SLC43A2 and TNXB. As this was the first epigenome-wide association study for SAD, it is worth noting that both genes have previously been associated with panic disorder. Further, we identified two DMRs associated with ELA within the SLC17A3 promoter region and the SIAH3 gene and several DMRs that were associated with the interaction of SAD and ELA. Of these, the regions within C2CD2L and MRPL28 showed the largest difference in DNA methylation. Lastly, we found that two DMRs were associated with both the severity of social anxiety and ELA, however, neither of them was found to mediate the contribution of ELA to SAD later in life. Future studies are needed to replicate our findings in independent cohorts and to investigate the biological pathways underlying these effects.Social anxiety disorder (SAD) is a psychiatric disorder characterized by extensive fear in social situations. Multiple genetic and environmental factors are known to contribute to its pathogenesis. One of the main environmental risk factors is early life adversity (ELA). Evidence is emerging that epigenetic mechanisms such as DNA methylation might play an important role in the biological mechanisms underlying SAD and ELA. To investigate the relationship between ELA, DNA methylation, and SAD, we performed an epigenome-wide association study for SAD and ELA examining DNA from whole blood of a cohort of 143 individuals using DNA methylation arrays. We identified two differentially methylated regions (DMRs) associated with SAD located within the genes  The etiology of social anxiety is influenced by genetic and environmental factors. One of the main environmental risk factors for SAD is early life adversity (ELA). Stressful experiences early in life, like childhood abuse and neglect, can lead to long-lasting behavioral and neurobiological changes ultimately contributing to an increased risk of SAD3. One mechanism that may explain how environmental factors can biologically contribute to psychopathological phenotypes is through epigenetics.Social anxiety disorder (SAD) is a psychiatric disorder characterized by intense fear in various social situations. Commonly, the fear of being the focus of attention and of negative evaluation play an essential role in the psychopathology and lead to severe impairments in daily life4. DNAm is affected by genetic and environmental factors5. Since DNAm can be influenced by environmental stimuli and change over time, it might play an important role in adapting organisms to a changing environment, especially during vulnerable time periods including prenatal developmental stages and childhood6. Several studies have already linked ELA with long-lasting differences in DNAm levels7. Furthermore, differential DNAm patterns are also associated with SAD, amongst other neuropsychiatric disorders9. Hence, DNAm differences associated with ELA might be involved in the association of ELA with SAD later in life.Epigenetic mechanisms can affect gene regulation without altering the DNA sequence. In human populations, the best-studied epigenetic modification is DNA methylation (DNAm), which refers to the covalent binding of a methyl group to the DNA. This occurs mostly, but not exclusively, on cytosines preceding a guanine, so called CpG sitesOXTR)11. Only a few broader epigenome-wide association studies (EWAS) have been conducted for anxiety disorders and these have mainly investigated panic disorder13. Differential DNAm in association with ELA has been shown for several candidate genes, such as SLC6A414, FKBP515, and NR3C116. In addition, studies investigating epigenome-wide DNAm in association with ELA, either in postmortem brain samples17 or peripheral tissues21, suggested differential DNAm in further genes such as ALS217, KITLG18, CYP2E119, and SLC17A321 at various stages in life. Altogether, these findings indicate that long-lasting DNAm changes may be involved in the outcome of adverse experiences early in life.Previous research investigating differential DNAm associated with SAD has exclusively focused on candidate genes, such as the oxytocin receptor gene  that measures five types of maltreatment: emotional and physical neglect and emotional, physical, and sexual abuse. Participants with at least a moderate score in one of the five categories were classified as participants with high levels of ELA24. Thus, four groups emerged: 1) control participants without SAD and low levels of ELA , 2) control participants without SAD and high levels of ELA , 3) participants suffering from SAD with low levels of ELA , and 4) participants suffering from SAD with high levels of ELA . We also assessed self-reported measures of alcohol consumption, smoking behavior, and psychotropic medication. All participants gave written informed consent to the experimental procedure prior to inclusion in the study. The study was performed in accordance with the Declaration of Helsinki and approved by the University of Tübingen local ethics committee.In total, 143 participants of Caucasian descent between the ages of 18–50 years took part in the study. All participants were assessed using the Structured Clinical Interview for DSM-IV (SCID) and 66 participants were found to be presently suffering from SAD as a primary diagnosis. Supplementary Table ®, Sarstedt, Sarstedt, GER), and stored at −80 °C. DNA was extracted using the QIAamp Blood Maxi Kit  according to manufacturer’s instructions. DNA was quantified using the Qubit® 2.0 Fluorometer  and stored at −20 °C.Blood samples were collected in EDTA tubes  according to manufacturer’s instructions. DNAm was determined using the Infinium MethylationEPIC Kit . To assess technical variation, one of the samples was run twice to generate a technical replicate. DNAm between those technical replicates had a Spearman correlation coefficient of rho >0.992 and a mean difference in methylation of 1.34% across all assessed CpG sites.Genomic DNA was bisulfite converted using the EZ DNA Methylation25. At first, the getQC function from the minfi package was applied to the raw DNAm data to ensure high quality data. The median intensities for each sample were above the default threshold26. Further, all samples had <1% of probes with a detection p-value > 0.05.Data processing and analyses were performed using the software R (Version 3.5.1)preprocessNoob function from the minfi package was applied27. Additionally, we excluded 59 single nucleotide polymorphism (SNP) probes, 19,632 sex chromosome probes, 53,315 cross-reactive and polymorphic probes28, 4081 poorly performing probes , and 98,983 known invariant probes  <0.02)29. Following these quality measures, 690,080 of the 866,150 original probes (79.7%) were left for further analyses.For background and dye bias correction, the 30. To reduce batch effects of different runs, chips, and rows, the ComBat function was applied31.To adjust for the bias originating from the two different bead types present on the BeadChip array, Beta-Mixture Quantile (BMIQ) normalization was performedestimateCellCounts.wmln function from the wateRmelon package was used to estimate the relative proportions of monocytes, granulocytes, CD4+ and CD8+ T cells, B cells, and natural killer cells33. This method is based on the commonly used Houseman reference dataset for 450 K array data34. As actual blood cell counts for monocytes, granulocytes, and lymphocytes  were also available, the statistical estimates were compared to these cell counts . Post-hoc pairwise comparisons were performed to investigate significant interaction effects.On an epigenome-wide level, we extracted differentially methylated regions (DMRs) using the package For the top DMRs associated with SAD, Pearson correlation was assessed to investigate the association of the mean DNAm level of the identified sites with the total LSAS score, a measure of social anxiety. Likewise, for the top DMRs associated with ELA, Pearson correlation was used to test for an association of the mean DNAm level of the identified sites with the total and the subscale scores of the CTQ, an assessment of ELA (Bonferroni correction for multiple testing). All DMRs are reported using genomic coordinates from the human assembly GRCh37 (hg19) genome reference.37. We only searched for genetic variants having an impact in “Middle Age”  using default parameters otherwise  and reported the number of different mQTLs for each DMR.To investigate the genetic influence on DNAm in the identified DMRs, we screened the reported CpG sites of each region for methylation quantitative trait loci (mQTLs) using an mQTL database39. Two different linear models were fitted with one factor of interest, either total CTQ or total LSAS score, and the covariates age and sex. Using DMRcate with the same parameters as previously described, DMRs were identified for both models. Using a hypergeometric test, we examined whether the overlap of CpG sites predicted by both models was significant. For each DMR predicted by both total CTQ and total LSAS score, the mean DNAm was included as a mediating variable in a mediation model with the independent variable total CTQ score and the dependent variable total LSAS score and the covariates age and sex. The direct and indirect effects were tested using a percentile bootstrap estimation approach with 5000 samples.To examine whether DNAm changes mediate increased social anxiety following ELA, a regression-based approach was performed using PROCESS (model 4)t(141) = 0.05, p = 0.96) nor in sex  with respect to SAD, significant differences in both age (t(85.8) = 2.60, p = 0.011) and sex  emerged with respect to ELA. The total score of the CTQ was not significantly different with respect to sex (t(141) = 1.01, p = 0.32), but it correlated positively with age 24. For the total score of the LSAS there was no significant difference with respect to sex (t(141) = 1.66, p = 0.10) and no correlation with age 22. Furthermore, there was no difference with respect to the amount of alcohol consumed the month before study participation, neither with respect to SAD (t(124.7) = 0.90, p = 0.37), nor with respect to ELA (t(116.0) = 0.42, p = 0.68). Similarly, there was no significant difference in the number of cigarettes smoked during the month before study participation, neither for SAD (t(74.1) = 1.91, p = 0.06), nor for ELA (t(116.0) = 1.09, p = 0.28). Finally, there was a significant correlation between the total score of the LSAS and CTQ .Table p-value < 0.05 with respect to SAD (see Supplementary Table SLC43A2 (pFDR-corr < 5e-4)  and the remaining three sites were located in exon 4 of TNXB (pFDR-corr < 3e-26) . Participants with SAD had decreased DNAm levels with a mean difference of 9.3% (SLC43A2) and 5.3% (TNXB) across the reported sites. Fig. Investigating the DNAm data from all participants using DMRcate, eight differentially methylated CpG sites were identified which had a DNAm difference >5% and an FDR-corrected SLC43A2 showed a significant negative correlation with the total LSAS score . In contrast, there was no significant correlation between TNXB DNAm levels and the total LSAS score   Fig. .SLC17A3, 8 kb upstream of exon 1 (pFDR-corr < 0.035) . Another two sites were within exon 2 of the SIAH3 gene (pFDR-corr = 0.001) . In both regions, DNAm was lower in participants with high levels of ELA with a mean difference of 8.7% (SLC17A3) and 10.6% (SIAH3) across the reported sites. Figure Investigating the DNAm data from all participants, twelve CpG sites were identified to be differentially methylated with respect to ELA  . SIAH3 DNAm levels showed a nominal significant, negative correlation with the total CTQ score  , and a significant, negative correlation with emotional abuse   Fig. . When in12) Fig. , and theC2CD2L gene, spanning intron 13 and exon 14 (pFDR-corr < 0.001)  and within the MRPL28 gene, spanning exon 2 and partially spanning intron 1 and 2 (pFDR-corr < 0.037) . Post-hoc t-tests showed that for the three CpG sites within C2CD2L, there was a significant difference in DNAm in participants suffering from SAD in association with ELA (t(64) = 3.78, p < 3.5e-4) but not in control participants (t(75) = 0.72, p = 0.47). Participants with SAD and high levels of ELA showed higher methylation in the C2CD2L region with a mean DNAm difference >9% compared to participants with SAD and low levels of ELA and the control participants. For the six CpG sites within MRPL28, there was a significant difference in DNAm in control participants in association with ELA (t(75) = 2.70, p = 0.009), but not in participants suffering from SAD (t(64) = 0.15, p = 0.88). Participants without SAD and high levels of ELA showed higher methylation with a mean difference in DNAm of >6% compared to control participants with low levels of ELA and participants suffering from SAD. Figure With respect to the interaction of SAD and ELA, 107 CpG sites in 21 DMRs were identified . Moreover, 122 regions were identified in association with the total LSAS score (pFDR-corr < 0.05). Two of these regions were overlapping; one region included eight differentially methylated CpG sites  0.8 kb upstream of ADAMTS16, and the other region included five overlapping CpG sites  located approximately 0.5 kb upstream of GALR1. Together, these 13 CpG sites represented a significant overlap (p < 6e-18).Investigating the DNAm data from all participants, 64 regions were identified where DNAm was significantly associated with the total CTQ score  and ADAMTS16 DNAm was a significant predictor of the total LSAS score . While the direct effect was significant , the indirect effect of ELA on SAD mediated by DNAm was not significant .To examine whether DNAm in these regions is mediating the increased risk for adult SAD after ELA, a mediation analysis based on the model shown in Supplementary Fig. GALR1, the total CTQ score was a significant predictor of DNAm  but GALR1 DNAm was not a significant predictor of the total LSAS score . While the direct effect of ELA on SAD was significant , the indirect effect mediated by methylation was not significant .For The present study represents a hypothesis-free EWAS for SAD, and, moreover, benefits from including ELA, one of the main environmental risk factors for SAD. Thus it afforded the investigation of the interrelations between SAD, ELA, and DNAm. We identified two DMRs associated with SAD, two DMRs associated with ELA, and several DMRs that exhibited differential DNAm associated with SAD interacting with ELA. An additional two DMRs where DNAm is associated with both the severity of ELA and of social anxiety were identified. However, there was no significant mediation effect of those epigenetic differences on the contribution of ELA to the severity of social anxiety later in life.SLC43A2 and one in the coding region of TNXB. SLC43A2 belongs to the solute carrier (SLC) family and encodes the essential amino acid transporter LAT4. Changes in DNAm in response to psychotherapy have been reported in this gene, specifically an increase in DNAm in response to cognitive-behavioral therapy (CBT) and, hence, it was discussed to be potentially involved in the treatment response of CBT in panic disorder patients40. Remarkably, the reported site, cg22273830, is one of the four sites in our identified DMR. Assuming aberrant DNAm in anxiety patients reverts to levels of healthy controls in response to therapy, this is in line with the decreased methylation levels in individuals with SAD observed in our study. In this context, it is tempting to speculate that our results add evidence to the hypothesis that hypomethylation in this DMR might be involved in the pathophysiology of anxiety disorders. TNXB encodes tenascin-X, which plays an important role in connective tissue structure41. Several studies also suggest a genetic and epigenetic association between TNXB and psychiatric disorders such as schizophrenia43 or anorexia nervosa45 and, strikingly, hypomethylation of a CpG site in intron 6 of TNXB has already been associated with panic disorder in an EWAS12. As the present study is the first EWAS for SAD, it is worth noting that we identified two genes that may be involved in the underlying mechanisms of SAD, which have already been associated with panic disorder in previous EWAS, pointing towards a more general association with anxiety disorders.For SAD, we identified two DMRs, one in an intron of SLC17A3 promoter and another region within SIAH3 that exhibited differential DNAm. SLC17A3 also belongs to the SLC family and encodes the sodium-phosphate transporter NPT4. A correlation analysis of our data revealed a nominal significant association of SLC17A3 DNAm with physical abuse but not with emotional abuse and neglect, which might indicate a more specific effect of physical childhood abuse on SLC17A3 DNAm. Interestingly, differential DNAm of the same CpG sites within the SLC17A3 promoter has already been reported in a small cohort for childhood abuse victims21, although with an opposite direction of effect. Notably from our mQTL analysis, DNAm in this region was found to be potentially influenced by a high number of mQTLs. This genetic contribution might interfere with environmental effects if, by chance, groups are unbalanced for genotype. Therefore, especially in small cohorts, differential DNAm patterns in this region have to be interpreted with caution. The SIAH3 gene encodes a member of the seven in absentia protein family and was suggested to be involved in the pathogenesis of neurodegenerative diseases46. When investigating different types of ELA, we specifically found a negative association of SIAH3 DNAm with emotional abuse and a nominal significant association in the same direction with emotional neglect. In contrast, no association with physical or sexual childhood maltreatment was observed. Since SIAH3 has not been previously associated with ELA or any stress-related disorders, it is important to validate this finding with future independent studies.Comparing participants with low and high ELA, we identified a region of the C2CD2L and MRPL28. C2CD2L encodes the transmembrane protein 24 (TMEM24) which concentrates in the endoplasmic reticulum47. Of interest, C2CD2L has been associated with substance abuse on a genetic, expression, and DNAm level49. Furthermore, in adipose-derived stem cells, C2CD2L hypermethylation at one of the CpG sites in the identified DMR has been associated with low birth weight50, which in turn is associated with prenatal stress51. MRPL28, a mitochondrial ribosomal gene, has been found to be upregulated in the hypothalamus of mice that experienced chronic social defeat stress, a condition which can lead to anxiety- and depressive-like states52. In peripheral blood of infants, MRPL28 hypermethylation at one of the CpG sites in the identified DMR has been associated with maternal asthma during pregnancy53, a maternal stressor which has consistently been associated with low birth weight54. Hence, hypermethylation in both genes might be associated with certain forms of prenatal stress. To date, an association with postnatal ELA has not been reported and it remains unclear how SAD might interact with potential effects. While for C2CD2L hypermethylation is only present in individuals suffering from SAD with high levels of ELA, MRP28L hypermethylation is only present in control participants with high ELA levels but not in any of the other groups. As individuals suffering from SAD with high levels of ELA showed increased C2CD2L DNAm compared to the remaining participants, there might be a cumulative effect of high levels of stress and anxiety during child- and adulthood on DNAm. In line with this, a previous study compared participants suffering from posttraumatic stress disorder (PTSD) with or without childhood abuse with control individuals without PTSD but trauma exposure. They reported a higher extent of DNAm differences in participants with PTSD and a history of childhood abuse compared to participants with PTSD but no history of childhood abuse55. For MRPL28, DNAm was found to be increased after high levels of ELA in control individuals but not in participants