a member of the GST family linked to CMT; however, its function remains unclear at the molecular level. The crystal structure of the complete human GDAP1 core domain reveals a GST-like fold, with a previously unseen mode for dimerization. The monomer-dimer equilibrium could be further linked to redox phenomena in the cell, and the function of full-length GDAP1 on the MOM may be regulated by the oligomeric state. The GDAP1 structure and the discovery of the first GDAP1 ligand not only provide information to map the CMT-related residue cluster and the corresponding interactions in detail, but also provides a template conformation for further functional studies and structure-assisted ligand design. Further studies on GDAP1-linked CMT should use the human GDAP1 crystal structure as a reference framework to explain the effects of mutations at the molecular level.


This study aimed to investigate the relationship between total serum testosterone level (TT) and metabolic syndrome (MetS) among adult female population. Subgroup analysis further stratified the population by menopausal status to address the potential hormonal difference in postmenopausal women.A total of 1966 participants from the National Health and Nutrition Examination Survey (NHANES) 2011–2012 cycle was included for analysis in this study. MetS was defined based on the National Cholesterol Education Program Adult Treatment Panel III guidelines. Serum TT was collected during the physical examination of the NHANES program and divided into quartiles (Q) in this analysis. Menopausal status was determined based on NHANES Reproductive Health Questionnaire. Logistic regression models were applied for analysis.The odds of MetS in Q2: 12.99–19.38 ng/mL , Q3: 19.39–28.38 ng/mL , and Q4: ≥28.40 ng/mL  were statistically lower compared to the reference Q1: <12.99 ng/mL. For the postmenopausal group, a significantly lower odds of MetS was observed in the Q2  and Q4 , while the odds of Q3  and Q4  were significantly lower than the reference Q1 in the premenopausal group.Elevated TT levels are associated with incremental reductions in the odds of metabolic syndrome among adult females. Although, serum testosterone level is associated with the occurrence of metabolic syndrome in both the postmenopausal and the premenopausal group, the patterns of the relationship are different. Metabolic syndrome (MetS) is a multiplex risk factor disease characterized by abdominal obesity, insulin resistance, hypertension, and dyslipidemia . In the Although the association between TT and MetS of the adult male population has been comprehensively studied in previous studies, indicating that low concentration of TT level is associated with a higher prevalence of MetS –6, such To our knowledge, only a few researches have examined the association between TT and MetS of the female population –11. ThusThis cross-sectional study analyzed data from the 2011–2012 National Health and Nutrition Examination Survey (NHANES) cycle. The Center for Disease Prevention and Control has conducted the NHANES program since the 1960s, aiming to provide nationally-representative vital health statistics. In each of the two-year cycle, the NHANES recruited approximately 10,000 non-institutionalized civilian resident population in the United States to collect health and nutritional status through home interviews and physical examinations in the Mobile Examination Center (MEC). All NHANES data collection protocols were approved by the National Center of Health Statistics Research Ethics Review Board [In total, 9756 women participated in the NHANES 2011–2012 cycle. Since this study tended to explore the association between TT level and MetS among the adult female population, male participants, and participants who reported less than 20 years old were excluded. Pregnant and breastfeeding participants were subject to the shift in hormone level as well as the effect on the metabolism and were thus excluded in this study , 14. DemThe diagnosis of MetS was determined based on the National Cholesterol Education Program Adult Treatment Panel III guidelines . The comBiospecimens, including blood, urine, oral rinse, and vaginal swabs, were collected during the MEC examination for laboratory analysis. The blood sample was used to assess the testosterone level as well as the indicators of MetS. The NHANES collected serum TT as an indicator of the testosterone level. Since the bound and unbound testosterone level were not separated in the database, this research used TT as an evaluation of the testosterone level. As indicated in the NHANES Laboratory Procedure Manual, routine quantitation of TT was based on the National Institute for Standards and Technology’s reference method, which is a validated isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method .Menopausal status was assessed based on the NHANES Reproductive Health Questionnaire, where RHD042 asked, “What is the reason that {you have/SP has} not had a period in the past 12 months?” Participants who answered “Menopause” or “Hysterectomy” were categorized as the postmenopausal group.2), and Fisher’s exact test when appropriate. Continuous variables were tested for normality using the