It has been known for several years that limiting phosphorus in the cow diet mitigates its excretion in manure, hence reducing the environmental phosphorus load after manure spreading. The quantity of phosphorus that could be applied in the field is regulated by law in several countries. This is not the same for trace minerals such as cobalt, copper, manganese, and zinc. Nevertheless, if overfed, these last minerals are excreted in manure in great quantities and could accumulate in the soil after manure spreading, which could lead to detrimental environmental effects. However, formulating cow rations according to the mineral requirements is challenging for nutritionists. The aim of this analysis is to compare dietary phosphorus, cobalt, copper, manganese, and zinc concentrations from 100 commercial Holstein dairy herds with the National Research Council recommendations. Phosphorus is included as a point of comparison, as its overfeeding has been well studied compared with other studied trace minerals. The results indicate that, at the median, phosphorus, cobalt, copper, manganese, and zinc were respectively fed 8%, 405%, 52%, 372%, and 65% over the recommendations. This suggests that most nutritionists are aware that precision feeding regarding phosphorus is important for dairy production sustainability. It also shows that the other studied minerals were fed in excess and that some attempts should be made to reduce the mineral concentrations of diets.This analysis is performed to obtain information on the current situation regarding phosphorus (P), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) concentrations in cow diets of commercial dairy herds in Québec, Canada, and to compare them with National Research Council recommendations. Data are collected on 100 Holstein dairy herds in Québec, Canada, and 4430 cows were involved. Rations are analyzed for selected minerals and cow requirements relative to the recommendations were calculated. Median percentages of mineral recommendations fulfilled by forage were 55%, 196%, 54%, 776%, 181%, and 44% for P, Co, Cu, Fe, Mn, and Zn, respectively. Daily dietary concentrations of P, Cu, Mn, and Zn decreased as lactation progressed, whereas Co and Fe were stable throughout lactation. Phosphorus was the mineral fed the closest to the requirements, cows below 21 days in milk were even underfed by 11%. All studied trace minerals were fed in excess for the majority of cows. Cobalt was fed on average 480% above requirements regardless of the stage of lactation. For Cu, Fe, Mn, and Zn, rations for cows below 21 days in milk were fed 23% , 930% (849–1019), 281% (251–314), and 35% (22–47) above the recommendations, respectively, and were closer to the requirements than after 21 days in milk. These results show that most nutritionists are aware that precision feeding regarding P is important to minimize detrimental environmental impacts of dairy production. However, some efforts should be made to limit trace mineral overfeeding to ensure environmental resiliency. It is well accepted that many minerals are required to support vital functions in mammals . For insUnderfeeding minerals could have detrimental effects on animal productivity and health , whereasA survey conducted in Wisconsin herds has shown that 50% and 94% of herds fed rations exceeding Zn and Cu requirements of the NRC, respectively . In a suAll procedures of this study were approved by the Animal Care committee from Université Laval, Québec, Canada following the guidelines of the Canadian Council on Animal Care .12 concentration in milk, to evaluate the Canadian dairy herd improvement model and to assess the nitrogen efficiency of eastern Canadian dairy herds [n = 98) or free-stall (n = 2) barns. The morning milking interval varied from 10.7 to 14.8 h. Total mixed ration (TMR) was used by 31 herds, automatic component feeding system (AFS) by 49, and manual component feeding system (MCF) by 20. This experiment was primarily conducted to evaluate the impact of diet management and composition on vitamin Bry herds ,25,26. THerd visits were conducted during 3 consecutive milkings. At the first visit, which was during the morning milking, all ingredients were sampled as well as the quantities of each ingredient given were recorded, as previously described . In summFeed samples were thawed, then placed in an air-forced oven at 55 °C for 48 h for dry matter (DM) determination and ground at 1 mm. Samples were sent to a commercial laboratory  for analysis by wet chemistry for crude protein , ADF (AFeed ingredients were divided into 9 distinct categories: (1) Forage; (2) Corn grain; (3) Other cereals; (4) Energy commercial supplements; (5) Fat supplement; (6) Soy products; (7) Protein commercial supplements; (8) Minerals and vitamins; (9) Feed additives. To be considered as energy or protein commercial supplements, feeds had to have at least 30% of non-fiber carbohydrates and 30% of crude protein on a DM basis, respectively. Other cereals included oat, barley, wheat, and mixed cereals, whereas feed additives included yeast, antitoxin, and sodium bicarbonate. The percentages of each category listed above in the diet were computed by dividing the daily quantity given of each ingredient or group of ingredients included in the category by the total ration offered on a DM basis using Proc SQL of SAS, version 9.4 . For AFS0.75) × (1 − ℮(−0.192 × (week of lactation + 3.67))); (3) within each herd, average daily gain during the first lactation for primiparous growth requirements = (herd average BW of second parity cows—herd average BW of primiparous cows)/herd average calving interval; (4) mature BW for primiparous P growth requirements = herd average BW of third and more parity cows; and (5) number of days of pregnancy = DIM at the time of sampling—(lactation plus dry period length—282 d of gestation), when (lactation and dry period length—282 d of gestation) was lower than 55 DIM, the cow was considered as non pregnant at the time of the visit and it was hypothesized that the reason for the end of the lactation cycle was due to culling instead of calving. For P, Cu, Fe, Mn, and Zn requirements from NRC, and P requirements from INRA and EAAP, they were first obtained as absorbed mineral requirements and an absorption coefficient was applied to obtain the daily dietary recommendations. According to the NRC models [Dietary requirements of P, Co, Cu, Fe, Mn, and Zn were calculated per cow on the basis of 3 references, i.e., NRC , INRA 3, and EAAp ≤ 0.05.Descriptive statistics were obtained with Proc UNIVARIATE of SAS. As for some herds, especially TMR herds, cows within the same feeding group, often based on DIM, theoretically received the same amount of minerals, it was decided to group cows according to DIM within each herd as follows: (1) at or below 21 DIM; (2) between 22 and 80 DIM; (3) between 81 and 199 DIM and; (4) at or above 200 DIM. These thresholds respectively represent fresh, early-, mid-, and late-lactation periods. Hence, in each herd, there were 4 averaged mineral dietary concentrations, one per DIM category. The DIM category can be considered as the experimental unit. Proc MIXED of SAS was used to assess the impact of the DIM category as the fixed effect on the characteristics of each group, on the dietary recommendations of mineral when a factorial approach is used, on the dietary mineral concentrations, and on the percentage of under or overfeeding minerals relative to the recommendations. A Tukey’s honestly significant difference test was performed when results reached significance or a tendency. Normality was visually assessed with residual plots. This condition was violated regarding dietary concentrations of Co, Cu, Fe, Mn, percentages of dietary Cu, Fe, Mn concentrations relative to the recommendations, and then log transformation was chosen to overcome this issue. Geometric means and a 95% confidence interval from back-transformed data were presented for these variables when indicated in Tables. Significance was declared at n = 1757; AFS, n = 2014; and MCF, n = 659). L, and 37.6% of aNDF on a DM basis. The predicted DMI averaged 23.2 kg/d. A total of 4430 Holstein cows were involved in that cross-sectional study  and averaged 226 (SD: 88) mg/kg of DM (p < 0.0001). No significant effect of DIM categories was observed for the Fe EAAP recommendation regarding the percentage of dietary concentration over the requirements (p = 0.17). Among the studied minerals, Fe was the most overfed according to the NRC and EAAP recommendations , whereas the opposite was obtained regarding INRA and EAAP requirements (The dietary Zn concentration was greater in cows below 21 than above 81 DIM (irements . About 9irements , with a As conducted, the study has some limitations that should be taken into account while interpreting results. For instance, a single sample of each ingredient has been taken for TM analysis in each farm. This assumes that samples were representative of what cows had received the day of the visit. This study also relies on the accuracy of mineral analyses of feed ingredients. In a previous study , authorsRegarding P, Cu, Mn, and Zn dietary concentrations per kg of DM, these decreased as lactation progressed. Among the studied minerals, P was the one closest to the precision feeding concept, especially in early lactation. Regarding other selected minerals, above 75% of cows received a ration with excess Co, Cu, Fe, Mn, and Zn. In addition, Fe and Co were among the most overfed minerals, regardless of lactation stage for Co. One herd was fed a dietary Cu concentration above the maximum tolerable level, and this should be avoided to prevent toxicities. Except for commercial products with mineral addition, forage was the major source of minerals, when disregarding ruminal interaction that could occur and mineral absorption efficacy. Forages were sufficient to fulfill the NRC Co, Fe, and Mn requirements for more than 50% of the participating herds. This paper shows that efforts should be made in commercial dairy herds in Québec, Canada regarding precision feeding of trace minerals. This is of major importance for animal health and also to ensure ecosystem resiliency and sustainability.