with SAD. One possible explanation might be a reversal of the long-lasting effect of ELA on DNAm in the presence of persisting clinically relevant levels of social anxiety. Additional studies are needed to investigate the underlying mechanisms of these effects. Nevertheless, the observed interaction effects highlight the importance of assessing ELA as an environmental factor influencing DNAm when investigating methylation changes in psychiatric disorders.Several DMRs were associated with an interaction between SAD and ELA. Here, the largest DNAm differences were located within the genes ADAMTS16 and GALR1. ADAMTS16 encodes a member of the ADAMTS  protein family56. It has previously been associated with hypertension58 and, interestingly, hypertension has been linked with both the exposure to ELA and the diagnosis of early-onset SAD59. Perhaps the association of ADAMTS16 DNAm with the severity of ELA and social anxiety in our data is related to chronic vegetative stress manifestation. Unfortunately, this hypothesis cannot be tested based on the present set of data since it did not include blood pressure measurements. GALR1 encodes the galanin receptor type 1. Interestingly, several rodent studies have shown that the neuropeptide galanin, as well as its receptor subtypes, might play a role in depressive- and anxiety-like behavior61. Upon our mediation analysis, however, we did not find significant evidence that DNAm, neither in ADAMTS16 nor in GALR1, statistically mediated the increased risk for SAD after ELA. One has to consider, though, that our study design may not have been ideally suited to investigate whether DNAm changes are involved in the manifestation of ELA in adult SAD. As the study design was primarily focused on interactions between ELA and SAD with the specific recruitment of four groups of participants with either high or low levels of ELA or social anxiety, the true degree of correlation between these two factors may have been underestimated in the present study with decreased sensitivity to detect potential mediators. To further investigate whether DNAm is a mediator of the manifestation of SAD in individuals with high levels of ELA, future studies should employ an unbiased recruitment strategy.Lastly, since ELA is a risk factor for anxiety disorders such as SAD, we investigated whether DNAm differences may mediate this association. We identified two DMRs associated with both ELA and social anxiety, located upstream of the genes 63. Although we were able to investigate an mQTL database37 and found many genetic variants affecting DNAm in some of the identified DMRs, especially within the SLC17A3 promotor region, our study would have benefitted from combining genome-wide SNP data with epigenome-wide methylation data to provide further insights into the role of genetic variants on DNAm in the context of ELA and SAD.Other limitations of our study include potential confounding factors or the lack of potential explanatory data. Despite a well-characterized sample, further confounders could exist like the experience of stressful life events in adulthood or dietary differences. Additional explanatory data such as genotype data were not assessed in our study. Integrative approaches have shown that DNAm in the majority of variably methylated regions was best predicted by the combination of environmental factors and genotype informationLastly, the biological interpretation of our findings is limited by several factors. Firstly, due to the lack of expression data, potential effects of observed DNAm differences on gene transcription remain unclarified. Secondly, due to the cell type and tissue specificity of epigenetic patterns, the DNAm levels assessed in whole blood, such as in the present study, and in the brain, where they can be assumed to be more directly linked to the pathophysiology of anxiety disorders, are not necessarily equivalent. Nevertheless, both ELA and SAD are associated with aberrant stress responsivity which can be argued to cause systemic effects. Another relevant limitation for this study is the relatively small sample size. Taking these limitations into consideration, however, we have demonstrated that several of our findings fit in well with previously reported results which in turn supports the reliability of our results. Still, future studies are needed to replicate our findings in independent, larger cohorts as a matter of course.In summary, the present study is the first to examine differential DNAm associated with SAD on an epigenome-wide level and to study its relation to ELA. We identified several DMRs associated either with SAD, ELA, or their interaction. Interestingly, the two identified DMRs with respect to SAD have previously already been reported in association with panic disorder. Hence, they may be involved in psychophysiological mechanisms underlying anxiety disorders in a more general domain. Furthermore, we identified two DMRs associated with both the severity of ELA and social anxiety but neither of them could be proved to play a mediating role in the contribution of childhood adversities to social anxiety in adulthood. Translational studies investigating the pathophysiological relevance of the identified DMRs are required to understand their role in disease vulnerability or pathogenesis.Supplementary Information


Background and Objectives: Khat chewing is considered as a daily habit that is practiced by more than five million people globally. The effect of khat chewing on the surface roughness and the color stability of natural teeth and the material used in the fabrication of dental prosthesis remains unknown. This study was conducted to explore and compare the effect of khat homogenate (KH) on the surface roughness (Ra) and the average color changes (ΔE*) amongst natural teeth and selected shades from different porcelain types, namely, feldspathic metal ceramic (MC) VM13, computer-aided design/computer assisted manufacture (CAD/CAM) feldspathic (Vitablocs Mark II), and multilayer zirconia (Ceramill Zolid PS) porcelains. Materials and Methods: Seventy samples were prepared from natural teeth, feldspathic MC, CAD/CAM Vitablocs Mark II, and zirconia porcelain. The Ra values were measured using a profilometer and expressed in micrometers, whereas the ΔE* values were measured using VITA Easyshade® V spectrophotometer for all samples before and after frequent immersion and thermocycling in KH for 30 days. The surface topography was used to assess the materials surfaces (glazed or polished) after KH immersion by using a white light interferometry machine. Results: Results revealed that the Ra and the ΔE* values of the different types of tested porcelain were influenced by KH. The order of surface roughness values was glazed or polished MC > polished Zircon > polished Vitablocs Mark II > natural teeth. The lowest ΔE* values were recorded for glazed Vitablocs Mark II and MC, and the values could be arranged as polished zircon > natural teeth > glazed zircon > polished MC > polished Vitablocs Mark II. P values were significantly varied (<0.001) among all the tested groups, except the zircon group (>0.05) for both Ra and ΔE*. Conclusions: KH significantly affected both surface parameter and color of glazed or polished porcelain materials and natural teeth. Dental porcelain has become an extremely important material in prosthodontic dentistry because of its many advantages, such as biocompatibility, durability, long live survival, and excellent aesthetic capabilities with long-term follow up. This material shows translucency, color brightness, and intensity properties as natural non-stained teeth ,2,3. As Khat or qat plant (Catha edulis) is a natural central nervous system stimulant used by millions around the globe. It is cultivated in the south of Saudi Arabia . Green kThe stability of dental ceramics in the oral environment is directly related to high surface polish, subcritical crack propagation, and chemical inertness of material atoms, thereby enabling them to resist degradation in the oral environment . Surface2 + (a1* − a2*)2 + (b1* − b2*)2 “× ½ [® V spectrophotometer device is reported to be more accurate and precise than the other instruments for prosthetic material color measurements [A spectrophotometer measures the reflectance or the transmittance of wavelengths from one object at a time without being affected by the subjective interferences of color ,25, wher*)2 “× ½ . Motro e*)2 “× ½ . Alghaza*)2 “× ½ . The VITurements ,30. Resistance to staining of a prosthesis is an important clinical requirement for fracture resistance and other mechanical properties . After aA dental practitioner usually performs some intraoral occlusal adjustments to the porcelain covering materials. Such adjustments include removal of a part of the glazed layer from the ceramic surfaces, leaving some pores in the ceramic material, and creating a rough surface. These effects can cause the discoloration of the prostheses that requires either re-glazing or polishing to maintain luster and a stable color ,33,34.Khat chewing usually has a bitter taste and causes mouth dryness. To overcome these issues, large quantities of soft drinks or beverages and sugar tablets, which can lead to cervical discoloration in both enamel and dentine for a long period, are consumed and may lead to staining of teeth, attrition, and cervical caries at the chewing side ,36. ThisIn this in vitro study, 70 samples were included to assess and measure the effect of KH on the surface roughness, surface topography, and color changes of glazed or polished feldspathic metal ceramic, machinable feldspathic or multilayer CAD/CAM zircon porcelain, and natural teeth. Seventy samples were prepared, fabricated, and divided into the four following groups.There was the natural teeth group, which consisted of 10 extracted maxillary caries-free central incisors collected from surgical clinics. The teeth were polished with rubber cups with fluoride-free prophy paste and then stored in 10% aqueous formalin solution at room