Shapiro-Wilk test. Mann-Whitney U test was applied to compare variables with abnormal distribution. Summary statistics presented the mean and standard deviation of the continuous variables (Mean ± SD), and frequencies and percent distributions of categorical variables (N%). Both unadjusted and adjusted logistic regression models were used to acquire the Odds Ratio (OR), 95% Confidence Interval (CI), and statistical significance of MetS and each component in the overall population. Additionally, subgroup analyses were performed to investigate both the postmenopausal and premenopausal groups using logistic regression models similar to the overall population analyses. The NHANES cycle oversampled a certain group of minorities to increase the representativeness of the sample. In the 2011–2012 survey year, the oversample domains were non-Hispanic black persons, non-Hispanic non-black Asian persons, Hispanic persons, and low- and non-low-income groups. The full sample 2-year MEC examination weight (WTMEC2YR) found in the NHAENS database was applied to all analyses in this study. All statistical analyses were performed using SPSS Statistics 23.0  and SAS 9.4 . A P value of less than 0.05 is defined as significant.The demographic characteristics of the study population were compared using independent samples t-test, Pearson’s chi-square test , which ranged from < 12.99 ng/mL in Q1, 12.99–19.38 ng/mL in Q2, 19.39–28.38 ng/mL in Q3, and ≥ 28.40 ng/mL in Q4. There were 571 women (28.6%) categorized in the MetS group and 1,395 (71.39%) women in the non-MetS group.After excluding males (n = 4856), participants aged less than 20 years old (n = 2080), pregnancy and breastfeeding (n = 10), and participants with missing data in age, sex, BMI, race, menopausal status, and serum TT level (n = 844), a total of 1966 participants were included in the analysis of this study. Of the included 1966 participants, the average age was 49 ± 17 years old, and the average BMI was 29.38 ± 7.33 kg/mWhen comparing the MetS group and the non-MetS group (referred to the control group), age, BMI, race, menopausal status, and TT levels were significantly different Table . Table 1P < 0.001). The MetS group indicated higher rates of overweight (27.32% vs. 28.67) and obese (63.57% vs. 30.97%) than that of the control group . Higher percentages of Hispanic and Non-Hispanic white participants were discovered in the MetS group than the control group . The menopausal status of the MetS group and the control group were statistically different , with the MetS group illustrating a higher proportion of participants classified as postmenopausal than the control group (66.9% vs. 43.1%). Additionally, the distribution of TT level was different between the MetS and control group, with more participants falling in the lowest quartile (34.85% vs. 21%) and the second quartile (26.9% vs. 24.52%) than the control group .The percentage of the mid-aged individual (50–59 years old) and elders (60–69 and 70 years old) were higher in the MetS group than the control group , Q3 , and Q4  showed a statistically significant lower odds of MetS when compared to Q1  was the reference group. For the overall population, participants in Q2 , Q3 , and Q4 , demonstrating a significant decrease in the occurrence of MetS. When analyzing the waist circumference, participants in Q3  and Q4  indicated a significantly lower odds of elevated waist circumference (> 88 cm) than the reference group in the unadjusted model. However, the significance did not retain after controlling for age, BMI, race, and menopausal status. Similarly, a statistically lower occurrence of elevated blood pressure was observed across all quartiles of the unadjusted model, but not in the adjusted logistic regression model.After adjusting for age, BMI, race, and menopausal status, the relationship remained consistent, with all three quartiles, Q2 , Q3, and Q4 , as well as the adjusted model, Q2 , Q3 , and Q4 . Decreased occurrence of low HDL-C (< 50 mg/dL) was detected among participants in Q2  and Q3  in the unadjusted model, while the Q2 , Q3, and Q4  in the adjusted model demonstrated an incremental reduction in the odds of low HDL-C. When analyzing the odds of high serum glucose (≥ 110 mg/dL) using the unadjusted model, the odds in Q2 , Q3 , and Q4  were significantly decreased as compared to Q1. However, only Q4  was found to decrease when controlling for covariates.The odds of having elevated serum triglyceride (≥ 150 mg/dL) significantly declined in all three quartiles of the unadjusted model, Q2 . In the logistic regression model adjusting for age, BMI, and race, both participants in Q2  and Q4  indicated significantly lower odds of MetS.Subgroup analysis was conducted to explore the effect of TT on MetS and its components in both the postmenopausal (n = 984) and premenopausal (n = 982) groups, as summarized in Table P < 0.001) and Q4  were significantly lower than Q1. Similarly, the odds of Q3  and Q4  were significantly lower than Q1 when adjusting for age, BMI, and race.In the premenopausal group, the results were consistent in both the unadjusted and adjusted logistic regression models. In the unadjusted model, the odds of Q3 and Q4  of