Autoantibodies to metabotropic glutamate receptor 5 (mGluR5) are known to be the cause of autoimmune encephalitis, particularly limbic encephalitis, closely related to Hodgkin’s lymphoma (HL). The involvement of peripheral neuropathy is rarely reported. In our case, mGluR5 antibody was found in a Guillain-Barré syndrome (GBS) patient accompanied by severe headache but without neuropsychiatric manifestations or HL. Presenting with severe headache, the patient developed progressive bilateral limb weakness, areflexia, and cranial nerve involvement consisting of eye movement disorder, restricted mouth opening and chewing, bilateral facial paralysis and bulbar palsy. Cerebrospinal fluid (CSF) revealed elevated CSF protein level and normal cell count, known as “albumino-cytological dissociation”. Oligoclonal IgG bands were found in both the CSF and serum. Electrophysiological studies revealed symmetrical sensory and motor neuropathy with a mixture of axonal and demyelinating features. Brain and spinal cord magnetic resonance imaging (MRI), as well as the electroencephalogram, were normal. The mGluR5 antibody was positive in both serum and CSF with a Cell-Based Assay (CBA). The patient responded well to intravenous gammaglobulin therapy, correlated with a reduction of mGluR5 antibody titer from 1:30 to 1:10 in the serum. After 6 months, the patient recovered completely without any sign of recurrence or neoplasm. This first case of mGluR5 antibody-associated GBS accompanied by severe headache shows that mGluR5-associated disorders are not limited to manifestations of limbic encephalitis and HL. As written by the pathologist Ian Carr in 1982, his daughter Jane lost memory gradually, with the final diagnosis of Hodgkin lymphoma (HL) . In 2014st day), conventional painkillers failed to provide significant relief. The pain developed continuously, and severe back and limb pains began to develop with numbness in both distal ends of extremities. On the 2nd day after initial symptoms, the patient started to have blurred vision, slurred speech, difficulty in opening the mouth, weakness in chewing, difficulty in swallowing (dysphagia), bitter taste on the tongue, choking on water, and difficulty in articulating words (dysarthria). On the 4th day after initial symptoms, he consulted the neurology outpatient clinic in our hospital. He denied a history of cranial traumatisms, infectious diseases, or other precipitating factors. He was given a systematic neurological examination, revealing multiple cranial neuropathies, including bilateral III, IV, V, VI, VII, IX and X nerve involvement. Ocular fundi were normal. The muscle strength of his limbs was grade 4-5/5, and the muscle tone and tendon reflexes were normal. Hypoalgesia was not found in the distal extremities, and the remaining physical and neurological examination results were not significant. Notably, massive red rashes and flakes were noticed on both cheeks. On the 4th day after initial symptoms (Admission Day), the lumbar puncture was performed, showing normal opening pressure (130 mm of water), elevated CSF protein level , and normal cell count (0×10^6/L), which is a classical phenomenon called “alb——umino-cytological dissociation”. Oligoclonal IgG bands were found in both CSF and serum, along with negative CSF cytologic examination. IgG and IgM of ganglioside antibodies in serum and CSF including sulfatide, GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GQ1b, GT1a, and GT1b were negative, and CNS demyelinating antibodies  and anti-neuronal antibody , Ri, Yo, and Hu) were also negative. The Cell-Based Assay (CBA) test found that mGluR5 antibodies were positive in serum (1:30) and CSF (1:10)  Figure 1th day, the patient had further deterioration. His muscle strength of lower limbs weakened to grade 3-4/5, with the development of urinary retention. He revealed symmetrical decreased tendon reflexes in the extremities. The brain and spinal cord MRI with gadolinium showed no alteration or meningeal enhancement. There were no abnormalities in complete blood count, erythrocyte sedimentation rate, C-reactive protein, procalcitonin, creatine kinase, creatine kinase-MB, and glycosylated hemoglobin. Hepatitis B, Hepatitis C, HIV, and syphilis-related antibody tests were all negative. The thyroid function test result indicated thyrotropin 0.22uIU/ml↓. Serological antibody testing including ANA, ENA and c-/p-ANCA was negative. Electrophysiological studies revealed symmetrical demyelinating neuropathy with dysfunction of motor and sensory nerve fibers, indicated by reduced conduction velocities, prolonged distal motor latency, increased F-wave latency, reduced motor and sensory-evoked amplitudes, and abnormal temporal dispersion.  and normal cell counts, and the mGluR5 antibody titer of the serum declined to 1:10 . After being discharged from the hospital, the patient was not given sequential long-term immunosuppressive treatment. Also, he experienced complete resolution of symptoms without any sign of recurrence and tumors during a 6-months follow-up.This is the first reported case of GBS associated with mGluR5 antibodies. Till now, only little mGluR5 associated encephalitis has been reported. All reported mGluR5-related disorders were summarized –6: (1) Mth day after initial symptoms), the CSF revealed the classic finding in GBS known as “albumino-cytological dissociation”   . Our patThere are no reports of mGluR5 antibody-associated GBS, only two cases reported that anti-mGluR5 encephalitis patients with cranial nerve involvement besides manifestations of limbic encephalitis , 12. GluSevere headache, extremity, and back pain are outstanding manifestations in our case. Prospective studies have demonstrated that extremity and back pain is severe and highly prevalent throughout the spectrum of GBS, while the headache in the setting of GBS is rare . To our This is the first case of mGluR5 antibody-associated GBS patient accompanied by severe headache, which warns that mGluR5-associated disorders are not limited to manifestations of limbic encephalitis and Hodgkin’s lymphoma, therefore further expanding the phenotypic spectrum of mGluR5-associated disorders. Meanwhile, this article suggests that the scope of antibody detection for ganglioside antibody-negative GBS patients can be expanded, and more work should be done to clarify the relationship between mGluR5 antibody and GBS.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the Second Xiangya Hospital of Central South University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.WY and CW had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. HZ and ZH contributed equally to this work in administrative, technical, or material support. CZ was the joint author. All authors have read and agreed to the published version of the manuscript.This research is supported by the grant of National Natural Science Foundation of China (No. 81801123) to Weiqian Yan and Natural Science Foundation of Hunan Province (No. 2021JJ41055) to Cheng Zhao.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the second experiment, both eyes were given NMDA, while one of the two was additionally given the cannabinoid agonist WIN 55,212-2. Ten days later, animals were perfused and the retinae were dissected as wholemounts and stained with Cresyl Violet. Quantitative analysis revealed that 70% of the neurons in the retinal ganglion cell (RGC) layer exposed to NMDA underwent cell death. The addition of the cannabinoid CB1/CB2 agonist doubled the number of neurons surviving the NMDA treatment. These data provide evidence that cannabinoids, either exogenous or endogenous, may be harnessed to provide protection from neurodegenerative diseases, including glaucoma, and from glutamate-induced, and potentially other forms of neurotoxicity, under chronic or acute conditions.Cannabinoid and glutamatergic signaling systems in the human retina coexist and greatly influence one another. Under glaucomatous conditions, excess levels of glutamate accrete in the retinal ganglion cell (RGC) layer. The present study tests the putative neuroprotective effect mediated by cannabinoids at the CB1 and CB2 receptors. In the first experiment, mice were given intravitreal injections of 160 nmol N-methyl- The optg system . Activatg system , and actg system ,11. CurrIn the past, treatment has focused on lowering intraocular pressure to reduce further damage to the optic nerve head . Howeverin vitro to be neuroprotective of cerebellar, hippocampal and neocortical neurons when challenged with toxic levels of glutamate [in vitro or in vivo. They have, however, been shown to reduce ganglion cell death following optic nerve crush [in vivo against NMDA excitotoxicity.Glutamate is the most common excitatory neurotransmitter in the brain, including the retina, and toxic to neurons , includilutamate . The neuve crush , suggestad libitum. All studies were conducted at Westmont College and were approved by the Review Board in accordance with the principles and procedures outlined in the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals.Experiments were performed on adult male and female C57/BL6 mice approximately 4 months of age, weighing 15–35 g. Mice were given food and water d-aspartic acid (the NMDA condition), and NMDA plus WIN 55,212-2 (the NMDA+WIN condition). WIN 55,212-2 is a CB1/CB2 receptor agonist [Three sterile solutions were administered by intravitreal injection: balanced saline solution (the Control condition), N-methyl- agonist ,38. The Mice were anesthetized by intraperitoneal injection of 0.017 ml/g body weight of a solution containing 1.75% tribromoethanol and 1.75% tertiary amyl alcohol. A topical application of 0.5% proparacaine hydrochloride was administered prior to each intravitreal injection. A small incision was made with a 22-gauge needle in the dorsal limbus, through which a Hamilton syringe was then passed to administer the solution into the vitreal chamber. All eyes were injected with 0.5 µl of solution, performed under visual control using a binocular operating microscope. Occasionally this procedure produced cataracts (less than 5%) due to damage to the lens; these mice were not used in the analysis. In the first experiment, the right eye was injected with saline vehicle while the left eye was injected with NMDA. In the second experiment, the right eye was injected with NMDA and the left eye was injected with NMDA+WIN. For all injections, one dose was applied on day 1.Previous studies have found WIN55,212-2 to affect brainstem activity when applied topically to the cornea  . Mice were perfused intracardially with 15 ml of 0.9% saline solution followed by 60 ml of 4% paraformaldehyde, and the eyes enucleated. The retinae were dissected and mounted on to gelatinized slides, ganglion cell layer up. Several radial cuts were made at the periphery, and the surface was cleaned and flattened with a fine brush. Each retina was immersed overnight in a solution containing 10% formaldehyde and 90% alcohol, being held flat under a coverslip and small weight. Retinas were subsequently hydrated and stained with 0.5–1% Cresyl Violet, dehydrated, cleared in hemo-de, and then mounted under coverslips.2. Morphologically distinguishable glial cells, endothelial cells, and immune cells were excluded from the cell count, but no attempt was made to distinguish between ganglion cells and displaced amacrine cells. Although RGCs predominate in the cells to be sampled, without specific staining methods for RBCs, we cannot rule out that cells other than RGCs were a part of our sample. Student’s one-tailed paired t tests were conducted to compare the effects of the treatments between the two eyes within each group, using a significance level of 0.05.The ganglion cell layer was imaged using a ×40 objective, and cells were counted within a square grid occupying 120 µm × 120 µm. Four such samples were taken at retinal loci midway between the optic nerve head and the retinal periphery in each quadrant of the flat-mounted retina. Individual cells intersecting the top and right edges of the grid were included in the counts, while cells intersecting the bottom and left edges were excluded. Quadrant counts were then averaged and corrected to obtain an average estimate of cell density for each eye, in cells per mm2, while paired eyes exposed to NMDA averaged 1898 cells per µm2. This difference is significant , being a 70% loss of cells during the 10-day treatment period. This compares well with the data of Li et al. (1999) [2 were seen in the NMDA-treated eyes, while