temperature (R)13 consisted of 20 samples. These samples were constructed by a conventional method using 0.4 mm-thick green wax with dimensions of 10 mm × 10 mm. The MC samples were invested, burned out, and casted with nickel-chromium dental casting alloy (Wiron(R) 99, Bego, Germany) following the manufacturer’s instructions. The metal samples were cut from their sprues, leaving 2 mm to facilitate handling of the samples during packing of different porcelain layers. The metal samples were sandblasted with aluminum oxide particle to remove the excess oxide layer. A feldspathic ceramic VITA VM(R)13  was used for porcelain build-up. The opaque layer on the metal substrate had a thickness of 0.3 ± 0.1 mm, while the body porcelain had a thickness of 2.0 ± 0.3 mm [The group of the feldspathic metal ceramic VITA VM± 0.3 mm . The felIn the group of machinable feldspathic porcelain Vitablocks Mark II , the samples were prepared by installing the blocks in a milling CAD/CAM machine  to produce 20 discs with dimensions of 10 mm × 10 mm and thickness of 2.0 ± 0.3 mm For the group of pre-sintered zirconia, Ceramill Zolid multilayer PS (preshaded) blocks were used to fabricate the samples by installing them in the milling CAD/CAM machine  to slice 20 disc-shaped samples with dimensions of 10 mm × 10 mm and thickness of 2.0 ± 0.3 mm. All samples were crystallized and sintered following the manufacturer’s recommendations The 70 prepared samples of the four groups were divided into two equal subgroups with 35 samples each. The natural teeth samples comprised the control group with five samples for each subgroup, and measurements were carried out without any surface treatments. The first subgroup samples (10 samples from each group) were obtained from the laboratory with glazed surfaces without further treatment, whereas the samples in the second subgroup (10 samples from each group) were polished with a porcelain polishing kit  to repreAll samples were tested using a profilometer device . The surface roughness was expressed in terms of roughness average (Ra), which is usually measured in micrometer (μm). For each sample, two readings were registered, and the mean values were calculated before immersion in KH and thermocycling. To measure the surface roughness of each sample, the stylus was moved above the surface of the samples twice in three different directions around the center of the sample for a distance of 4 mm according to the profilometer’s instructions to record all peaks and recesses that characterized the surface. This was considered as Ra value before treatment. Ra measurements followed the ISO 11562 recommendations to standardize gaining of the results .® V, VITA, Germany). The spectrophotometer was used to measure the Commission International D’ Eclairage CIE-Lab values of the samples to provide a numerical representation of 3D color measurements. These measurements were previously used in studies assessing the shades of both porcelain and teeth. L*, a*, and b* were measured twice, and the mean value was obtained. The means of color data with the standard deviations of tooth surfaces were calculated as previously described [E* before KH immersion using the equation mentioned earlier. Color was measured for all assigned samples by the same operator under the same settings and gray background. Color measurements were made using Vita Easy shade probe spectrophotometer  values after immersion and thermocycling. All procedures for sample preparation, fabrication, finishing and polishing, surface and color measurements, sample immersion, and thermocycling were carried out by the same operator. The Ra values indicate the average of surface roughness before and after immersion, while ΔE* values refer to the average color changes, which were calculated by the aforementioned equation.After 30 days, the surface roughness and the colors of the samples were measured after KH immersion and thermocycling with the same profilometer and VITA EasyshadeA sample from each type of porcelain in glazed or polished surface was scanned after KH immersion and thermocycling. The surface topography of six samples was represented graphically via white light interferometry  under 50× magnification with back scan and length parameters of 20 µm in VSI/VXI mode to obtain a 3D rendering of the sample surfaces. Vision 64 software , which is part of the GT-K1 system, was used to copy the surface topography parameters.E* differences of natural teeth and porcelain sample restorations (VM(R)13, Vitablocs Mark II, Ceramill Zolid) in terms of glazing and polishing were recorded and then compared before and after KH immersion. Microsoft Excel 13 software was used to input the data, which were analyzed using Statistical Package for Social Science (SPSS) version 22.0 . ANOVA test followed by Bonferroni test were conducted to detect any significant difference between and within the groups at p > 0.05.The average Ra values and the mean color changes ΔDuring statistical analysis, the 70 samples were represented by letters from a to g, in which (a) represented natural teeth before and after KH immersion and thermocycling;  were used for glazed and polished MC, Vitablocs Mark II, and zircon, respectively. p < 0.05, p < 0.05, E* were calculated by the aforementioned equation. The ΔE* values could be arranged as follows: natural teeth > zircon polished or glazed > polished MC > polished Vitablocs Mark II > glazed Vitablocs Mark II and MC. The ΔE* values significantly differed between natural teeth and all tested glazed or polished porcelain  has recommended that khat should be considered as a controlled drug. This in-vitro study was designed to assess the effect of KH immersion with thermocycling on the surface roughness and the color stability of the most used types of porcelain materials in the fabrication of the dental prosthesis. The surface roughness of glazed surfaces was compared with that of polished porcelain surfaces using a manual polishing kit . The preFew studies in relation to KH and dental materials have been conducted. An in-vitro study investigated the effect of khat extract on the color of composite materials . They coThe smoothness, and the surface quality of the prosthetic material are important to achieve the desired aesthetic appearance and long-term clinical success , becauseThe Ra value was used as a roughness parameter in the current study because it is the most commonly used parameter for surface assessment, thereby facilitating a convenient comparison with other studies . The effE* measurements of VITA Easyshade® V spectrophotometer devices are more precise and accurate than those of other digital instruments [The CIE Lab system was chosen in the current study for assessment of chromaticity and to evaluate the average color differences, because it is well suited for the determination of small color differences ,50. The truments ,30. E* were higher in polished samples than in glazed samples. This result supported our findings, even though we used KH immersion, because khat could have an acidic effect on the teeth, resulting in a discoloration effect of KH on teeth surfaces after a certain period of chewing. KH has the fibrous nature of khat leaves; thus, it may cause some mechanical alterations in porcelain surfaces due to the friction mechanism and the hardness of khat [E* (2.12) was slightly high , Saba et al. (2017), Colombo et al. (2017), Alp et al. (2018), and Sarikaya and Guler (2011) evaluated the effect of coffee ,45,48,50 of khat ,58. Howetly high , and thiE* of 2.8 is clinically acceptable [E* values of ≥1 to ≤3.3 can be detected by the eyes and are clinically acceptable [E* ranged from 0.28 for glazed samples of Vitablocs Mark II to 2.25 for polished samples of zirconia porcelain  e.max-tested samples immersed in different beverages with thermocycling [E* values of 0.23–2.52 units, except for samples of natural teeth after (3.45) KH immersion and thermocycling. Therefore, the behavior of all tested samples could be considered acceptable. Algazali et al. performed a clinical in vivo study and found that Δceptable . Al Ghazceptable ,50,60. Torcelain . Similarocycling . This reE* values of monolithic zirconia as well as the feldspathic ceramic materials group; the ceramic became brighter, opaquer, less glossy, and rougher after rinsing with the whitening mouthwash. Additionally, long-term use of specific mouth wash may cause deterioration of optical and surface properties of high-translucency CAD/CAM prosthetic materials. Lee et al. concluded that ultrasonic scaling resulted in significant changes in the surface roughness and showed scrapes as well as surface deterioration of the Vitablocs Mark II ceramic materials after scaling [E* values on the same materials.A group of in vitro studies was carried out by Lee et al. to assess the effect of different types of mouth wash in relation to different type of feldspathic and zirconia CAD/CAM ceramic materials ,62. They scaling . The resIntraoral restoration surfaces can be prepared using various glazing and mechanical polishing techniques. The techniques recommended by manufacturers involve the use of different glazing procedures combined with crystallization or after crystallization and polishing for all porcelain to maintain and obtain color stability, translucency, and surface roughness ,56,60. ALimited studies have used white light interferometer microscope images to examine the surface topography of both traditional and recent ceramic materials after application or immersion of any beverage or stained materials. In this study, the polished samples of zirconia F showed E* of other brands of dental porcelain.Considering that this study was the first to analyze the effect of KH on different types of glazed or polished porcelain and natural teeth, it involved some limitations. First, the current study was an in vitro study and allowed staining on both sides of the material. In clinical situations, the material is cemented to a tooth structure and is exposed to KH and light on the outer surface only. Moreover, the color of the samples is a combination of gray background and the sample’s color, and the average color values may change in oral cavity where different backgrounds exist. The results of this study should be simulated in further clinical studies to mimic the effect of actual khat chewing, which is usually associated with soft drinks and smoking on Ra and ΔE* for both glazed or polished porcelain materials and natural teeth with significant differences among all groups and subgroups, except between natural teeth and zircon in terms of Ra measurements. The limitations of this study could be addressed by further clinical studies to mimic the effect of khat chewing habit on Ra and ΔE* of different brands and recent types of dental porcelain materials.This in vitro study shows that KH exerts significant effects on the overall surface roughness of the examined sample surfaces. The effects were higher on natural teeth and zirconia than on feldspathic porcelain, whether manually packed or machinable by CAD/CAM. KH showed acceptable changes in Δ