the unadjusted model, while significance was detected in all Q2 , Q3  and Q4  when controlling for age, BMI, and race. Similarly, a reduction was also observed in the Q3  and Q4  in the uncontrolled analysis of the premenopausal group. After adjusting for age, BMI, and race, only the third quartile Q3  showed a reduction in high serum triglyceride odds.When investigating the odds of elevated waist circumference (> 88 cm), no statistical difference was found across all quartiles when compared to the reference, which remained consistent in both the postmenopausal and premenopausal group. In the postmenopausal group, a significantly reduced occurrence of elevated serum triglycerides (≥ 150 mg/dL) was detected in Q3  of the postmenopausal group showed a lower OR of reduced HDL-C (< 50 mg/dL) using the unadjusted model, while both Q2  and Q4  showed a decreased OR after adjusting for age, BMI, and race. In the premenopausal group, a reduced occurrence of low HDL-C level was detected in both the Q3  and Q4  of the uncontrolled model and the Q3  and Q4  of the controlled model.Comparing to the reference group, the Q4  was significantly lower than the reference group in the unadjusted model. For the high serum glucose, the odds of all three quartiles were not statistically different from the reference in the postmenopausal group. However, discrepancies were detected in the unadjusted model of the premenopausal group, with Q3  and Q4  odds being significantly lower than the reference group. When adjusting for age, BMI, and race, the odds of Q3  and Q4  were significantly lower than the reference.No difference in the odds of high blood pressure was discovered in the postmenopausal group. In the premenopausal group, only the OR of Q4  in the adult female population, this study analyzed data from the 2011–2012 NHANES. Age, BMI, race, and menopausal status are associated with MetS , 18, 19,As the TT level increased, we detected an incremental reduction in the odds of MetS independently from age, BMI, race, and menopausal status. When comparing to the reference TT level (< 12.99 ng/mL), the TT level at ≥ 28.4 ng/mL indicated the most decrease in the OR, followed by the TT level at 19.39–28.38 ng/mL and TT level at 12.99–19.38 ng/mL, corresponding to 46.4%, 42.5%, and 31.3% reduction in the occurrence of MetS. The inverse relationship between TT level and MetS discovered in our study is in accordance with previous observational studies in the male population –23.In addition to the odds of MetS, we analyzed the impact of TT on the components of MetS to consider the underlying mechanisms of TT on the development of MetS in the female population. Interestingly, TT level was only associated with certain components of the MetS, including serum triglycerides, HDL-C, and serum glucose. Decreased odds of high serum triglyceride level and low HDL-C level were detected in an incremental pattern. TT level at 12.99–19.38 ng/mL, 19.39–28.38 ng/mL, and ≥ 28.40 ng/mL corresponded to a 34.9%, 45.9%, and 51.2% decline in the occurrence of elevated serum triglyceride. Similarly, a 28.3%, 38.2%, and 48% reduction in the odds of low HDL-C level was found at TT level 12.99–19.38 ng/mL, 19.39–28.38 ng/mL, and ≥ 28.40 ng/mL, respectively. For high serum glucose levels, only the highest TT level showed a significant decrease (47.5%) in the OR compared to the reference.The results of the present study indicate that higher serum TT ameliorates lipid and glucose metabolism-related MetS components more profoundly than other components, such as hypertension and waist circumference, in the female population. Nevertheless, previous observational studies in the male population revealed that TT level was correlated with all MetS components, including reduced total cholesterol, reduced systolic and diastolic blood pressure, lower blood glucose, and lower waist circumference , 24, 25.To address the potential hormonal disparities between postmenopausal and premenopausal participants, subgroup analysis stratified by menopausal status was performed in this research. The postmenopausal and premenopausal groups are associated with MetS odds as the TT level elevates. In the postmenopausal group, a decline in the odds of MetS was discovered at both TT level 12.99–19.38 ng/mL (31.1%) and ≥ 28.40 ng/mL (39.4%.), while no significant decrease in OR was detected at TT level 19.39–28.38 ng/mL. When investigating the components of MetS in the postmenopausal group, only high serum triglyceride and low HDL-C were related to the increase of TT level. TT level at 12.99–19.38 ng/mL, 19.39–28.38 ng/mL, and ≥ 28.40 ng/mL resulted in 32.1%, 41.9%, 61.9% reduction in the occurrence of high serum triglyceride. The odds of decreased HDL-C were 34.4% and 38% lowered when the TT level increased to 12.99–19.38 ng/mL and ≥ 28.40 ng/mL as compared to the lowest TT level.In the premenopausal group, TT level at 19.39–28.38 ng/mL and ≥ 28.40 ng/mL were related to lower occurrences of MetS. However, the reduction tended to be decremental, with the third quartile illustrating a 56.1% reduction and the fourth quartile demonstrating a 53.6% decrease. Elevated serum triglycerides, reduced HDL-C, and high serum glucose were