an average of 4555 cells per µm2 were counted in the paired eyes receiving NMDA and WIN 55,212-2 simultaneously. This result was also significant , indicating that a single application of WIN 55,212-2 was sufficient to nearly double the number of neurons surviving exposure to NMDA. Thus, while NMDA results in a substantial loss of neurons in the ganglion cell layer, the loss is reduced to only 28% following NMDA + WIN treatment.. (1999) , who fouIn 2, and that for the NMDA-injected eye treated with topical WIN55,212-2 was 4088 µm2. These data were statistically significant . The higher value of RGC survival rate in the control eye of the mice administered topical WIN55,212-2 in the contralateral eye may reflect systemic crossover of the cannabinoid  from the contralateral eye that received topical WIN55,212-2.Next we used a CB1 receptor antagonist, AM251A , to dete2 receptor [i/o have been found to be neuroprotective [An endogenous cannabinoid signaling system is conserved in vertebrate retinas, including human ,27,29, areceptor , providereceptor . Indeed,receptor . Cannabiotective . Althougotective  where brin vivo test of a cannabinoid agonist to protect against glutamate excitotoxicity correlates with several in vitro studies on the efficacy of these agents. These studies all found that cannabinoids provided neuroprotection of cultured cortical, cerebellar, and hippocampal neurons [in vivo study suggests that cannabinoids possess great therapeutic potential for slowing the neurodegenerative process of glaucoma and other neurodegenerative disorders that involve excitotoxicity.Our  neurons . The can neurons  to elici neurons . Some st neurons ,34, sugg neurons . Additio neurons . The neu neurons . Muller  neurons ,56, Mull neurons . Ciliary neurons . Endocan neurons , act to  neurons , and to  neurons . The mecFor a substance to have a role as a neuroprotective agent in glaucoma, it would ideally be delivered topically to the eye and usable repeatedly . MinimalSubtleties in the neuroprotective effects observed in our study will require further work. As an example, the neuroprotective actions of WIN55,212-2 at the CB2 receptor may occur only at the onset of neuroinflammation, and not in later phases of the neurodegenerative sequalae . And WINi protein are significantly reduced [in vitro model of neurodegeneration [Neurodegenerative diseases increase with age as the cannabinoid signaling systems in brain decline . For exa reduced ,71. Data reduced  suggest  reduced . Thus reneration . The Magneration  found thneration , moleculneration  and a loneration  may occuneration . Therefoneration ,76, and neration , in concin vivo mouse model. This further indicates the potential for therapeutic applications of cannabinoids in neurodegenerative diseases, including glaucoma.In summary, we have demonstrated that the cannabimimetic drug, the CB1 and CB2 receptor agonist WIN55,212-2, acts to protect RGCs from NMDA-induced excitotoxicity in an


Vitis vinifera by-products: two pomace extracts, white (WPE) and red (RPE), a canes extract (CE), and their combination (CoE), were evaluated, to be included in freeze-drying mouthwashes formulations. The cytocompatibility and anticancerous potential of the four extracts were tested on three cancerous cell lines, as well as the cytoprotective activity against nicotine-induced cytotoxicity and the antioxidant potential determined on a human gingival fibroblasts (HGF) cell line. Additionally, the anti-inflammatory activity and the antimicrobial activity against several microorganisms from the oral microbiome were tested. Freeze-dried mouthwashes with CoE were prepared and characterized, both as lyophilizates and after reconstitution. The four tested extracts showed the highest cytotoxicity on MDA-kb2 cell line. The antioxidant potential was demonstrated for WPE, RPE, CE, and CoE, both in non-stimulated and H2O2 stimulated conditions. The four extracts reduced the levels of proinflammatory cytokines  in a dose-dependent manner, confirming their anti-inflammatory activity. The antimicrobial activity of tested extracts was shown against pathogenic bacteria from the oral microbiome. Mouthwashes of CoE with poloxamer-407, xylitol, and different ratios of mannitol were prepared by freeze-drying leading to porous formulations with interesting mechanical properties and reconstitution times.In this study, the biological activities of four extracts from  Vitis vinifera L. (grapevine) is a plant belonging to the Vitaceae family, which is extensively used in the food and wine industry. This plant and its by-products, mainly grape skins and seeds, pomace, leaves, and canes, are important sources of polyphenols with potential biologic activity such as cardioprotective, antioxidant, anticancer, anti-aging, anti-inflammatory, and antimicrobial effects [ effects . Among t effects .Vitis spp. is often reported in the scientific literature, being attributed mainly to their metal chelating properties, to their ability to scavenge the free radicals, to reduce the hydroperoxide formation or to inhibit the lipid oxidation [Vitis polyphenolic extracts is ascribed rather to the total active ingredients content than to a single component [Vitis vinifera L. include their external use for medicinal purposes in skin cancer prevention [The antioxidant activity of polyphenols extracted from xidation . The antomponent . Due to evention  or for cevention , in skinevention , in skinevention , or to revention .Vitis extracts was reported [The effectiveness of polyphenols from grape extracts in reducing chronic inflammation is also mentioned in several scientific publications, as a result of their ability to modulate the inflammatory pathways along with the decrease of the reactive oxygen species levels . Thus, treported ,10.An important purpose of oral care products is to control the dental plaque, a biofilm in which a complex microbial community develops. Among the antimicrobial substances used to control dental plaque, chlorhexidine is currently used, along with triclosan, essential oils, or stannous and zinc salts. Undesirable effects like tooth staining, diarrhea, vomiting, but also dysbiosis, and bacterial resistance to antiseptic agents, namely chlorhexidine and triclosan, were observed ,12. To rStreptococcus mutans is considered the main microorganism identified in the early development of dental caries, being identified in about 90% of isolates from human caries [Porphyromonas gingivalis, Tanerella forsythia, and Treponema denticola, which form the “red complex”. Other pathogens involved in the progression of the disease are Prevotella spp., Fusobacterium spp., and Parvimonas micra which form the “orange complex”. Porphyromonas gingivalis is considered the main etiological factor which disrupts the relationship between normal and pathogen microbiota of oral cavity, initiates the destructive cascade and leads to inflammation and bone destruction, as it was shown in a murine model for periodontal disease. Porphyromonasgingivalis specific CD4+ T cells predict the onset of the disease, but also its progression, determine the clonal expansion kinetics and the cytokine expression. Treponema spp. Fusobacteriun spp. are pathobionts rather than trigger pathogens [Staphylococcus aureus, Escherichia coli, Candida albicans, and Pseudomonas aeruginosa was previously reported [Vitis vinifera L. against Staphylococcus aureus, Escherichia coli, Candida albicans, and several oral pathogens such as Streptococcus mutans, Enterococcus faecalis, Porphyromonas gingivalis, and Klebsiella spp. was demonstrated [Up to date, the use of grape products in oral care has been extensively reported in the scientific literature. The polyphenols from grape products and by-products can suppress the growth of microorganisms in dental plaque, one of the most important causes of dental caries and periodontal disease. n caries . Anotherathogens . The antreported . In addinstrated .V. vinifera by-products, by investigating the anticancerous, antioxidant, anti-inflammatory, and antimicrobial activity of white pomace extract (WPE), red pomace extract (RPE), and canes extract (CE) from V. vinifera. The second step of the study focused on the preparation of an oral care product, namely a lyophilized mouthwash containing V. vinifera extracts that may be used in the prevention and treatment of different oral health conditions. Thus, the mechanical structure and the reconstitution time of the lyophilizates were investigated. The viscosity and texture profile of reconstituted mouthwashes were further investigated and compared with a commercially available mouthwash. Following the analysis of the results, an optimal formulation was chosen.The present study aimed to evaluate the bioactivities of Vitis vinifera were obtained from the experimental fields of Murfatlar winery company . The red pomace (RP) sample was a mixture of equal parts of vegetable product from Pinot Noir, Feteasca neagra, Cabernet Sauvignon, and Mamaia varieties. The white pomace (WP) sample was a mixture of equal parts of Muscat Ottonel and Sauvignon Blanc varieties, while the canes (C) sample was a mixture of equal parts of all the above varieties. The plant material was dried at room temperature, while being aerated by palletizing to facilitate water removal and avoid microorganism contamination, once every 24 h. Further, it was ground  for 5 min and passed through a 200 µm sieve  [The pomace and canes of some varieties of Germany) . 2O2) 30% solution, N-acetyl-l-cysteine (≥99%), phosphate buffer, resazurin, 2,7 dichloro-fluorescein diacetate (DCFH-DA), and Lipopolysaccharides isolated from E. coli were purchased from Sigma Aldrich . Dulbecco’s modified Eagle medium (DMEM) was purchased from Gibco , and fetal bovine serum (FBS) from Sigma Aldrich . ELISA kits for the quantification of IL-6 and IL-8 were acquired from Biolegend , while IL-1β kit was obtained from Biogems . To test the antimicrobial activity, the following growth media purchased from Oxoid, UK, and prepared according to the manufacturer’s instructions were used: Nutrient agar and Müeller–Hinton agar.Dimethyl sulfoxide (DMSO) (≥99%), hydrogen peroxide , Poloxamer 407 (PX) , xylitol , and distilled water.The materials used in the second phase of lyophilized mouthwash preparation were mannitol ParteckV/V), at a solid: solvent ratio of 1:10 (w/V). After cooling, the samples were filtered through filter paper in a graduated flask, then centrifuged (1930× g) for 20 min and the supernatants were recovered [The extraction conditions were previously optimized through the Design of Experiments methodology . Brieflyecovered . So finaFor the preparation of the lyophilized mouthwash, the combined extract (CoE) was concentrated through rotary evaporation  under reduced pressure at 50 °C and 80 rpm until 33.0313% of the initial weight, and further used for the formulation of the mouthwashes. The extracts used for cell culture experiments were concentrated following the above-mentioned steps and further lyophilized. For each lyophilized extract, a 100 mg/mL stock solution was prepared in dimethyl sulfoxide (DMSO) that was further diluted to obtain the working solutions. These solutions were diluted in cell culture medium to obtain the desired concentrations, ranging from 400 to 10 µg/mL. The characterization phenolic of the WPE, RPE, and CE extracts using LC/MS method was previously reported . The pol2, cells were routinely cultured at 37 °C in a humidified incubator with 5% CO2 supplementation and the medium was changed every 2–3 days. Cells were routinely harvested at 70–80% confluence for subculturing or further use in experiments.The cancerous cell lines A549 (human lung adenocarcinoma), T47D-KBluc (human breast cancer), and MDA-kb2 (human breast cancer) were purchased from American Type Culture Collection . A549 cells were maintained in DMEM medium supplemented with 10% FBS, T47D-KBluc cells in RPMI-1640 supplemented with 10% FBS and MDA-kb2 cells in Leibovitz’s L-15 Medium supplemented with 10% FBS. Normal human gingival fibroblasts (HGF) were purchased from CLS Cell Lines Service, Eppelheim, Germany, and were maintained in DMEM supplemented with 10% FBS. Except for the MDA-kb2 cells that were cultured without additional COexcitation = 530/25; λemission = 590/35, using Synergy 2 Multi-Mode Microplate Reader. The experiments included three biological replicates, each one including six technical replicates and negative control (cells exposed to culture medium containing 0.2% DMSO). The results were expressed as