Health-related quality of life (HRQoL) serves as a direct measure of individuals’ health, life expectancy and the impact that the utilization of health care has on quality of life. The purpose of this study is to assess the HRQoL of people living with HIV (PLHIV), and to ascertain its association with the social inequalities and clinical determinants among people living with HIV in Benishangul Gumuz Regional State, Ethiopia.A cross-sectional study was conducted between December 2016 and February 2017; 390 people at two referral hospitals and three health centers participated in the study. The Patient-Reported Outcomes Measurement Information System Global Health Scale  was used to measure key HRQoL domains. Global Physical Health (GPH) and Global Mental Health (GPH) summary scores were employed. GPH and GMH summary scores below 50 (the standardized mean score) were determined as poor HRQoL. Bivariate and multivariate logistic regression analyses were used to identify factors associated with GPH and GMH summary scores.This study included 259 (66.4%) females and 131 (33.6%) males. The GPH summary scores ranged from 16.2 to 67.7 with a mean of 48.8 (SD = 8.9). Almost 44.6% of the study population has a GPH summary score of below 50; the GMH summary scores ranged from 28.4 to 67.6 with a mean of 50.8 (SD = 8.1). About 41.8% of the study population has a GMH summary score of below 50. Unemployment, household food insecurity and comorbidities with HIV were associated with both poor GPH and poor GMH summary scores. Age below 25 years and being a member of Christian fellowship were inversely associated with poor GPH. The least wealth index score and CD4 count below 350 cells/mL were also associated with poor GMH.Overall, socioeconomic inequalities and HIV-related clinical factors play an important role in improving the HRQoL of PLHIV. Many of these determinants are alterable risk factors. Appropriate strategies can improve the holistic management of chronic HIV care and maximize PLHIVs’ HRQoL. Such strategies require the adoption of comprehensive interventions, including policies and programmes that would improve the health, wellbeing and livelihood of PLHIV. Quality of life is a broad multidimensional concept that usually includes subjective evaluations of both positive and negative aspects of life ; and hasWorldwide, HIV/AIDS continues to be a major public health problem. By the end of 2018, an estimated 37.9 million people were living with HIV and AIDS globally, and 1.7 million people were newly infected by HIV . To contSocial inequalities in HRQoL affect the treatment outcome and life expectation of people living with HIV –15. SociHRQoL serves as a direct measure of individuals’ health, life expectancy and the impact that the utilization of health care has on quality of life . A few sA cross-sectional study was conducted among PLHIVs at two referral hospitals and three health centers in HIV clinics and pharmacy refill in Benishangul Gumuz Regional State, Ethiopia. These health facilities were purposely targeted as they are utilized by the majority of PLHIVs in that region. Data were collected from December 2016 to February 2017. The sample size for this study was determined using the formula for the estimation of single proportion, n = Ethiopia , the marA structured questionnaire was used for face-to-face interviews with the study participants. Before data collection commenced, the study tool was piloted with a group of health care providers at the selected facilities over a period of 2 weeks to ensure clarity and consistency. The result assisted in ensuring the quality and consistency of the instrument. Data collectors were given training on the objectives of the study and how they could approach patients to gain their confidence. Interviews were conducted while participants were waiting to see ART service providers or to have prescriptions filled. Data collection forms were reviewed daily for completeness. The collected data were entered into a database and processed.HRQoL is the perceived physical and mental health of an individual or a group over time . In thisThe GPH and GMH summary scores are based on PROMIS Global T-score values generated based on the principal component analysis conversion, a method that is used to summarize and group together correlated variables and confirm the presence of a single component. T-score distributions are standardized such that 50 represents the average (mean), and the standard deviation around that mean is 10 points. A high score always represents more of the concept being measured, and poor HRQoL was defined as one standard deviation or more below the PROMIS population norm (PROMIS® 2010:1). The sampling adequacy of the study was assessed by examining the Kaiser–Meyer–Olkin test (KMO test) sampling adequacy study . The KMOTo assess the HRQoL of people living with HIV and ascertain its association with the social inequalities and clinical determinants, we used socioeconomic indicators such as education, income, asset possession, employment status, nutritional status, household food insecurity and sociodemographic data. Sociodemographic data included age, gender, ethnicity and marital status. Clinical determinants included CD4 count, period on ART and history of opportunistic infections. Clinical and laboratory data were collected by means of interviews and medical record reviews.Food insecurity is defined as “the economic and social condition of limited or uncertain access to adequate food” [te food” . The domte food” .Asset possession refers to a household’s possession of assets. Relevant information was elicited by asking participants a series of 13 questions about household assets and housing characteristics. A wealth index was generated using principal component analysis by combining socioeconomic variables and household characteristics [eristics .Malnutrition is the condition that occurs when the body does not get enough nutrients [2). Participants with a BMI of less than 18.5 kg/m2 were considered malnourished. Inclusion of independent variables was based on literature reviews conducted, data availability and theoretical relevance [utrients . This iselevance .SPSS 24.0  was used for data analysis. The GPH and GMH were analyzed based on PROMIS Global T-score values. To arrive at a bottom-line, T-score distributions are standardized such that 50 represents the average (mean), and the standard deviation around that mean is 10 points. A high score of above the PROMIS population norm of 50 represent more of the concepts measured; but a score below 50 indicates poor of the concepts being measured . For thiFor data analysis and sample characterization, descriptive statistics were used, with simple frequency, measures of central tendency and variability. Binary logistic regression analysis was used to find associations between the independent variables and the outcome variable. The outcome variables were dichotomized in relation to PROMIS cut-off measures employed. All the variables showing a significant association in a bivariate analysis at p < 0.25 were entered into a stepwise backward Wald multivariate linear regression model to determine social inequalities and disease-related factors associated with the HRQoL. In the final multivariate analysis, the test was two-sided and p value < 0.05 was considered statistically significant.Table 2.The socioeconomic characteristics of participants were as follows: 36.4% had no formal education. Most of the participants were poor as measured by the wealth index (a proxy measure for asset possession), with 60% of them in the least relative wealth tertile, 19.5% in the middle, and only 20.5% in the highest relative wealth tertile. More than a quarter of participants were unemployed (31%). A large number of the participants (67.7%) lived on a poor mean monthly income of 1,260 Ethiopian Birr , which is below the World Bank absolute poverty threshold of US$1.90/day. The vast majority of the study participants (76%) were food insecure and (60%) had a BMI of less than 18.5 kg/mAll participants had been on ART follow-up for a mean duration of 2.49 years (SD = 0.77 years), while 7.7% of them had been on ART for more than 10 years. The mean CD4 count was 559 (SD = 319.6) cells/mL, with a range of 60 to 1,914 cells/mL. More than a quarter of them (33.6%) were frequently ill, with comorbidities in the previous three months that varied from pneumonia and tuberculosis to numerous opportunistic infections. Most of the participants were followed up at a public hospital (74%) and at a health center (26%).The GPH summary score ranged from 16.2 to 67.7, with a mean of 48.8 (SD = 8.9). Almost 44.6% of the study population had a GPH summary score of below 50. The GMH summary score ranged from 28.4 to 67.6, with a mean estimated at 50.8 (SD = 8.1); 41.8% of the study population had a GMH summary score of below 50.Sociodemographic and economic inequalities, and clinical determinants