associated with the increasing TT level in the premenopausal group. The occurrence of high serum triglycerides was 44.0% lower than the reference at TT level 12.99–19.38 ng/mL but not at other TT levels. TT level at 19.39–28.38 ng/mL and ≥ 28.40 ng/mL led to a 50.3% and 55.8% incremental reduction in the odds of decreased HDL-C level, as well as a 45.2% and 71.4% increase in the occurrence of high serum glucose.Although TT serves as an independent predictor of MetS in both the postmenopausal and premenopausal groups, the findings of this research demonstrate disparities in the patterns and odds of the MetS components, as well as odds of low HDL-C level. Additionally, TT is a stronger predictor of the odds of high serum triglycerides in the postmenopausal group as compared to the premenopausal group. Conversely, a more potent relationship exists between TT and the odds of hyperglycemia in the premenopausal group when comparing to the postmenopausal group.The analysis of the components of the MetS uncovers a more detailed impact of TT on MetS. Furthermore, we use population-based nationally representative data from the NHANES database, making the results of this research generalizable to the U.S. population. To our knowledge, this is the first study that uses generalizable large sample to analyze the relationship between TT level and MetS. Although a correlation is unearthed, this observational study cannot establish a causal relationship between the predictor and the outcome. Hence, the direction of the relationship between TT level and MetS is not ascertained in our research. Notably, research has provided compelling evidence that MetS patients are predisposed to lower TT levels due to hyperglycemia and obesity , 28, impFor males, the TT level below 300 ng/dL is considered low, according to the American Urologic Association , 32, illMoreover, Sex hormones-binding globulin (SHBG) and human serum albumin binding protein, the testosterone cognate binding proteins, are essential in regulating circulating testosterone distribution . TestostThe efficacy of testosterone therapy in ameliorating the components of MetS has been justified in multiple studies of the male population , 25. NevOur findings show the permissive role of serum testosterone in the pathogenesis of MetS in the adult female population. In this research, an increased serum TT level is accompanied by an incremental decrease in the odds of MetS, with TT level at ≥ 28.40 ng/mL showing the greatest reduction, 46.40%, as compared to TT level at < 12.99 ng/mL. Two components of the MetS, elevated serum triglyceride and decreased HDL-C level, are strongly associated with the rising of TT. Serum TT levels affect MetS events in both postmenopausal and premenopausal groups, but the patterns are different between the two groups. Future studies are necessary to determine the optimal range of TT level for women to minimize the development of MetS and capture more integrated mechanisms for clinical implications.


In vivo application of GM-CSF lead to enhanced production of diphtheria-specific antibodies as well as more diphtheria-specific CD4+ T cells following vaccination with multivalent tetanus/diphtheria vaccine. In contrast, the humoral and cellular immune response to the tetanus component was unaltered. Furthermore, application of GM-CSF resulted in more splenic CD11b+ dendritic cells (DCs) with a higher MHC-II expression. GM-CSF also induced a stronger recruitment of CD11b+ DCs to the injected muscle. Most remarkably, GM-CSF was able to boost the diphtheria-specific immune response to the multivalent vaccine in aged mice. This study demonstrates that local administration of GM-CSF is able to improve immune responsiveness to the diphtheria component of multivalent tetanus/ diphtheria vaccine in young and old mice. This information could be useful for the future design of vaccines for the elderly.Multivalent tetanus and diphtheria toxoid containing vaccines belong to the most frequently applied vaccines. However, there is an imbalance in the degree of protection against the two antigens with insufficient long-term protection against diphtheria, particularly in the elderly population. We have previously reported a positive correlation between granulocyte macrophage-colony stimulating factor (GM-CSF) and the production of diphtheria-specific antibodies. Therefore, in the present study we analyzed the effects of  Diphtheria vaccines belong to the most frequently applied vaccines worldwide, with a long history dating back to the 1920s, when Glenny and Hopkins inactivated the diphtheria toxin and showed a high immunity index of the toxoid [+ T cells, and peripheral diphtheria-specific antibodies in adults [Mycobacterium tuberculosis, and many more [Our group has a long-standing history studying tetanus and diphtheria vaccinations in persons of different ages and we have repeatedly shown good protection against tetanus, while protection against diphtheria was insufficient –5. Studin adults ,13. GM-Cn adults ,12,15. Tany more –22.in vivo effect of GM-CSF on immune responses to diphtheria and tetanus immunization in young and aged mice using multivalent vaccines containing diphtheria