relative values compared to the negative control (100%). The cytotoxicity of the four extracts was evaluated by Alamar Blue (AB) assay using the protocol previously described  after an2O2 in HBSS or HBSS for 2 h. The conversion of CFH-DA to the fluorescent compound dichlorofluorescein (DCF) by ROS was optically measured using Synergy 2 Multi-Mode Microplate Reader at an λexcitation = 485/20 and λemission = 528/20. The potency of the WPE, RPE, CE, and CoE to mitigate the induction of oxidative stress was compared to a standard antioxidant (20 mM solution of N-Acetyl Cysteine (NAC) treatment).The ability of the WPE, RPE, CE, and CoE to reduce the oxidative stress in HGF-1 cells was evaluated using the ROS sensitive dye 2′,7′-Dichlorofluorescin Diacetate (DCFH-DA) as previously described . Briefly50 values . After the incubation period, cells were washed with PBS and further incubated with a resazurin solution of 200 µM for 4 h. For each condition, three biological replicates, each one with six technical replicates were performed and included a negative control (medium containing 0.2% DMSO). The results are presented as relative values compared to the negative control (100%)The cytoprotective effects of the WPE, RPE, CE, and CoE against nicotine-induced toxicity were measured using the AB assay described above based on a previously reported protocol . HGF celThe WPE, RPE, CE, and CoE anti-inflammatory activities were firstly evaluated by quantifying three pro-inflammatory cytokines  from cell culture supernatants using ELISA assays. HGF cells were simultaneously exposed to 100 ng/mL LPS and three non-cytotoxic concentrations of the WPE, RPE, CE, and CoE  for 24 h. To rule out an additive cytotoxic effect of LPS in the presence of the WPE, RPE, CE, and CoE extracts, the cytotoxic effects of the mixtures on HGF cells were first evaluated. Following the 24 h exposure to the mixtures of LPS with extracts, the concentrations of IL-8, IL-6, and IL-1β were measured using commercially available ELISA Kits from a standard curve fitted based on a four-parameter logistic curve according to the manufacturer’s instructions.Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Klebsiella sp., and Citrobacter sp., which can be components of the normal oral microbiota or the periodontopathic microbiota. The antimicrobial activity was tested through a qualitative diffusimetric method, adapted to the disk/well method, which is considered a reliable method with large applicability in clinical practice to test the efficacy of most antimicrobial substances [Vitis vinifera L. pomace and canes and a control sample—the solvent used for extraction. Incubation was performed at 37 °C for 18–24 h, thereafter the diameter of the inhibition zone was measured. The diameter of the inhibition area is correlated with the sensitivity of bacterium to the tested extracts [The following microorganisms were chosen to carry out the test: bstances . AccordiMouthwashes were designed as freeze-dried structures obtained by the lyophilization of CoE solutions that were meant to be reconstituted with a certain volume of water before use. Five solutions (coded F1 to F5) were prepared by using CoE, PX, mannitol, and xylitol. The CoE solutions were poured into 2 mL and 4 mL PVC blister sockets. The amount of CoE needed for each blister socket (0.10 g) was calculated from the concentration that showed the highest anti-inflammatory and antioxidant effects during the in vitro studies. PX, a nonionic surfactant was added at two different CoE:PX ratios, 1:1 and 2:1 respectively, as shown in The textural characteristics of freeze-dried mouthwashes were determined using Brookfield CT3 Texture Analyzer . The samples were carefully removed from alveolae and placed on the rigid test surface (TA-BT-KIT fixture). The measurements were performed during a compression test by using the TA10 probe. During the test, the cylinder probe performed the compression of the sample with a constant speed of 0.1 mm/s and a load of 10 g, until a penetration depth of 5 mm. For each formulation, the determinations were performed in triplicate and the mean value ± standard deviation was reported. The texture profile data was collected using TexturePro CT Software V 1.9  and the following parameters were calculated: hardness of the sample measured as the load value recorded at highest peak during compression, the rigidity of the sample measured at 3 mm depth, and fracturability calculated as the load value recorded at the first fracture. Generally, these characteristics are indicative of the brittleness of the samples.The reconstitution time was measured by placing the sample in 10 mL distilled water, the necessary volume for the reconstitution of the mouthwash. The time elapsed until the complete disintegration of samples when no solid residue was perceived, was measured using a digital stopwatch. The analyses were performed in triplicate and the average times ± standard deviation was reported. ®—F6) containing the same nonionic surfactant was also analyzed to establish the desired quality profile of the final product. Back extrusion test was performed by applying a load of 1 g with a constant speed of 1 mm/s while the probe penetrated the sample to a target distance of 20 mm. During the compression of the sample, several parameters that define the peak force and area of work were recorded. The negative part of the force–time graph describes the sample adhesiveness. The measurements were performed in triplicate and the mean value ± standard deviation was reported.Brookfield CT3 Texture Analyzer  equipped with the TA-DEC probe and TA-BT-KIT was used to determine the reconstituted mouthwash texture. The purpose of the instrumental texture analysis was to provide objective measurements that indicate sensory attributes of the formulation , the varThe viscosity of the reconstituted mouthwashes was determined at 23 ± 0.2 °C and 200 rpm by using a rotational rheometer Brookfield DV-III Ultra  equipped with spindle LV-1. The measurements were performed in triplicate and the mean value ± standard deviation of each determination was reported.p values less than 0.05 were considered statistically significant.The data are presented as mean values ± standard deviation (SD) of at least three biological replicates. The normally distributed data sets were analyzed using one-way analysis of variance (ANOVA) with a post hoc Holm–Sidak test. Unless stated otherwise, data analyses and graphical representation were done using the SigmaPlot 11.0 computer software . Results with a Vitis vinifera extracts was evaluated on three cancerous cell lines including mammary (MDA-kb2 and T47D-KBluc) and pulmonary (A549) derived cancerous cell types in parallel with normal human fibroblasts. In comparison with the normal cell type where only WPE and CoE significantly decreased the viability at the highest tested dose of 400 µg/mL, higher toxicity was observed in cancerous cells  that did not induce statistically significant toxicities after an exposure of 24 h. The assay was performed in two conditions, namely H2O2-stimulated and non-stimulated conditions. In non-stimulated conditions, similar to what was observed for the standard antioxidant NAC, exposure to the extracts resulted in a statistically decrease in ROS that was dose-dependent  was evaluated at 24 h and 48 h using AB assay. For all the extracts tested, a cytoprotective dose-dependent effect was observed starting from the lowest tested concentration of 100 µg/mL at both time points evaluated  in an inflammation-induced cellular model. The pro-inflammatory response was induced in HGF by exposing the cells to a non-cytotoxic dose of lipopolysaccharides (LPS) isolated from E. coli. Exposure of HGF to 100 ng/mL LPS induced a robust inflammatory response, the levels of all three pro-inflammatory cytokines displaying a statistical increase  and 5% (F2) . It was Freeze-drying led to five porous formulations of 25 mm diameter × 15 mm height  and of 18 mm diameter × 14 mm height (F4) which were characterized through texture analysis, for hardness, rigidity, and fracturability . DiffereF3 contained lower ratios of PX when compared to F1 and had very porous and brittle structures that made the extraction from alveolae difficult. Consequently, a significant decrease in hardness and fracturability was recorded, so apparently, PX also had a positive effect on the mechanical properties. F4 had a similar composition to F3, but a volume of 2 mL was poured in each alveolae. This led to slightly increased hardness, rigidity, and fracturability, yet no significant differences when compared to F1. So, the volume of the freeze-dried structures does not seem to have an important impact on the mechanical characteristics. F5 displayed the highest values that define the mechanical profile of the samples; adding 10% of xylitol led to significantly higher mechanical parameters, but the products presented tacky surfaces, thus difficult to handle.Reconstitution time was tested in the volume of water necessary for one application, of 10 mL, and varied between 4.82 and 30.56 s. Significant differences were found between all formulations F2–F5 when compared to F1, because except for F1, all formulations disintegrated rapidly, in less than 10 s. That indicates that the changes performed on the initial formulation, like mannitol increase or adding xylitol led to faster reconstitution, but also consolidated structures with increased mechanical strength. The composition of the lyophilizates determines the texture parameters which are good predictors of the sensory characteristics of the product. So, the texture analysis was performed on the reconstituted samples and finally compared to the ones of a commercial mouthwash. As shown in The low influence of the formulation factors was expected because the added excipients were all highly soluble in water with no impact on texture, but a slight increase of viscosity was obtained when adding higher ratios of polyols . MoreoveVitis vinifera extracts were selected for their vasoconstrictor and tonic effects and were compressed with surfactants, disintegrants and/or effervescent agents [Attempting to formulate solid mouthwashes is not a novel approach, several patents and scientific articles reported the preparation of soluble, dispersible, effervescent tablets used as oral rinses after a prior water dissolution ,59. Somet agents ,61. The t agents . This eft agents . Lately,t agents .Up to this point of the study, the effect of the extracts was demonstrated in vitro, and an optimal formulation was chosen for its potential application, a freeze-dried mouthwash. Of course, this optimization strategy has several limitations, conclusions cannot be established outside the studied formulation domain; however, the best results were obtained for formulation F2, so further studies will follow to evaluate the in vivo effects of the product. Vitis vinifera by-products represent a valuable source of polyphenols that may lead to various health benefits. The current study evaluated the bioactivities of several V. vinifera by-products, namely white pomace, red pomace and canes extract. The anticancerous potential of the four extracts was evaluated on three cancerous cell lines including mammary and pulmonary-derived cancerous cell types, in parallel with normal human fibroblasts. The highest cytotoxicity of the tested extracts was observed in the case of MDA-kb2 cell line, while in the case of A549 and T47D-KBluc cell lines, the anticancerous activities of the extracts were modest. All the extracts displayed an antioxidant potential, anti-inflammatory properties, and an improved protective effect against nicotine-induced cytotoxicity. In addition, the tested extracts had an observable antimicrobial effect on several strains existing in normal oral microbiota or periodontopathic microbiota. The results obtained during in vitro study supported the inclusion of the combined extract, prepared as a mixture of equal parts of the individual extracts, in a lyophilized mouthwash formulation. Following the characterization of the five formulations, an optimal freeze-dried mouthwash with good structural and mechanical properties was chosen. Up-to-date, a limited number of lyophilized compositions for oral care have been reported. As an element of novelty, the current research provides scientific evidence to argue the suitability of these innovative mouthwash formulations in the prevention and the treatment of different oral health conditions, such as periodontal disease.