associated with HRQoL of PLHIV were explored using bivariate logistic regression analysis and subsequent multivariable logistic regression analyses. Age, ethnic group, religious affiliation, place of residence, income, food security status, wealth index and current CD4 count have a statistical association with the GPH quality of life domain; while gender, age, marital status, education level and duration on ART were among the factors not showed statistical association with GPH in the bivariate analysis. Table In multivariate analysis, age, religious affiliation, employment status, household food security status and comorbidities have a statistically significant association with GPH. Those PLHIVs aged below 25 years was found 0.4 times less likely to be associated with poor GPH . Unemployed PLHIVs were found almost twice more likely to have poor GPH than employed individuals . In addition, food-insecure participants were twice as likely to have a poor GPH . Coexistence comorbidities with HIV in the last three months is found associated with high odds of being in the state of poor GPH .In bivariate logistic regression analysis, employment status, income, household food security, BMI, wealth index, CD4 cell count and comorbidities with HIV were found to have a statistically significant association with GMH . Food-insecure people living with HIV were around 6.43 times more likely to have poor GMH than their food-secure counterparts . Participants in the third tertile of the wealth index  were almost twice as likely to have poor GMH than those in the second tertile of the wealth index . It was also shown that those PLHIVs who reported to have a current CD4 count below 350 cells/mL were 1.91 times likely to have poor GMH than those whose CD4 count was above 350 cells/mL . Likewise, those PLHIVs who were sick with HIV/AIDS-related comorbidities were found 1.52 times more likely to have poor GMH than those without the conditions .Our study used the Patient-Reported Outcomes Measurement Information System® (PROMIS®) instrument to assess the HRQoL among PLHIV in Benishangul Gumuz Regional State, Ethiopia, and attempted to demonstrate the importance of factors in the sociodemographic, economic and clinical variables to quality of life. Overall, PLHIVs in this study have shown to have poor GPH  in comparison to GMH . Affiliation to the Christian religion group, unemployment, household food insecurity and comorbidities with HIV were identified as the most important socioeconomic and clinical determinant factors of poor global physical health related quality of life among PLHIVs. Participants aged younger than 25 years were found to have good GPH summary scores in comparison to participants older than 25 years of age.Our findings correspond with the reports of Abera et al.  that theSeveral studies have shown that certain sociodemographic factors, namely religious affiliation, is associated with quality of life among PLHIVs , 44, 45.Low levels of socioeconomic status, which are characterized by unemployment and food insecurity, are strongly associated with poor GPH score domains of HRQoL. Our study shows that unemployed PLHIVs are nearly twice more likely to have poor GPH. Likewise, it has been shown that unemployment leads to poor HRQoL among PLHIV in both developed and developing countries , 18, 50.Unemployment and food insecurity have also been found to be predictors of poor global mental HRQoL in our study. Like previously reported studies , 53, 54,This study also shows that the third tertile on the wealth index (or the poorest PLHIVs in terms of asset possession) has a strong association with GMH, which is consistent with reports by Weldsilase et al.  and AbebIt has been found that there is a strong correlation between HIV-related clinical outcomes, such as CD4 < 350 cells/mL, and poor GMH summary scores. Comorbidities with HIV are strongly associated with both poor GPH and poor GMH summary scores. Like our study, studies in Nigeria , SoutherThe limitations of this study include its cross-sectional nature, which may preclude conclusions on causality. Hence the exact direction of the associations between predictor variables and HRQoL cannot be established. Recruitment of participants from the outpatient clinics and exclusion of hospitalized PLHIV may have altered the HRQoL findings. Finally, other confounding factors such viral load, clinical stage, alcohol use and smoking were not considered in the questionnaires. For further exploration, we would take these factors into consideration.The study reveals that socioeconomic inequalities are important determinants of improved HRQoL among PLHIVs. Factors such as being older than 25 years, unemployment, household food insecurity and comorbidities with HIV are significantly associated with poor physical HRQoL. Conversely a Christian religious affiliation is associated with higher global physical health-related quality of life. Unemployment, food insecurity, least wealth index score, recent CD4 count below 350 cells/mL and comorbidities with HIV are associated with poor global mental HRQoL. Many of the predictors are alterable risk factors. Appropriate strategies can improve the holistic management of chronic illness and maximize PLHIVs’ HRQoL. Improving the HRQoL of PLHIVs requires comprehensive intervention strategies, including policies and programmes that will improve the health, wellbeing and livelihood of PLHIVs. For instance, age-specific physical health strengthening activities for adults over 25 years of age improve physical HRQoL. Faith-based behavioral and physical intervention strategies improve religious determinants of poor physical HRQoL, and tackling the issue of unemployment and food insecurity improves the physical HRQoL , 45, 51.


Pregnancy dramatically changes the metabolic status of bitches. As a consequence, malnutrition negatively affects both reproductive parameters and survival rates of puppies. Therefore, before mating and conception, as well as during pregnancy and lactation, it is necessary to satisfy the bitches’ specific nutritional requirements. The present study aimed to compare the effects of two diets, Control (CTR) and Experimental (EX), which differ mainly as regards ingredients, energy, and protein contents, and which were administered to bitches of medium and large size from two months before the expected onset of proestrus up to 30 days after delivery. Bitches’ weight, body condition score, litter size, milk yield and quality, and puppies’ growth were evaluated. Considering the performance of the bitches and the puppies, it would seem that both the diets are adequate for bitches at these stages of their life cycle. In fact, after 30 days of lactation, all tested bitches showed a healthy status. Moreover, both the percentage of newborns mortality and puppies’ growth kinetics fell within the normal physiological range for the species.p < 0.01) higher in the EX group than in the CTR one. Concerning the size effect, the percentage of weight gain on the initial body weight was double in medium-sized dogs compared to large dogs (p < 0.01). The number of puppies per litter was significantly higher (p < 0.05) for the EX group compared to the CTR one. Concerning puppies’ weight, the CTR group showed a significantly (p < 0.01) higher body weight from the 21st day of life due to the significant (p < 0.01) higher daily weight gain during the suckling period. Considering the performance of bitches and puppies, both diets seem useful for these stages of their lives. In fact, after 30 days of lactation, all tested bitches showed a healthy status and both the percentage of newborns mortality and puppies’ growth kinetics fell into the normal physiological range for the species.The study aimed to compare two diets, Control (CTR) and Experimental (EX)  administered to medium and large-sized bitches from two months before the expected proestrus and up to 30 days after delivery on mothers’ weight, body condition score, litter size, milk quality, and puppies’ growth. No differences were found for body weight during pregnancy, even if the BCS after delivery was significantly ( The impact of nutrition during pregnancy and early lactation on reproduction has been already recognized for some species ,2. By coIn all mammals, the metabolic status changes dramatically during pregnancy, and as a consequence, any mistake may negatively affect the reproductive parameters both in terms of deficiencies and excesses ,4. For tThe assessment of a specific feeding plan for the bitches should be carried out routinely. In bitches, during the last three weeks of pregnancy, energy requirements for maintenance increase progressively from 1.25 to 1.50 times to sustain fetus development. Before and during pregnancy, certain macro- and micro-nutrients play specific roles in hormone production, placentation, and fetal development , therebyDuring lactation, the bitches’ daily energy requirements increase up to 2–4 times the levels required for maintenance ,11,12, cThe lactation efficiency depends on the fat content in the diet. Increasing lipid percentage from 12 up to 20 on a dry matter basis (DM) causes the milk fat to increase by about 30% . This laThe present investigation aimed to compare two diets  administered to medium and large-sized bitches from two months before the expected onset of proestrus up to 30 days after delivery. Mothers’ weight, body condition score BCS, litter size, milk yield and quality, and puppies’ growth were monitored. We hypothesized that a specific diet, formulated with a higher concentration of macro and micro-nutrients might be able to improve bitches’ performance and puppies’ growth.All the procedures used in the study were approved (PG/2020/0044625) by the Ethical Animal Care and Use Committee of the University of Naples Federico II following local and national law regulations and guidelines .Twenty-two privately owned pluriparus 5 years old bitches were recruited for the study at a private veterinary clinic three months before the expected onset