toxoid, tetanus toxoid, acellular pertussis, and inactivated polio virus, to model the human immunization schedule.The goal of the present study was to investigate the 22.1ad libitum, and kept under specific pathogen free conditions. All animal protocols were approved by the Federal Ministry of Science, Research and Economy of Austria, and carried out in accordance with the Austrian law for animal protection and the institutional guidelines at the University of Innsbruck.Eight week and 17 month old C57BL/6JRj male mice were purchased from JANVIER LABS . Mice were provided standard food and water 2.2Bordella pertussis (25 μg pertussis toxoid (PT), 25 μg pertussis filamentous haemag-glutinin (FHA), and 8 μg pertactin), and three types of inactivated polio viruses  per single human dose (0.5 mL), or 30 μL of diluted Boostrix Polio® containing per single human dose (0.5 mL) not less than 2 IU diphtheria toxoid, and not less than 20 IU tetanus toxoid, three purified antigens of Bordella pertussis  and the same three types of inactivated polio viruses as included in Infanrix® IPV at the same dosages (GlaxoSmithKline) i.m. into the Musculus biceps femoris of the right or left leg. For all vaccinations, Infanrix® IPV from the same batch was used. The same was true for Boostrix Polio®. Both vaccines were diluted 1:2.4 with phosphate buffered saline . GM-CSF-treated mice received 100 ng of recombinant mouse GM-CSF  diluted in 20 μL PBS with 0.2% Bovine Serum Albumin , on the day of vaccination and the consecutive three days, in the same injection site as the vaccine. The control mice received a placebo consisting of 20 μL PBS with 0.2% BSA. Two different vaccination schemes were applied. Mice received either three shots of Infanrix® IPV within 9 weeks, and one shot of Boostrix Polio® 9 months thereafter  and serum was separated according to manufacturer’s instructions.Mice were vaccinated with either 30 μL of diluted Infanrix® IPV containing not less than 30 IU diphtheria toxoid, and not less than 40 IU tetanus toxoid, three purified antigens of ereafter . Alternaereafter , and spl2.3ELISA analysis was performed as previously described in detail . Adjustm2.44CL, 10 mM KHCO3, 0.1 mM EDTA pH 7.4; all Merck KGaA) at RT.Spleen, lymph nodes, and muscle tissue were digested with Liberase™ Research Grade  DNAse I (Roche) for 30 min, smashed through a Falcon® 70 mm cell strainer  and washed with RPMI1640 with L-Glutamine and 25 mM Hepes (Lonza) supplemented with 10% fetal calf serum . Muscle tissues of the mice were scaled before cell isolation. Isolated cells from the spleen underwent erythrocyte lysis by incubating them for 5 min in 10 mL lysis buffer  at 37 °C for 6 h, with 10 μg/mL Brefeldin A (Merck KGaA) added after the first hour of stimulation. Live/dead staining was done with Zombie Violet™ Fixable Viability Dye (Biolegend). Surface staining was done with anti-CD3-BV510™  and anti-CD4-PE/Cy7 . Intracellular staining was done with anti-IFN-γ-PE , anti-TNF-α-FITC , anti-IL2-APC , anti-IL4- FITC , anti-IL6-FITC , anti-IL10-APC .Cytokine production of diphtheria- or tetanus-specific CD4To study DCs, live/dead staining was done with Zombie Violet™ Fixable Viability Dye and isolated cells were stained subsequently with anti-CD3-PerCP-Cy™5.5 , anti-CD11b-APC-Cy™7 , anti- CD11c-BV510 , anti-CD19-PerCP-Cy™5.5 , anti-CD40-PE-Vio770 , anti- CD80-APC , anti-CD86-APC , anti-CD274-PE , anti-MHC-II-FITC , and anti-NKp46-PerCP-eFluor™710 .All flow cytometry data were acquired using the FACSCanto™ II cytometer (BD Biosciences), exported as FCS3.0 files and analyzed using the FlowJo® software version 10.0.7 .2.6+ DCs was determined using CountBright™ absolute counting beads  as described in the user’s manual, 140 μL of cell suspension from lymph nodes and 190 μL of cell suspension from muscle tissue were stained with antibodies for flow cytometry. Immediately before the cells were acquired at the flow cytometer, 20 μL of counting beads were added. The CD11b+ DC concentration per mL and mg muscle was calculated according to this formula: The total number of CD11bThe DC concentration per lymph node was calculated according to this formula: 2.7Group wise comparisons of diphtheria- and tetanus-specific antibodies were performed with two-way ANOVA for repeated measurements, using treatment and time as independent factors and antibodies as dependent variable. The main effect of treatment was reported.p-values were adjusted with Bonferroni-Holm correction.A general linear model for repeated measurements was used to study the main effect of age on the production of diphtheria- and tetanus-specific antibodies following vaccination. Treatment and age were thereby used as ‘between-subjects’ and time as ‘within-subject’. Only the main effect of age was reported. Further subgroup analysis was done with two-way ANOVA for repeated measurements using treatment and time as independent factors and antibodies as dependent variable. Only the main effect of treatment was reported. Because of multiple comparisons, Mann-Whitney U test was applied for all other statistical comparisons. The level of significance for all tests was considered α = 0.05.IBM® SPSS® Statistics Ver.24.0.0.0  was used for