Extracellular matrix (ECM) synthesis and metabolism abnormalities may influence the pelvic supporting system and lead to the occurrence and development of pelvic organ prolapse (POP). Genetic polymorphisms of such related genes have been increasingly studied. This study aims to explore the association between the single-nucleotide polymorphisms (SNPs) of genes encoding ECM processing enzymes , ECM degrading enzymes  and their tissue inhibitors of metalloproteinase (TIMPs), and POP.We conducted an association study including 48 women with POP at stages III and IV and 48 women without prolapse in Chinese groups. SNPs were identified using the target region sequencing technique. We performed Fisher’s exact tests to assess the association between SNPs and POP in the unadjusted model and logistic regression analysis in the adjusted model, adjusting for delivery and pregnancy.TIMP2 SNP rs2277698 , ADAMTS13 SNP rs149586801 , and ADAMTS1 SNPs rs370850 and rs422803 , rs402007, rs428785, rs434857, and rs445784 , and POP in the adjusted model.There was a significant association between TIMP2, ADAMTS13, and ADAMTS1 might be candidate genes for POP. Our results provide preliminarily new evidence for future investigation of these genes in the pathophysiology of POP.The online version contains supplementary material available at 10.1007/s00192-021-04917-5. Pelvic organ prolapse (POP) is a common pelvic disorder among older women. It is characterized as the downward bulging of uterus, bladder, rectum, etc., urinary and fecal inconvenience, and sexual dysfunction, which negatively affect women’s quality of life and social activities . The preCOL1A1) [COL3A1) [LAMC1) [MMPs) [The etiology of POP is complicated. Race/ethnicity, advancing age, obesity, higher parity, and menopause are known risk factors for POP. In addition, it is well demonstrated that POP has the characteristics of familial aggregation, as the risk of POP in women increases if their mothers or sisters have suffered from the disease, indicating a genetic contribution to POP . PreviouCOL1A1) , 5, coll[COL3A1) , laminin [LAMC1) , and mat) [MMPs) , which aMMP1, 3, and 9 genes, which could alter the expression of these genes and increase the risk of POP [MMP9 SNPs  and found rs17576 to be associated with POP. Wu et al. [MMP9 SNPs in an association study and found two SNPs, rs3918253 and rs3918256, associated with prolapse. Wang et al. [MMP10 SNP rs17435959 was associated with POP in a Chinese group of 91 cases and 172 controls. Besides these limited studies, other MMP members have not been studied in POP.MMPs are a family of multiple catabolic proteases involved in the degradation of collagen fibers and other components of ECM. Several previous studies have identified the existence of polymorphisms in the promoter regions of k of POP –11. Chenk of POP  detectedu et al.  further g et al.  demonstrTIMP2, which has been considered to be involved in connective tissue disorders and vascular diseases.The bioactivity of MMPs is regulated by tissue inhibitors of metalloproteinase (TIMPs), which are also crucial factors of ECM remodeling. Allen-Brady et al.  carried ADAMTSs, known as a disintegrin and metalloproteinase with thrombospondin motif, are a family of proteinases involved in procollagen processing. They can cleave the N-terminal of the peptide chains of procollagen molecules, which are the precursors of mature collagen . Thus, AMMP1, 3, 9, and 10, SNPs from other MMP members, TIMPs, and ADAMTSs would be associated with POP. Using a target region sequencing technique, we (1) confirmed the previous findings on MMPs and (2) investigated the susceptible loci of some of the other MMP, ADAMTS, and TIMP family genes for POP. We believe that these results might offer novel insights into the molecular mechanisms of POP development in Chinese women.As the fine balance between the synthesis and degradation of ECM components is essential to the integrity of the pelvic floor supportive structures, and given the previous limited findings on some of the MMP studies, and also given the fact that there has been no research on the association between the single-nucleotide polymorphisms (SNPs) of TIMPs/ADAMTSs and risks of POP, we have carried out a case-control association study in a group of Chinese women. We supposed that besides This was part of a case-control association study in which we recruited women from Peking Union Medical College Hospital (PUMCH) in Beijing from October 2016 to May 2017. Cases were POP patients diagnosed at Pelvic Organ Prolapse Quantification (POP-Q) stages III and IV from the Department of Gynecology and Obstetrics, and control women were diagnosed with no prolapse and have not suffered from prolapse surgery from the Physical Examination Center. All the participants signed the informed consents, and the study was approved by the Ethics Committee of PUMCH.We excluded women with the well-known connective tissue diseases, including Marfan syndromes, Ehlers-Danlos syndromes, rheumatoid arthritis or scleroderma, and women with neurological diseases including sclerosis or stroke. As the genetic variants vary between different race/ethnic groups, all the samples were limited to Chinese ancestry. Finally, we recruited 48 cases and 48 controls subjected to the next genotyping. Sociodemographic data and physical information were collected as summarized in our previous study .Genome DNA was extracted from the peripheral venous whole-blood samples using a Puregene Blood Kit . The target region sequencing approach was used to achieve a comprehensive assessment of the specific genes; 1–2 μg DNA of each blood sample was sequenced by Agilent Liquid Capture System  at Novogene  based on the Illumina HiSeq 4000 platform to provide a more than 200× deep of sequencing.http://broadinstitute.github.io/picard) were used to sort the files and mark the duplicated reads. Samtools mpileup and bcftools were used to call variants, insertions, and deletions. The SNP quality control included SNPs with read depth > 4, mapping quality > 30, and the variant quality > 20. Variants with a minor allele frequency (MAF) > 5% remained. SNPs were assessed by PolyPhen-2, SIFT, MutationTaster, and CADD software, respectively, for the prediction of their functional effects. ANNOVAR was applied to provide the annotations of the position, type, and conservative prediction of the altered alleles and other information.The valid sequencing data landed in a BAM file by Burrows-Wheeler Aligner (BWA) software based on the reference genome (UCSC hg19). Samtools and Picard . The linkage disequilibrium (LD) graphs indicating r2 between SNPs were drawn by Haploview .Odds ratio (OR) and 95% confidence interval (CI) were shown for each of the variants with OR < 1 indicating a protective effect and OR > 1 indicating a risk effect . P < 0.0P = 0.663) and body mass index (BMI)  between case and control groups. However, the case group had approximately ≥ 1 delivery  and pregnancy  in contrast to the control group. In the case group, 41 women (85.42%) were diagnosed at POP-Q stage III and 7 women (14.58%) were at stage IV as previously described [We have described sample characteristics in our previous study. Briefly, we matched age  and degradation  in Chinese women. These genes are listed in Table P values < 0.1 in the unadjusted model and 16 SNPs with P values < 0.1 in the adjusted model that were considered to be significant or suggestively significant  after the adjustment, which was an intronic SNP. For other MMP members, we have identified two new MMP13 SNPs, rs3758853 and rs78356340, which were intronic SNPs, which showed a suggestively significant association with POP in the adjusted model   and a suggestively significant association with POP in the unadjusted model . TIMP3 SNP rs9862 showed a suggestively significant association with POP in the adjusted model . rs2277698 and rs9862 were synonymous SNPs that did not result in amino acid changes. TIMP4 SNP rs10433537 showed a suggestively significant association with POP in both the unadjusted and adjusted model , which was an intronic SNP . There was also a suggestively significant association between rs1055432 and POP in the adjusted model . rs1055432 was a synonymous SNP that did not lead to the amino acid alteration  in the adjusted model, which was an intronic SNP . They also showed a trend toward significance with POP in the unadjusted model . rs402007, rs428785, rs434857, and rs445784 were respectively significantly associated with POP in the adjusted model . rs402007 was in the 5’UTR region of the ADAMTS1 gene. rs428785 was a missense SNP. rs434857 and rs445784 were synonymous SNPs that did not lead to amino acid changes. There was also a trend toward significance for the SNP rs436525 and POP in the adjusted model . rs436525 was also a synonymous SNPs without amino acid changes (Table ADAMTS1 gene were described. rs436525 was in good LD with six other SNPs (r2 > 0.8), and rs370850, rs422803, rs402007, rs428785, rs434857, and rs445784 were in perfect LD between each other (r2 = 1)  Fig. . The misMMP9 and 10 polymorphisms and further analyzed the association of some other MMP genes, TIMPs, and some ADAMTS genes with risks of POP for the first time. Besides MMP9 and 10, our study provided novel evidence that MMP13, TIMP2, 3, 4, and ADAMTS1, 13, 14 might be possible candidate genes for POP.POP is a common female pelvic disorder due to the decline of the pelvic floor supportive tissues. The imbalance of synthesis and degradation of collagen and other ECM components plays a role in the pathogenesis of POP. Such regulation of ECM stability mainly relied on MMPs, which can cleave collagen, elastin, and proteoglycans, etc., and their inhibitors TIMPs . ADAMTSsMMPs act on ECM breakdown in multiple physiological and pathological processes, including embryonic development, tissue remodeling, wound repair, angiogenesis, inflammatory processes, and tumor progression . AccordiMMP1, 3, and 9 in our target region sequencing approach, we did not verify our data in relation to the previous polymorphisms in the promoter regions of MMP1, 3, and 9. However, we compared our results to the previous MMP9 and 10 studies [MMP9 SNPs, rs3918242, rs17576, and rs2250889, and found that rs17576 G allele was a risk site for POP. The authors recruited 92 POP patients at POP-Q stage ≥ II and 152 control women at stage 0–I of Taiwanese women. Wu et al. [MMP9 SNPs and found rs3918253 and rs3918256 associated with POP in non-Hispanic white women in 239 cases with stages III and IV and 197 controls with stages 0–I. In our study, we did not find the previously reported MMP9 SNPs rs3918253 and rs3918256 by Wu et al. [MMP9 SNPs rs3918242, rs2250889, rs3918278, rs2274755, rs17577, rs2236416, and rs3787268 by Wu et al. and Chen et al., we confirmed that these SNPs also showed no association with POP in our study. Besides validating the previous study, we additionally identified a MMP9 SNP rs3918254 that had a suggestively significant association with POP, which was an intronic SNP. Wang et al. [MMP10 SNP rs17435959 genotype G/C was distributed differentially between 91 women with POP at stage ≥ II and 172 control women at stage 0-I of Chinese ancestry. However, we did not find a significant association in the MMP10 gene in our study. The inconsistency of these studies may partially be attributed to the differences in the recruitment criteria and the characteristics of the women, for example, the race, age, BMI, parity, etc., which were key influencing factors for POP, or in the detection methods.The role of MMPs in the development of POP has been primarily explored in their expression changes as well as their susceptible loci for POP. Previous analysis demonstrated that women with POP showed higher expression levels of MMP1 and MMP8 compared with the control women . The exp studies –14. Chen studies  conducteu et al.  additionu et al.  in signiu et al.  reportedu et al.  and our MMP9 and MMP10, we have sequenced multiple MMP genes and aimed to find the association between these genes and POP. However, we did not observe any positive associations among MMP1, 2, 3, 8, 10, and POP, respectively. Additionally, we have found two new MMP13 SNPs, rs3758853 and rs78356340, which showed borderline significant associations with POP and may have a risk effect for POP.Studies on other MMP polymorphisms have not been conducted in POP. In this study, besides TIMP1, 2, 3, and 4 genes for the first time. We found a significant association between a TIMP2 SNP rs2277698 and POP. Our data suggested that it may show a protective effect for POP development. We also reported a TIMP3 SNP rs9862 and a TIMP4 SNP rs10433537 that had a borderline association with POP, which should be further investigated in future research.There were few studies focusing on TIMP family genes and POP. TIMP2 expression level in the POP group was lower than that in the control group . TIMP3 aADAMTS1 SNPs rs370850, rs422803, rs402007, rs428785, rs434857, and rs445784 and POP, which may be risk factors related to POP, and a significant association between ADAMTS13 SNP rs149586801 and POP, which showed potential protective effects for this disease. We also found three additional SNPs, ADAMTS1 SNP rs436525, ADAMTS13 SNP rs1055432, and ADAMTS14 SNP