of proestrus and they were equally divided into two groups . During the trial, 4 owners decided to leave the experimental protocol, consequently, we could follow only 18 bitches up to 30 days after delivery. The breeds of the dogs were as follows: Bernese Mountain dog (2), French Bulldog (2), English Bulldog (3), Dobermann (1), Argentine Dogo (2), Pitbull (1), Great Danes (1), Golden Retriever (1), Hovawart (1), Labrador Retriever (2), German Shepherd (1), Whippet (1). Control group included 4 dogs of medium size (body weight <30 kg) and 5 of large size (body weight >30 kg) with a mean body weight after delivery equal to 32.6 ± 13.5 kg, mean BCS  equal to 2.7 ± 0.3.Experimental group consisted of 2 bitches of medium size and 7 of large size, with a mean body weight after delivery equal to 39.7 ± 14.8 kg mean BCS  equal to 3.4 ± 0.4.Taking into consideration their initial body weight, the groups were constituted as follows:Two diets differing as regards ingredients (starch and protein sources), energy, protein, and folate levels, named respectively CTR  and EX , were used . Both diBoth diets were supplemented with a mix of vitamins and microminerals in proportion to their specific energy level. In addition, both diets were supplemented with different levels of folate .Each group was fed one of the diets starting two months before the expected onset of proestrus until the puppies were weaned. During the trial, the daily individual rations were calculated according to NRC  indicatig for 20 min to obtain serum. The bitches were weighed the day after delivery and after 3 weeks of lactation when blood was collected from the cephalic vein and centrifuged at 1500× Serum chemistry analyses were performed by an automatic biochemical analyzer AMS AUTOLAB  using reagents from Spinreact  to determine: blood urea nitrogen (BUN), creatinine (CREA), glucose (GLU) total proteins (TP), albumin (ALB), bilirubin (Bil T), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), cholesterol (COL T) and triglycerides (TRI), chlorine (Cl) sodium (Na), calcium (Ca), and phosphorus (P).Reactive oxygen metabolites (d-ROMs test) and biological antioxidant potential (BAP test) were also measured on serum aliquots using reagents from Diacron International s.r.l.  validated for canine species.After delivery, 15 mL of milk were collected three times  from different nipples. Milk samples were transported into pre-cooled boxes (3.0 ± 1.0 °C) to the Laboratory of Chemical analysis at the Department of Veterinary Medicine and Animal Production within 3.30 h of collection to determine fat and protein content (procedure 991.21 and 2000.18) [The puppies were weighed daily by the owners during the first 10 days of life and then weekly until 30 days of age. From birth, puppies were clinically monitored every week until the end of weaning (60 days after birth) by checking heart rate, respiratory rate, rectal temperature, urinalysis, and fecal exams. The data were analyzed by one-way ANOVA  according to the following model: When significant differences were found in the ANOVA, means were compared using Tukey’s test.Litter size data were analyzed using the Wilcoxon non-parametric test.The correlations between serum and milk parameters and between milk composition and puppies’ performance were also evaluated .p < 0.01) higher in the EX group than in CTR one. During the first month of lactation, no differences in body weight or BCS were found between groups. In p < 0.01). No differences between size groups were shown for the body condition score. All the bitches showed an increase in body weight during pregnancy and a decrease in the first month of lactation.Concerning the size effect, the percentage of weight gain on the initial body weight was double in medium-sized dogs compared to large-sized dogs (p < 0.01) than for larger dogs. The weight loss observed during lactation was higher (p < 0.01) for the bitches of the EX group which, after 30 days of milk production, showed a mean bodyweight lower than it initially was, whilst the LWi of the CTR group was higher than it initially was . On the other hand, the body condition score trend was quite similar for both treatments. The weight gain registered during pregnancy for medium size bitches was significantly higher (p < 0.05 for EX and CTR one).Significant differences were observed for the number of puppies born per litter (litter size)  higher bodyweight from the 21st day of age due to the significant (p < 0.01) higher values of daily weight gain during the suckling period 0–28 d (124.87 vs. 97.97 g/d) compared to EX group. Regarding the effect of dog size, significant differences (p < 0.01) were registered every week, except for the fourth one. However, the weight gain registered throughout the trial was similar for both large and medium-sized puppies. During the first week of life puppies of the EX group doubled their body weight, whereas CTR group puppies increased their weight by 1.39 times (p < 0.01). The dogs’ size did not affect the relative body weight gain during the first week of life .Perinatal mortality (puppies died during the first week) was on average in the two groups equal to 6.8% . In p < 0.05) than the control group. Medium-sized bitches were higher in Ca*P and P (p < 0.05) than the large-sized ones.Concerning bitches’ biochemical parameters evaluated after 3 weeks of lactation , only a p < 0.05), although the percentage of protein in the milk was not correlated with BUN of the bitches’ serum. Milk composition and, in particular fat percentage, was significantly and negatively correlated with puppies’ weight gain . Regarding blood parameters, Ca and d-ROMs levels were significantly related to puppies’ weight at 21 d .A significant correlation between serum parameters and milk composition was observed. More particularly, the percentage of milk fat was negatively related to serum glucose and cholesterol levels , thus limiting excessive weight at the end of pregnancy (into the range of 5–10%) indicated as ideal by Greco . On the Must it be taken into account that the use in the trial of different dog breeds, instead of a specific breed, could represent a limit for the study. Nevertheless, the use of a cohort of dogs heterogeneous, in terms of size and breeds, could reveal general effects not segregated on race specificity. Middelton et al. , evaluatOur data underline some significant variations in milk composition during the suckling period. Regardless of the nutritional treatment, the large-sized bitches showed higher milk protein and fat contents throughout the lactation period. Only a few data is available regarding the composition of dog milk. Our results are in contrast with those reported by Lönnerdal et al.  who founThe performance registered in puppies  during the first month are in line with the range indicated by Fascetti  who suggNo statistical differences were detected between the groups as regards all biochemical parameters, thus suggesting that both diets were well tolerated. The same can be said as regards the evaluation of oxidative status. The d-ROMs levels observed in both groups fell in the normal range values for the canine species proposed by Pasquini et al.  and by SFolate supplementation is recommended in humans during pregnancy to prevent malformation, particularly neural tube defects. Elwood & Colquhoun  reportedThe higher level of Ca/P and phosphorus registered in the control group suggests that the higher concentration of calcium and phosphorus in the experimental diet was not sufficient to satisfy the bitches requirement, although these bitches may have compensated for this shortage with their organic reserve during lactation. Usually, breeders and practitioners tend to over-supplement bitches’ diets during gestation and lactation with different minerals, such as calcium. This practice could be inappropriate for the bitches’ health. Even if the Ca requirement at these stages could be higher than adult maintenance ones, oversupplying Ca is not beneficial: when the Ca administered from the beginning of pregnancy is over supplemented, serum calcium concentration remains high and results in a down-regulation of parathyroid activity . It is wThis mechanism could explain the similar levels of serum calcium in EX and CTR groups, despite the higher Ca concentration in the EX diet than the CTR ones. Further studies are needed to investigate the causes of the increase in serum Ca/P and P levels observed in the dogs in the EX group.In any case, the significant correlation between bitches’ serum calcium level and puppies’ weight gain observed in this trial seems to confirm the role of this mineral in healthy fetal development.In conclusion, the experimental diet seemed to guarantee higher performance in terms of litter size and correct weight gain during pregnancy than the control one. Nevertheless, milk composition and weight loss after 30 d of lactation were more favorable for the control group. This data is probably due to the higher milk yield necessary to maintain the growth of a higher number of puppies in the experimental group. This hypothesis seems confirmed by the higher relative weight gain registered during the first week of life in the CTR group. The supplementation in the EX diet, mainly regarding Ca, P, DHA, EPA, and folate did not lead to significant health differences in bitches or puppies. Importantly, the EX diet failed to improve the oxidative status thus suggesting that different doses or substances should be tested to achieve such a goal. Moreover, Ca and P supplementation did not optimize their serum levels, suggesting that the influence of these two minerals intake on mineral metabolism in bitches needs to be further studied.This study has some limitations: (1) the use in the trial of different dog breeds instead of a specific dog breed; (2) the categorization of dogs’ size only in terms of body weight (>30 kg and <30 kg); (3) grouping of breeds into CTR and EX groups it’s not completely balanced; (4) the limited sample size (9 dogs per group).