the general linear model and GraphPad Prism® Ver.5.01  was used for all other statistical analyses and graphical representations.33.1in vivo application of GM-CSF influences the production of diphtheria- and tetanus-specific antibodies after vaccination. Young mice were vaccinated i.m. with commercially-available multivalent vaccines containing diphtheria and tetanus toxoid, according to the vaccination scheme shown in The first objective was to test if After the first three vaccine shots, GM-CSF-treated mice had higher levels of diphtheria-specific antibodies when compared with the placebo group. Subsequent to reaching an antibody-plateau 14 days after completing the primary vaccination series, the placebo-group exhibited a faster decline of diphtheria-specific antibodies than the GM-CSF group . Both tri.m. with Infanrix® IPV with simultaneous application of GM-CSF or placebo, and were sacrificed either 7 days after the second, or 7 days after the third shot . Diphtheria-specific antibody concentrations in old GM-CSF treated mice were still lower than that of young GM-CSF treated mice. Again, GM-CSF application had no influence on the production of tetanus-specific antibodies, as there was no difference in tetanus-specific antibody levels between the placebo- and the GM-CSF-treated group in both, young and aged mice (GM-CSF- and placebo-treated young mice had similar diphtheria-specific antibody titers until the third vaccine shot. Thereafter the GM-CSF group had significantly more diphtheria- specific antibodies compared to the placebo group . Exactlyged mice . Interes4+ T cells and diphtheria-, but not tetanus-specific antibodies. We were thus wondering whether in vivo administration of GM-CSF would affect the immune responsiveness to the diphtheria-component in multi-valent vaccines. We now showed for the first time that in vivo application of recombinant GM-CSF boosted the cellular as well as the humoral immune response to diphtheria, while the response to tetanus was unaltered. Remarkably, we also succeeded in improving the relatively low immune response to diphtheria in GM-CSF treated-aged mice to the same level as the one of placebo treated-young mice. However, there was still a difference between young and old GM-CSF treated mice.Multivalent vaccines have the advantage of a reduced number of injections and doctor’s visits for immunizations. In the case of multivalent tetanus/diphtheria vaccines, it has been demonstrated that adults are more often unprotected against diphtheria than against tetanus –5. This Chlamydia trachomatis, and HIV [C. trachomatis when applied at the same site as the antigen [Mice had increased diphtheria-specific antibody concentrations when GM-CSF was applied in addition to a multivalent tetanus/ diphtheria vaccine. GM-CSF has not yet been combined with diphtheria vaccine or multivalent vaccine, but with monovalent vaccines where GM-CSF was either incorporated into the vaccine formulation or applied together with the vaccine. GM-CSF induced increased antibody titers to H5N1 influenza virus,  and HIV ,26,22. G antigen . In this antigen . This al antigen . Due to + T cell response. Mice vaccinated with GM-CSF had increased proportions of diphtheria-specific CD4+ T cells producing IL-2, IL-6, and TNF-α compared to placebo-treated mice, whereas again, there was no difference in the proportion of tetanus-specific CD4+ T cells. IL-6 is beneficial for the generation of T follicular helper cells and thus indirectly supports the production of high affinity antibodies by plasma cells [+ T cell responses [GM-CSF also stimulated the diphtheria-specific CD4ma cells –31. Furtma cells . Both ILma cells . Our datesponses ,20.+ DCs in the spleen of GM-CSF-treated mice, which also had a higher expression of MHC-II. Upregulation of MHC-II expression is known to occur with DC maturation, and is essential for antigen presentation to CD4+ T cells [+ DCs to the injected muscle immediately after vaccination was also increased by GM-CSF treatment, indicating that GM-CSF exerted a strong chemotactic effect on DCs when co-applied with diphtheria toxoid. This is in accordance with previous reports which emphasized the importance of GM-CSF as a stimulator of antigenic response by supporting the recruitment and migration of leucocytes, and in particular DCs, to the local administration site [GM-CSF is also a good stimulator of DCs ,35. Henc T cells . The recion site ,19.The fact that GM-CSF is improving the diphtheria-, but not the tetanus-component of the multivalent vaccine is of interest, as there is no urgent need to boost protection against tetanus either in young or in elderly adults –5. HowevIn summary, our study demonstrates that local administration of recombinant GM-CSF can improve the immune response to the diphtheria-component in a multivalent tetanus and diphtheria toxoid containing vaccine, while the response to the tetanus-component remains unaltered. The exact mechanism is still unclear, but it is most likely DCs that are primarily affected. Our results are also promising in terms of future vaccine development customized for the elderly, in which only single components of multivalent vaccines may need to be improved.