rs4747097, which have suggestively significant associations with POP. Additionally, ADAMTS1 SNP rs428785 is a missense SNP and can lead to a substitution of alanine to proline, which might exhibit a benign and tolerant effect predicted by PolyPhen-2 and SIFT. Therefore, ADAMTS1, ADAMTS13, and ADAMTS14 could be novel candidate genes for POP, and this needs further validation.The ADAMTS protease family plays crucial roles in ECM remodeling and tissue morphogenesis as well as in inflammation and other physiological and pathological processes . This faMMP9 and 10, but also analyzed the association between MMP1, 2, 3, 8, and 13 and POP for the first time. Additionally, the polymorphisms of TIMP and some ADAMTS family genes have been first studied in POP, suggesting new susceptible SNPs. Third, we focused on unrelated Chinese women from mainland China in this candidate gene association study, choosing extreme symptoms of severe POP with POP-Q stages III and IV, tying to improve the detection of the potential variants.Our study had several strengths. As previous studies were few and focused on certain limited SNPs of a gene, we sequenced several MMP, TIMP, and ADAMTS family genes by target region sequencing technique to assess the entire genes with their coding regions. Second, we not only validated the data of the previously identified SNPs in MMP, TIMP, and ADAMTS polymorphisms for future investigation of the involvement of these candidate genes in the etiology of POP. The genetic contributions to POP remain poorly understood. Additional work needs to be done to provide further validation of POP predisposition variants in a variety of different populations to establish the role of these genes in the pathogenesis of prolapse [However, the main limitation of our study was the small sample size. Since our nationwide epidemiological survey in mainland China found that the prevalence of POP at stages III and IV was 2.04% , this maprolapse .MMP9 and 13, TIMP2, 3, and 4 and ADAMTS1, 13, and 14 genes in Chinese groups, preliminarily revealing the underlying mechanisms for the pathophysiology of this common disease.MMP, TIMP and ADAMTS family genes are crucial for ECM synthesis, modification, and metabolism. In this study, we provide initial evidence that the genetic variants in these genes may have an association with POP. We identified several susceptible SNPs in the Table S1All of the variants with a minor allele frequency of 5% (XLS 66 kb)


Small-for-gestational-age (SGA) infants have been associated with increased risk of adverse perinatal outcomes (APOs). In this work, we assess the predictive ability of the ultrasound-estimated percentile weight (EPW) at 35 weeks of gestational age to predict late-onset SGA and APOs, according to six growth standards, and whether the ultrasound–delivery interval influences the detection rate. To this purpose, we analyze a retrospective cohort study of 9585 singleton pregnancies. EPWs at 35 weeks were calculated to the customized Miguel Servet University Hospital (MSUH) and Figueras standards and the non-customized MSUH, Fetal Medicine Foundation (FMF), INTERGROWTH-21st, and WHO standards. As results of our analysis, for a 10% false positive rate, the detection rates for SGA ranged between 48.9% with the customized Figueras standard (AUC 0.82) and 60.8% with the non-customized FMF standard (AUC 0.87). Detection rates to predict SGA by ultrasound–delivery interval (1–6 weeks) show higher detection rates as intervals decrease. APOs detection rates ranged from 27.0% with FMF to 7.9% with the Figueras standard. In conclusion, the ability of EPW to predict SGA at 35 weeks is good for all standards, and slightly better for non-customized standards. The APO detection rate is significantly greater for non-customized standards. Screenings for fetal growth abnormalities are essential components of antenatal care, and fetal ultrasound plays a key role in the assessment of these conditions ,2,3. SmaThese fetuses are the leading cause of stillbirth ,7,8, andSeveral studies customized or not to maternal and fetal physiological variables have been proposed to predict SGA ; these fFurthermore, new standards have recently been published by EFW , includiSince the controversy arose about the most appropriate method to predict SGA, and the lack of comparative assessment for the cited approaches, the objective of this study is to compare the ability of EPW—according to six growth standards, by ultrasound at 35 weeks, including population, population-customized, and international references—to predict late-onset SGA, defined as a birth weight below the 10th percentile at term delivery. The secondary objective is to determine whether the ultrasound–delivery interval influences the detection rate of SGA newborns.This was a retrospective cohort study of births assisted at the Miguel Servet University Hospital (MSUH), between March 2012 and December 2016. The inclusion criteria were as follows: live singleton pregnancies controlled in MSUH from the first trimester of gestation; fetal ultrasound assessment at gestational age of 35 (range 34–36) weeks; and deliveries between 37 and 42 weeks of gestational age of fetuses without stillbirth associated with malformations or chromosomal abnormalities. Of the 19,310 consecutive deliveries assisted in our hospital in the period studied, the 9585 cases that fulfilled the specific inclusion criteria—such as data availability to estimate percentile weights by standards—were considered for the analysis. Study participants’ selection samples are detailed in The last menstrual period was adjusted by first trimester ultrasound . UniversEFW was calculated with the formula of each standard to which it was built. We used Hadlock et al.’s  formula,For the calculation of the EPW, we collected in the study the maternal age and body mass index (BMI) at the beginning of pregnancy, parity, maternal and paternal height, maternal ethnic origin, smoking habits, infant gender, birth weight, and ultrasound EFW. We also collected perinatal outcomes in order to analyze APOs in SGA infants at delivery, defined as the occurrence of a 5-min Apgar score < 7, instrumental or cesarean delivery for non-reassuring fetal status, arterial cord blood pH < 7.10, and stillbirth.EPWs were calculated according to 6 different customized and NC growth standards, including population, population-customized, and international references. For the customized standards, the methodologies of Hadlock et al.  and GardTo assess ultrasound weight measures in the third trimester, EPWs were estimated between 34 and 36 weeks of gestational age. The WHO EPW was calculated by interpolation of the 5th, 10th, 25th, 50th, 75th, 90th, and 95th percentiles.As a gold standard for the analysis, SGA was defined as a birth weight below the 10th percentile, using a growth reference for the Spanish population based on 9362 birthweights . We did Data were descriptively analyzed using medians and interquartile ranges for continuous variables, and absolute and relative frequencies for categorical variables. The ability of EPW provided by the six standards to predict SGA was analyzed using the area under the receiver operating characteristic curve (AUC) . SensitiIn addition, we built logistic regression models to estimate the OR and 95% confidence interval that correspond to an increase of 1% in the EPW at 35 weeks, as a predictor for SGA at delivery, performing a subanalysis for different ultrasound–delivery intervals (1–6 weeks).We analyzed the diagnostic ability of the EPW 10th percentile and SGA birthweights to detect the following adverse perinatal outcomes: 5-min Apgar score < 7, instrumental delivery for non-reassuring fetal status (NRFS), cesarean delivery for NRFS, arterial cord blood pH < 7.10, and stillbirth. Comparison between APOs predicted by standards was performed using a proportion test.Analyses were performed using R version 3.6.2 language programming  .n = 902).EPW distributions are detailed in n = 139) APOs, 28.6% (n = 12) 5-min Apgar scores < 7, 19.9% (n = 32) instrumental deliveries for NRFS, 26.8% (n = 71) cesarean deliveries for NRFS, 17.7% (n = 45) neonatal acidemia (pH cord blood pH < 7.10), and 26.3% (n = 5) stillbirth.Regarding APOs, p-values of the comparisons of the standard AUC values and sensitivity for a 90% specificity are shown in p-values of all comparisons are illustrated in Regarding APO prediction by EPW < 10, in We have demonstrated the utility of EPW by ultrasound at 35 weeks (range 34+0–36+6 weeks) as a predictor of SGA fetuses at delivery at term. Adjusting the percentile threshold points, the growth standards showed a similar good predictive ability, but with a significant advantage for the non-customized MSUH and Fetal Medicine Foundation standards, and a disadvantage for both the customized MSUH and Figueras standards, for SGA fetuses.In our results, we found an underestimation of 10th percentile, by ultrasound at 35 weeks (range 34–36 weeks) with the WHO (7.5%) and FMF (2.7%) standards, and an overestimation with the Figueras (18.1%) standard; for that, we can conclude that these standards have a lack of calibration for our study population. The MSHU (NC (11.9%) and customized (12.2%)) and INTERGROWTH-21st (12.7%) standards fit better to the 10th percentile, with a minimum error, probably for the exclusion of premature deliveries.When we analyzed the APO-predictive ability of the six standards by percentile weight <10 at 35th week of gestational age, the customized Fetal Medicine Foundation and WHO standards showed the greatest diagnostic ability, with statistically significant differences from the rest of standards. The main reason for this lies in the greater proportion of 10th percentile EPW for the Fetal Medicine Foundation (21.2%) and WHO (12.6%) standards, In any case, with similar proportions of EPW < 10, the non-customized MSUH and INTERGROWTH-21st standards show a better APO-predictive ability than the customized MSUH and Figueras standards. A previous study did not find any significant differences between the customized and non-customized standards when analyzing the predictive ability of EPW to detect APOs; by contrast, using EPW > 90th percentile, we detected significant differences .There is no international consensus on performing a universal ultrasound in the third trimester; two international guidelines—the RCOG , and theThe EPW at third trimester ultrasound over 32 weeks has been shown to be a good predictive model (AUC > 0.85) for the detection of SGA at delivery in several studies, although with detection rates limited for late-onset SGA births ,38,39. FBlue et al., in 2018 , compareIn two studies by Odibo et al. in 2018  and 2019Reboul et al., in 2017 , found tIn clinical practice we can say that more important than the choice of the growth standard is its calibration before clinical use, both by ultrasound and delivery, in the reference population. The physiological and non-pathological characteristics of each population are those that will allow us to calibrate the standard to be used.There are several factors for which ultrasound in the third trimester presents limitations when predicting SGA and FGRs at delivery, and some of them are unavoidable—especially the systematic error of ultrasound at the time of EFW calculation . With thAccording to our results, it would be appropriate to raise the ultrasound-estimated weight percentile cutoff point above 10 for fetal growth control. This is because the 10th percentile has been shown to be insufficient, and with low predictive capacity for SGA at delivery and, therefore, fetuses that can potentially be IUGR even before delivery can escape control and, thus, increase their morbidity and mortality. Our recommendation, in the ultrasound during the third trimester, between 35 and 36 weeks, could be to raise the cutoff point at least from the 10th to the 20th percentile for strict control of fetal growth.Our study has several strengths, including the wide sample size close to 10,000 pregnancies. Ultrasound measurements were performed in routine clinical practice; thus, weight estimations were more concentrated over specific weeks of gestational age. Limitations of our investigation are that our data came from a single hospital, and their retrospective nature could limit the generalization of our standards. Furthermore, the information of the ultrasound was available to the obstetricians, which could mean a bias in the management of the pregnancies. A small percentage of labors are inductions of labor or cesarean sections programmed by IUGR, and they could act as confounding factors in the study. Similarly, other cases of early termination due to other causes have not been taken into account.In summary, even with limited detection rates, the growth standards showed a similar good predictive ability, with a statistically significant improvement by the use of non-customized standards, for SGA at delivery by ultrasound at 35 weeks. Generally, a shorter ultrasound delivery interval for the different standards was correlated with better prediction rates for small gestational age cases. When focusing on the use of EPW < 10th percentile at week 35 for the prediction of APOs, non-customized standards also demonstrated an advantage over customized standards.