Viruses interfere with their host’s metabolism through the expression of auxiliary metabolic genes (AMGs) that, until now, are mostly studied under large physicochemical gradients. Here, we focus on coastal marine ecosystems and we sequence the viral metagenome (virome) of samples with discrete levels of human-driven disturbances. We aim to describe the relevance of viromics with respect to ecological quality status, defined by the classic seawater trophic index (TRIX). Neither viral (family level) nor bacterial  community structure correlated with TRIX. AMGs involved in the Calvin and tricarboxylic acid cycles were found at stations with poor ecological quality, supporting viral lysis by modifying the host’s energy supply. AMGs involved in “non-traditional” energy-production pathways  were found irrespective of ecological quality, highlighting the importance of recognizing the prevalent metabolic paths and their intermediate byproducts. Various AMGs explained the variability between stations with poor vs. good ecological quality. Our study confirms the pivotal role of the virome content in ecosystem functioning, acting as a “pool” of available functions that may be transferred to the hosts. Further, it suggests that AMGs could be used as an ultra-sensitive metric of energy-production pathways with relevance in the vulnerable coastal zone and its ecological quality. Coastal marine ecosystems are frequently under the intense pressure of a wide range of anthropogenic activities, despite their high productivity and provision of goods and services with great socioeconomic imprint ,2. IndeeWithin this context, studying the structure of microbial plankton has recently emerged as a complementary approach to characterize ecosystem status e.g., ,4). Micr. Micr4])While it is nowadays recognized that prokaryotes  rely upon viruses in multiple ways , it is rHere, our aim is to examine the whole viral metagenome (virome) in direct relation to human-driven chemical alterations in the coastal zone. More specifically, we attempt to detect differences in viral diversity and AMG content that reflect overall good vs. poor ecological quality. Based on AMG composition, we aim to assess the potential role of viruses in regulating critical host functions under continuously changing conditions in polluted sites. We chose to detect these differences along the Greek coastline under the European Water Framework Directive WFD, EC 2000) for two reasons: firstly, because the coastline belongs to the top-priority regions for conservation planning in the Mediterranean Sea ,18,19. S00 for tw−1 FeCl3 to achieve viral particle flocculation within the following 6–12 h [Surface seawater (2 m) was collected from 14 coastal stations in the Ionian and South Aegean seas a within g 6–12 h  and filtSamples were grouped according to the yearly estimated trophic index (TRIX) of the surface waters (2 m), using the formula described in  that con1,12 = 2.65, p < 0.05, To statistically substantiate the grouping of the particular sampling stations based on the annual surface TRIX into “Good” versus “Moderate–Poor–Bad,” we tested their similarities on physicochemical properties, using only the measurements of the specific sampling campaign  profiler was deployed (Sea-Bird SBE19) to measure temperature and conductivity and to further calculate salinity and density. The concentration of chlorophyll was determined fluorometrically based on  Supplem. The samp < 0.05 [Viral dsDNA metagenome preparation started by firstly resuspending the 1μm polycarbonate filters in an ascorbic acid buffer . Subsequp < 0.05  and VirSp < 0.05 . VirSortp < 0.05 ), while p < 0.05 , while tp < 0.05  was usedNormalization of AMG copy numbers was done using the viral-abundance results of flow cytometry. Firstly, a normalization factor  was calcShannon–Wiener’s diversity index was calculated using the PRIMER DIVERSE routine ,40. The Principal coordinates analysis was applied to coordinate the data . Signifi1,12 = 1.76, p > 0.05). Irrespective of ecological quality, Podoviridae  where industrial activity is the largest source of pressure on the total Greek coastline, and this outcome resembled that of other polluted areas too [T4-like viruses that were detected in higher abundances).al scale ,48. Siph contigs a. The hiPhycodnaviridae was reported at Good stations reaching on average 5% ± 2% of the assigned reads compared to MoPoB, which was 2% ± 2% (Phycodnaviridae detection was confirmed by the flow cytometry enumeration of high DNA virus-like particles (VLPs) that mostly infect eukaryotic phytoplankton [p < 0.05). The detection of high DNA VLPs and Phycodnaviridae suggests the presence of eukaryotic algae in the Good group of stations [Minor taxonomic differences were detected in two “rare” viral families: a higher percentage contribution of  2% ± 2% . Phycodnplankton . Indeed,stations , which wPhycodnaviridae, the biotechnologically and ecologically important Inoviridae family was also not broadly distributed; it reached up to 0.5% of the total assigned reads , with the concentration of NO3 being the most important . In our study, the average concentrations of NO3 as well as other nitrogen forms  and particulate organic carbon concentration (POC) increased with decreasing ecological quality  did not allow us to draw a firm conclusion about the relation between nutrients and viral community patterns in the examined coastal sites. In other words, while the relation detected here between the viral community and dissolved NO3 was surely expected considering previous macro-diversity studies that revealed the strong dependence of the viral community patterns on the environment [A suite of abiotic variables a explain quality . Howeverironment ,53, we wThe need for such validation is particularly important considering that the input of dissolved nutrients at the impacted sites is intense, but frequently the detected concentrations of nitrogen and phosphorus are quite low, reaching oligotrophic levels . This paradox may indicate (1) fast consumption (2) fast integration into particulate forms and/or (3) removal toward deeper layers in the water column for the dissolved nutrients. These processes have been reported for other regions that receive large inputs of nutrients such as aquaculture farm zones ,55. The To sum up, we found that viral taxonomy did not differ according to the ecological quality of the sampling stations at the family level, rejecting our first hypothesis. However, it seems absolutely necessary to evaluate the same hypothesis using viral-genera variations that would probably reveal additional differences. At the family level, viral populations seemed to be uniformly distributed, confirming viromics surveys larger than ours, and highlighting the strong connectivity within the oceans ,56,57,581,11 = 4.64, p < 0.05). The Shannon–Wiener diversity index of AMGs was on average 5.13 ± 0.06 with small variations between the ecological quality groups ; the highest numbers of AMGs were counted along with the highest organic matter loading  based on the SIMPER test.Stations were significantly different in their total AMG content based on seawater ecological quality  were significantly more abundant in stations with poor ecological quality , the latter observation for the rest energy-production AMGs participating in the Calvin cycle inhibition, suggests that a redirection toward accumulation of ATP, NADPH, and Ru5P for subsequent enhanced dNTP synthesis [rpe, icd, eno, gltA and sucA, sucB, sucC and sucD  eventually support viral lysis by modifying the energy supply to the host. It is noteworthy though that the genes involved in the TCA cycle were exceptionally abundant in the Amvrakikos gulf (3 stations). This gulf receives large amounts of dissolved nutrients due to riverine biogenic inputs and human activities (agriculture and mariculture), which would not support this “starvation” response. However, the fast consumption of the added nutrients and their fast integration into particulate forms (assimilation in the food web), as well as their removal to the deep may lead to the unexpectedly low levels of dissolved nutrients  and, furkos gulf d, i.e., kos gulf . acc, mcm, and fum were significantly higher at MoPoB than at Good  cycle AMGs currence . The precurrence , indicatpetF, encoding for ferredoxin, was more abundant at MoPoB than at Good stations . At the phylum level, significant differences were found between the two groups of stations . The abiotic variables that explained bacterial community patterns at this level were the concentrations of NO2, NO3, NH4 and SiO4 as well as water conductivity , while at the family level the variables were the same and in addition, the concentrations of Chl α, total nitrogen, and particulate phosphorus , similarly to the test for the viral community presented above. Bacterial community composition at the genus and family levels did not differ between stations based on ecological quality (TRIX range)  compared to Good (81% ± 5%), followed by a similar pattern of Actinobacteria: 6% ± 6% and 3% ± 2% at MoPoB and Good, respectively  than MoPoB  but a highly variable one at Good . Shannon–Wiener diversity based on the bacterial operation-taxonomic units (out) data was on average 5.11 ± 0.60 (family level), with most of the lowest values found at MoPoB . The domectively b. Actinond waste . On the 1% ± 1%, b, confir1% ± 1%, . On the Caulobacteraceae, Oxalobacteraceae, Hyphomicrobiaceae, Microbacteriaceae, Saprospirae, SAR324, Methylophilaceae, and Thiohalorhabdales were detected at low percentage contributions throughout the coastal zone, indicating the widespread presence of sewage-related and heavy-metal-tolerant taxa. Several pathogens were also detected at all stations, including Streptococcus, Lactobacillus, Campylobacterales, HTCC2188, HTCC2207, Bacteroides, Clostridia, Enterobacteriaceae, and Dechloromonas [Bacterial taxa indicative of pollution and, more specifically, of raw anthropogenic inputs  were fouoromonas . Our study confirmed that bacterial as well as viral community structure does not depict the ecosystem’s ecological status based on classic ecological indices such as the TRIX. This lack of dependence between community composition and trophic status  may lead to improper management of polluted regions. In fact, newly developed ecosystem-management tools consider the microbial community composition  and reseattP copies were putatively assigned to bacterial hosts based on exact matches with the bacterial attB  or throial attB ; Spirochet al. [Nowadays, one of the key questions in marine ecosystem monitoring and conservation is which biological component(s) should be monitored for the detection and management of pollution. As Nõges et al.  recentlyWe detected genes involved in redox balance, sulfur-, and fatty acid- based metabolism as well as anaerobic metabolism in areas that would be characterized as having good ecological status based on classic indices . Although we do not provide information on actual viral gene expression, the potential for host-metabolism redirection remains at a dormant-like state within the available pool of viral functional genes. This pool can act as an alternative ultra-sensitive metric of energy-production pathways  in vulnerable ecosystems where the physicochemical conditions change rapidly. As discussed above, such an approach requires further investigation with a virome dataset larger than the presented one, as well as confirmation of respective bacterial functions with transcriptome analysis and elimination of the possibility to confuse bacterial with viral AMGs. We also found that neither the viral nor the bacterial taxonomy at the family or genus level differed based on the ecological quality of the ecosystem. “Typical” bacterial and viral taxa of polluted sites were found under all levels of ecological quality. Thus, taxonomic differences cannot yet be evaluated in an ecological context, while the diversity of AMGs can uncover the direction of energy production and organic matter circulation. Transcending the era of trying to answer “who does what?,” studying the pool of available metabolic functions within both the viral and host metagenomes may be more meaningful than studying the mere community structure, especially under the scope of the drastic anthropogenic impacts on the marine ecosystem.