Object: Primary osteoporosis (PO) is the most common bone disease, which is characterized by decreased bone mass, damage of bone tissue microstructure, increased bone fragility, and is prone to fracture. Gut microbiome may be involved in bone metabolism of PO through gut–brain axis regulation of immune system and endocrine system, however, the specific mechanism is still unclear. The purpose of this study was to characterize the gut microbiome of patients with PO and its possible role in the occurrence and development of the disease.Methods: Fecal samples were collected from 48 PO patients and 48 healthy controls (HC). The composition of gut microbiome community was analyzed by 16s rDNA amplification sequencing, and the difference of gut microbiome composition between PO patients and HC individuals was compared. PICRUSt was also used to predict the biological function of gut microbiome in patients with PO, and to explore its possible role in the occurrence and development of this disease. The classification model is constructed by random forest algorithm so as to screen the key biomarkers.Result: The diversity of gut microorganisms in PO patients was significantly higher than that in HC group (p < 0.05) and there was significant difference in microbial composition in PO group. The abundance of Dialister  and Faecalibacterium  were significantly enriched which were the key flora related to PO. Although no significant correlation between bone mineral density and the richness of microbial communities are found, PICRUST results show that there are a wide range of potential pathways between gut microbiome and PO patients, including genetic information processing, metabolism, environmental information processing, cellular processes, human diseases, and organic systems. Notably, the discriminant model based on dominant microflora can effectively distinguish PO from HC (AUC = 93.56).Conclusions: The findings show that PO is related to the change of gut microbiome, especially the enriched Dialister and Faecalibacterium genera, which give new clues to understand the disease and provide markers for the diagnosis and new strategies for intervention treatment of the disease. Osteoporosis, the most common bone disease, which is characterized by decreased bone mass, damage of bone tissue microstructure, increased bone fragility and prone to fracture  and a group of age-and sex-matched HC (48 cases). Based on the in-depth study of the microbial components related to the disease, the differences between the composition of gut microbiome in patients with PO and HC group were obtained, and these data were correlated with clinical parameters. In order to understand the community structure deeply, the function of microbial community in patients with PO was predicted. This study provides a theoretical basis for the relationship between gut microbiome and the incidence of PO, and provides a new strategy for the treatment of patients with PO.The prospective study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University, with all participants signing informed consent forms. A total of 96 stool samples from inpatients in our hospital in 2019 were prospectively collected in this study. All participants were orthopedic inpatients, and the patients were selected according to the exclusion criteria: (1) secondary osteoporosis; (2) incomplete case data or patients and their families could not cooperate to complete the questionnaire survey; (3) age <40 years old; (4) antibiotics, probiotics, probiotics, or symbiosis in the 2 months before stool samples were collected; (5) patients with severe malnutrition, infection, drug use, or alcohol abuse; (6) patients with digestive system diseases; (7) patients with severe systemic diseases, tumors, or other serious primary diseases; (8) previous lumbar and hip surgery; (9) fecal samples were not up to standard . The bone mineral density of spine (BMDs) and bone mineral density of hip (BMDh) were measured by dual energy X-ray absorptiometry   and high efficiency high fidelity enzyme were used to amplify the 16S rDNA V3-V4 region of bacteria. Two percentage of agarose gel was used to detect PCR products. After the PCR products were fully mixed, the PCR products were purified by agarose gel electrophoresis with 1 × TAE concentration of 2%. The target bands were cut and the products were recovered by GeneJET gel  recovery kit. Then the library was constructed by Ion Plus Fragment Library Kit 48 rxns library construction kit , and the constructed library was detected by Qubit quantitative and library detection. After the library is qualified, Ion S5TMXL  is used for computer sequencing according to the recommendation of the reagent.First, the low quality part of reads was cut by Cutadapt (V1.9.1), then the sample data were separated from the obtained reads according to Barcode, and the Barcode and primer sequences were truncated to obtain Raw reads. Finally, the Reads sequence was compared with the species annotation database, the chimera sequence was detected and removed, and the Clean Reads were obtained.All the Clean Reads of all samples were clustered by Uparse software (Uparse v7.0.1001). The sequences were clustered into Operational Taxonomic Units (OTUs), with 97% identity and the sequences with the highest frequency were taken as the representative sequences of OTUs according to the principle of algorithm. Then the species annotation of OTUs sequence was analyzed by Mothur method and SILVA132 SSUrRNA database (Set the threshold to 0.8–1), and the taxonomic information was obtained. At the same time, the bacterial community composition of each sample at each classification level  was calculated. After that, the phylogenetic relationship of all OTUs sequences is obtained by using MUSCLE (Version 3.8.31) software for fast multi-sequence alignment. Finally, the sample with the least amount of data is taken as the standard to homogenize the data of each sample. The homogenized data were followed by sample complexity analysis  and sample comparative analysis .Alpha Diversity is used to analyze the diversity of microbial community in Within-community. The diversity analysis of single sample can reflect the richness and diversity of microbial community