Brain metastases are common in melanoma and are often associated with significant morbidity and mortality. Although many new treatments for melanoma have been approved in recent years, including immune checkpoint inhibitors and BRAF/MEK inhibitors, limited data are available for survival for patients with brain metastases treated with these novel therapies. The aim of this retrospective study was to evaluate current surgical, radiation, and systemic therapies over the past 10 years in melanoma patients with brain metastases. Our study noted increased overall survival in patients treated with craniotomy and CTLA-4 checkpoint inhibitors, while whole brain radiation was associated with poorer overall survival.Brain metastases commonly develop in melanoma and are associated with poor overall survival of about five to nine months. Fortunately, new therapies, including immune checkpoint inhibitors and BRAF/MEK inhibitors, have been developed. The aim of this study was to identify outcomes of different treatment strategies in patients with melanoma brain metastases in the era of checkpoint inhibitors. Patients with brain metastases secondary to melanoma were identified at a single institution. Univariate and multivariable analyses were performed to identify baseline and treatment factors, which correlated with progression-free and overall survival. A total of 209 patients with melanoma brain metastases were identified. The median overall survival of the cohort was 5.3 months. On multivariable analysis, the presence of non-cranial metastatic disease, poor performance status (ECOG 2–4), whole-brain radiation therapy, and older age at diagnosis of brain metastasis were associated with poorer overall survival. Craniotomy  and treatment with a CTLA-4 checkpoint inhibitor  were the only interventions associated with improved overall survival. Further studies with novel agents are needed to extend lifespan in patients with brain metastases in melanoma. Of all malignancies, melanoma has the highest propensity to migrate to the brain, which is associated with a poor prognosis ,2,3. AboDespite recent advances in the treatment of advanced melanoma, brain metastases remain a significant source of morbidity and mortality, with a poor median overall survival. In a recent report, the weighted median overall survival with single-agent chemotherapy, immunotherapy alone, or targeted therapy were between five to nine months only . Due to Many studies have suggested that novel therapies have efficacy against brain metastases in the clinical trial setting, including BRAF/MEK targeted therapy and immunotherapy ,11,12,13After IRB approval, a total of 445 patients were identified with the assistance of TriNetX by searching EPIC at the University of Iowa, using the search terms “melanoma” and “dexamethasone.” All patient charts and imaging were manually reviewed, identifying 209 patients with melanoma brain metastases. Inclusion criteria included all identified patients treated with brain metastases at the University of Iowa from 8 January 2008 through 8 January 2019. Patients were excluded if treatment modalities used over the course of the disease could not be identified. Patient characteristics, including sex, ethnicity, histopathological subtype, location of primary, and BRAF status, were identified and recorded. All patients were staged according to the guidelines set by the AJCC 8th edition . Dates oCox regression models were used to assess the effects of clinical, pathological, and treatment variables on progression-free survival (PFS) and overall survival (OS). For PFS, time was calculated from diagnosis of brain metastasis to progression of brain metastasis by imaging Repsonce Evaluation Criteria in Solid Tumors RECIST 1.1 criteria , initiatA total of 209 patients with brain metastases secondary to melanoma were identified. The demographic and baseline characteristics at the time of diagnosis of brain metastasis are presented in Upon diagnosis of brain metastasis, 108 (51.7%) had at least one metastasis greater than 2 cm in greatest diameter. The 87 patients with a single metastasis were categorized further, with locations of metastases described in Of all the patients diagnosed with brain metastasis, 57 patients were treated with adjuvant therapy, which included—interferon (21 patients), GM-CSF (16 patients), PD-1 inhibitors (7 patients), CTLA-4 inhibitors (6 patients), seviprotimut-L (5 patients) and talimogene laherparepvec, transgenic lymphocytes/interferon, dabrafenib/trametinib, and a combination of IL-2, cisplatin, vinblastine, and dacarbazine (1 patient each).A total of 82 patients (including 31 previously treated with adjuvant therapy) were treated for metastatic melanoma before the discovery of brain metastases. Of these patients, 48 had one line of therapy, 19 had two, 11 had three, three had five, two had four, and one patient had six. Of these patients, 25 were exposed to ipilimumab before the development of brain metastases, 29 were exposed to a PD-1 checkpoint inhibitor, and 25 were treated with a BRAF ± MEK inhibitor.Most patients (133) were treated with systemic therapy after the diagnosis of brain metastases. First-line therapies consisted of PD-1 inhibitors (45 patients), cytotoxic chemotherapy (37 patients), BRAF/MEK inhibitors (22 patients), ipilimumab (18 patients), a combination of ipilimumab with nivolumab (3 patients), and other therapies (8 patients). Of the treated patients, 77 were treated with a single line of therapy, 36 had two lines of therapy, 13 had three lines of therapy, and 7 had four lines of therapy. Initial salvage therapies after progression included PD-1 and/or CTLA-4 checkpoint inhibitors in 22 patients, BRAF/MEK inhibitors in 13, chemotherapy in 17, and other therapies in 4. In total, 63 patients were eventually treated with a PD-1 inhibitor after diagnosis of brain metastasis, 41 with BRAF/MEK inhibitor, and 24 with ipilimumab.In total, 74 patients (35.4%) underwent craniotomy with tumor resection, of which 64 patients had the tumor resection within one month of diagnosis of brain metastasis. Patients were selected for surgery based on evaluation and discussion by neurosurgeons and radiation oncologists. Two separate patients received a brain biopsy for diagnosis, and an additional two patients underwent ventriculostomy for ventricular obstruction. Surgical resections were performed on 50 patients with a single metastasis, nine with two metastases, four with three metastases, eight with four metastases, and three with five or greater metastasis. Of patients, who underwent craniotomy, 64  had a primary brain lesion greater than 2 cm in diameter. Of the 66 patients, of which operative reports were able to be obtained, 63 tumors were noted to be gross total resections.Regarding radiation therapy, 116 had SRS, including all but one of the patients treated with craniotomy. WBRT was used for 107 patients, including 44 patients who underwent both SRS and WBRT.The median OS survival of the entire cohort was 5.2 months see .. ConcerOf the 74 craniotomy patients, 14 died prior to brain imaging surveillance after craniotomy. A total of 23 patients had stable surveillance imaging, of which 12 were still alive as of this analysis. The remaining 37 patients had intracranial progression after craniotomy with a median time to progression of 119 days and, of which 2 were alive as of the time of analysis. Progression was secondary to new intracranial metastases in 21 patients, development of leptomeningeal disease in 3 patients, and local recurrence in 7; progression was otherwise due to progression of unresected lesions.In the 107 patients treated with WBRT, 35 died prior to surveillance imaging. A total of 16 had stable disease, of which 4 were alive at the time of analysis. The remaining 56 had intracranial progression, with a median time to progression of 59.5 days. In 25 patients, progression was determined by new metastases, and development of leptomeningeal involvement in 4 patients, while the remainder was noted to have increased size of previously noted lesions only.Of the 116 patients treated with SRS, 13 died prior to additional imaging. A further 26 patients had stable disease, with 13 still alive as of the time of analysis. A total of 77 progressed, with a median time to progression of 103 days. New metastases were identified in 60 patients on the first progression after SRS, and 4 developed the leptomeningeal disease, while the remainder only had local progression.The univariate and multivariable analysis for OS is presented in PD-1 and CTLA-4 checkpoint inhibitors were evaluated separately in the above analysis. Assessment of a synergistic effect of the PD-1 and CLTA-4 inhibitors, when administered in combination, was not found to be statistically significant, which may be a result of a small sample size.Here we present a cohort of 209 melanoma patients with intracranial metastases. Historically, patients with brain metastases have had a very poor prognosis, with previous retrospective studies identifying as few as 2.4% surviving greater than three years, with the cause of death in 94.5% attributable to brain metastases .Our study confirms the findings of previous epidemiological studies remarking on the higher prevalence of brain metastases among male patients. Interestingly, a surprisingly high percentage (12.4%) of patients had melanoma of unknown primary even after excluding the patients who never had a dermatology evaluation. A retrospective review in 2017 by Utter et al. on melanomas of unknown primaries found that 79% of patients with melanoma of unknown primary developed brain metastases [In this series, ipilimumab was found to be associated with improved OS, while PD-1 checkpoint inhibitors were not associated with improved outcomes in the multivariable analysis and were associated with poorer PFS. Recent prospective clinical trials by Long et al.  and TawbTreatment with ipilimumab was also found to be associated with better outcomes than BRAF/MEK inhibitors. Prospective studies by Dummer et al., McArthur et al., and Long et al. have found the response of melanoma brain metastases to single-agent BRAF therapy, although median progression-free survival and median overall survival were short ,10,26. SAdditionally, our study confirms that craniotomy with surgical resection resulted in better outcomes, which is consistent with previous studies . HoweverAlthough SRS resulted in better outcomes in the univariate analysis, this survival advantage was not found in the multivariable analysis. In our study, WBRT was associated with decreased OS. This is in agreement with current National Comprehensive Cancer Network guidelines, which do not recommend upfront WBRT for melanoma patients with brain metastasis . MoreoveOne limitation of this study is the retrospective design, which is common to other retrospective studies, including the risk of confounding bias and inability to determine causation. Adjustment for potential confounders, such as ECOG and age, in the multivariable analyses, was done. However, there may be additional confounding factors not captured in the study, which may limit interpretations. These findings should be further validated by additional external cohorts and prospective clinical trials.This study reported on the baseline characteristics and impact of treatment on melanoma brain metastases. Importantly, multivariable analysis systemic treatment with ipilimumab was associated with better OS, while WBRT was associated with worse OS. However, even with these promising results with ipilimumab, OS remains poor. Further studies with novel agents and combinations are warranted to identify efficacious treatment for brain metastases in melanoma.