in the sample. The composition of microbial community in two groups of different samples was compared and analyzed by Beta Diversity. First of all, according to the species annotation results of all samples and the abundance information of OTUs, the OTUs information of the same classification is combined to obtain the species abundance information Profiling Table. At the same time, using the phylogenetic relationship between OTUs, the Unweighted Unifrac, is further calculated to obtain a distance matrix. Then, the Weighted Unifrac distance is further constructed by using the abundance information of OTUs to Unweighted Unifrac. Finally, the differences of microflora between PO group and HC group were analyzed by means of Principal Component Analysis (PCA), Principal Co-ordinates Analysis (PCoA), Non-Metric Multi-Dimensional Scaling (NMDS), Unweighted Pair-group Method with Arithmetic Means (UPGMA) and Beta diversity index.P-value between each data and microbial abundance. Random forest algorithm was used to construct the classification model and screen the Biomarker that plays an important role in grouping. Based on the analysis of random forests with species abundance, different numbers of species were selected according to different classification levels to construct a random forest model. The important Biomarkers were screened by MeanDecreaseAccuracy and MeanDecreaseGin, and then cross validation conducted for each model (default 10-fold).Spearman correlation analysis was used to analyze the relationship between clinical data and microbial richness, so as to obtain the correlation and significant T-test was carried out between the two groups to find out the species with significant differences between the two groups at each classification level (p < 0.05). The MetaStat method was used to test the species abundance data between groups to get the p-value, and the q-value was obtained by correcting the p-value. Finally, the species with significant differences were selected according to the q-value. LEfSe (LDA Effect Size) was used to compare the two groups to find out the Biomarker with statistical difference between the two groups, which is an analytical tool for discovering and interpreting high-dimensional biometric markers , emphasizing statistical significance and biological relevance, and is able to identify the characteristics of different abundance and associated categories.Anosim analysis was used to test whether the difference between groups was significantly larger than that within groups, so as to judge whether the grouping was meaningful or not, which is based on the rank of Bray-Curtis distance value for inter-group difference significance test, using R vegan package anosim function. PICRUSt can perform metagenome function prediction based on KEGG database according to 16S rDNA sequencing data, which, based on the OTU tree in the Greengene database, and the genetic information on the OTU, infers the gene function spectrum of their common ancestors, and the gene function spectrum of other untested species in the Greengenes database so as to construct the archaeal and bacterial domain full spectrum. The gene function predicts the spectrum, and finally the sequence composition obtained by sequencing is matched with the database to predict the metabolic function of the flora.p < 0.01)  and HC group . The patients of the two groups were matched with regard to the age, gender, body mass index, and eating habits. There was a significant difference in BMD of lumbar vertebrae and hips between patients with osteoporosis and healthy controls ( < 0.01) .P = 0.004) and ACE  and observed_species  suggested that the intestinal microbial diversity in primary osteoporosis was significantly higher than that in HC group (P < 0.001), permutational MANOVA  suggested that there were significant differences between the two groups. Importantly, there was also a illustrious discrepancy in the composition of gut microbiome between the PO group and the HC group  in the Bray-Curtis-based PCA diagram ; at the level of class, order and family, the abundance of Erysipelotrichia group was significantly lower than that of HC group . In addition, at the level of family, Ruminococcaceae and Prevotellaceae were significantly enriched in PO group, while the abundance of Lachnospiraceae in HC group was significantly lower than that in HC group. The abundance of Faecalibacterium , unidentified_Prevotellaceae  and Dialister  increased significantly at genus level in patients with PO. The microflora abundance of Subdoligranulum , Blautia , and unidentified_Erysipelotrichaceae  decreased, and the difference was statistically significant . Compared with HC group, the homologous genes of PO patients increased significantly, including amino acid metabolism; cofactor and vitamin metabolism; nucleotide metabolism; terpenoid and polyketone metabolism; replication and repair, translation, folding, classification, and degradation in genetic information processing; transport and catabolism in cell processes; cell growth and death; biological system; endocrine system; immune system; digestive system; and immune system disease microbial function , and at genus level, Haemophilus was negatively correlated with BMDs and BMDh (p < 0.05), Bifidobacterium was negatively correlated with BMDh (p < 0.05). However, based on the analysis of the key differential flora above, there was no significant difference in the flora associated with BMD at each taxonomic level between PO patients and HC groups (p > 0.05).Since BMD is the basis for the diagnosis of osteoporosis and the severity of the disease can be judged, a correlation analysis was made between the microbial population change of the PO patient and BMD. For example, at the phylum level, Euryarchaeota and actinobacteria were negatively correlated with BMDh  Bioprjoect database with project number PRJNA590676, The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study.GP and FL designed the study. ZXu and ZXi analyzed the data, created figures, and wrote the manuscript. JS and SH collected clinical data. YC, CL, XS, BX, LT, and CG collected stool samples. All authors read and approved the final manuscript.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.