GRIN3B (p = 0.021), rs3983721 in GRIN3A (p = 0.00326), rs2129575 in TPH2 (p = 0.033), rs6583954 in CYP2C19 (p = 0.033), and rs174699 in COMT (p = 0.036). These were all associated with heroin-using and craving scores with and without adjustments for age, sex, and body mass index. We combined five variants, and the ensuing dose-response effect indicated that heroin-craving scores increased with the numbers of risk alleles (p for trend = 0.0008). These findings will likely help us to understand the genetic mechanism of craving, which will help in predicting the risk of relapse in clinical practice and the potential for therapies to target craving in heroin addiction.Addiction is characterized by drug-craving, compulsive drug-taking, and relapse, and results from the interaction between multiple genetic and environmental factors. Reward pathways play an important role in mediating drug-seeking and drug-taking behaviors, and relapse. The objective of this study was to identify heroin addicts who carry specific genetic variants in their dopaminergic reward systems. A total of 326 heroin-dependent patients undergoing methadone maintenance therapy (MMT) were recruited from the Addiction Center of the China Medical University Hospital. A heroin-use and craving questionnaire was used to evaluate the urge for heroin, the daily or weekly frequency of heroin usage, daily life disturbance, anxiety, and the ability to overcome heroin use. A general linear regression model was used to assess the associations of genetic polymorphisms in one’s dopaminergic reward system with heroin-use and craving scores. Results: The most significant results were obtained for rs2240158 in  Heroin addiction, a chronic relapsing disease characterized by compulsive drug-seeking, drug abuse, tolerance, and physical dependence, is a major public health concern worldwide . It is aCOMT) [POMC), tryptophan hydroxylase 2 (TPH2), and brain-derived neurotrophic factor (BDNF) [OPRK1, OPRL1, PDYN, cannabinoid receptor 1 [COMT), tryptophan hydroxylases 1 (TPH1 and 2 TPH2), methadone-metabolizing enzymes , and cognitive function-related genes . Previous linkage and association studies of polymorphisms have reported several genetic variants associated with drug abuse, addiction, and related phenotypes ,6. TheseCOMT) ,11, proor (BDNF) . Physiolr (BDNF) . To idenCraving is an integral symptom that is considered to be central to the motivational drive in heroin addiction . We explThe study protocol was reviewed and approved by the Institutional Review Board of China Medical University Hospital (CMUH) (DMR101-IRB1-218) and was in compliance with the Declaration of Helsinki. Informed consent was obtained from all subjects involved in the study. In total, 316 heroin-dependent patients undergoing methadone maintenance therapy (MMT) were recruited from the Addiction Center of the CMUH. As assessed by senior psychiatrists experienced in heroin dependence, all the recruited patients met the criteria from the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, for heroin abuse and dependence, and had been receiving MMT for at least 6 months at a dosage that had been unchanged for at least 4 weeks at the CMUH. We selected from the extreme margin of the specific phenotype range  to maximize the power of the study. For each patient, the following clinical information was recorded: sex, weight (in kg), height (in cm), liver function test results, comorbidities, and daily methadone dose.We designed a 14-item heroin-use and craving (HUC) questionnaire to evaluate the behavior of heroin users undergoing MMT. Each question was scored from 0 to 4. The questionnaire was divided into two parts: Part I , assessing the urge for heroin and whether the individual can shift their attention away from heroin, and Part II , investigating the daily or weekly frequency of heroin usage, daily life disturbance, anxiety, and ability to overcome heroin use. In essence, the higher the score, the more severe the heroin craving was. The score of each item was calculated according to the manual.OPRK1, OPRL1, PDYN, CNR1, COMT, TPH1, TPH2, CYP1A2, CYP2B6, CYP2C19, GRIN3A, GRIN3B, and GRM6. Single nucleotide polymorphism (SNPs) with minor allele frequency >0.05 were selected on the basis of the previous findings and population data in the SNP databases of the National Center for Biotechnology Information (Han Chinese) in Beijing, China. In total, 44 SNPs were genotyped at the National Center for Genome Medicine, Taiwan, by using Sequenom iPLEX matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry technology. DNA was extracted from 3 to 10 mL of whole blood using a QIAamp DNA Blood Mini Kit  according to the manufacturer’s protocol. Duplicate samples were randomly selected for quality control, and the concordance rate was >0.99 for all SNPs assayed.This study focused on the 13 aforementioned genes encoding various metabolic components: t test for continuous variables in both sexes. Single SNP association analyses were conducted with a general linear model, under dominant or recessive model assumptions. We performed QTL mapping for HUC. The general linear model was used to assess associations of genetic polymorphisms in the reward pathway with heroin-use and craving scores, making adjustments for sex, age, and body mass index (BMI). Each SNP genotype frequency distribution was examined for Hardy-Weinberg equilibrium using the chi-square one degree of freedom goodness-of-fit test. Demographic characteristics and clinical parameters were evaluated with a chi-squared contingency table for categorical variables and a The demographic details and heroin use history of the subjects are summarized in GRIN3B, rs3983721 in GRIN3A, rs2129575 in TPH2, rs174699 in COMT and rs6583954 in CYP2C19) were significantly associated with HUC questionnaire scores. Two SNPs (rs174699 in COMT and rs6583954 in CYP2C19) were significantly associated with urge for heroin. Another two SNPs (rs2240158 in GRIN3B and rs3983721 in GRIN3A) were significantly associated with ability to overcome heroin use . The genetic risk allele scores (combined gene effects) were significantly associated with HUC questionnaire scores, urges for heroin, and ability to overcome heroin use . We found that five SNPs were significantly associated with HUC questionnaire score, as well as the urge for heroin and the ability to overcome heroin use; in addition, GRIN3A, GRIN3B, CYP2C19, TPH2, and COMT, contributing to susceptibility to heroin addiction. All the variants identified are from non-coding regions. As expected for a complex genetic disorder, each variant was shown to have a small effect on the risk of heroin addiction. The R-squared ranged from 3% to 5% for each genetic variant. This explained a variance in the combined effects of multiple genes on heroin-using and craving scores, and ability to overcome heroin use, of 6.3%  receptors, which belong to the superfamily of glutamate-regulated ion channels, and are involved in the physiological and pathological processes of the central nervous system. Some genetic variations in the glutamatergic system have been reported to contribute to vulnerability to drug addiction [GRIN3A and GRIN3B may play roles in heroin addiction, acting as dominant-negative modulators of NMDA receptors.The variants in the ehaviors ,18,19. Wddiction ,20. GRINTPH2 have previously been associated with heroin addiction in African Americans and in Hispanics, and are also associated with phenotypes related to smoking status, neuropsychiatric disorders, higher reward dependence, and personality traits [Several variants in y traits ,21. TPH COMT gene is associated with urge for heroin, which plays a critical role in heroin dependence [COMT gene SNP rs174699 was associated with urge for heroin. Individuals with the CC genotype would thus be expected to exhibit a stronger urge for heroin use than individuals with the CT or TT genotypes. COMT plays an important role in the regulation of synaptic dopamine levels, and in the reinforcement mechanism of drug dependence. The COMT SNP rs174699 was selected as a possible marker of genetic predisposition to addiction.Our findings indicate that the pendence ,22. The CYP2C19 gene encodes a CYP450 enzyme that contributes to methadone metabolism, dependent on treatment dose, plasma concentration, and the side effects of methadone [CYP2C19 gene SNP rs6583954 was found to be associated with heroin-using and craving score, and the ability to overcome heroin use. Individuals with the T allele would thus be expected to exhibit a stronger ability to overcome heroin use than individuals with the C allele. Variations in the CYP2C19 gene may influence the steady-state plasma concentrations of methadone and the plasma concentrations of methadone [CYP2C19 gene may potentially serve as an indicator for ability to overcome heroin use.The ethadone . Methadoethadone , while tThere are some limitations in this study that need to be considered. The results were compared to those of previous studies. Some variants from previous reports were not genotyped, and the phenotype and population were often different. We cannot determine if the negative findings in this study are true negatives, because the SNP coverage of some of the genes may be insufficient to exclude real associations. Based on known or inferred biologically functional genes, we used a candidate gene approach that allows for scanning a limited number of SNPs and is limited by current knowledge. Our study had only limited phenotypic and genotypic information. It is thus necessary to genotype more novel susceptibility genes identified by genome-wide association studies (GWAS) ,25,26,27This study suggests an extension to the list of susceptibility genes and variants underlying heroin addiction. Although the variants identified in this study are suggested for assessing the degree of urge for heroin and the ability to overcome heroin use, they may uncover a genetic basis for craving and heroin-addictive behavior. These findings will likely help us understand the genetic mechanism of craving, which will be helpful in predicting the risk of relapse in clinical practice as well as the potential applicability of therapies for targeting craving in heroin addiction.