CA6 gene on oral microbiota and specifically the acidophilic and caries-associated Streptococcus mutans in 17-year old Swedish adolescents (n = 154). Associations with caries status and secreted CA6 protein were also evaluated. Single Nucleotide Polymorphisms (27 SNPs in 5 haploblocks) and saliva and tooth biofilm microbiota from Illumina MiSeq 16S rDNA (V3-V4) sequencing and culturing were analysed. Haploblock 4  CCC associated with low prevalence of S. mutans (OR (95% CI): 0.5 ), and caries ), whereas haploblock 4 TTG associated with high prevalence of S. mutans ) and caries ). The TTG-haploblock 4 (represented by rs12138897(G)) was characterized by S. mutans, Scardovia wiggsiae, Treponema sp. HOT268, Tannerella sp. HOT286, Veillonella gp.1 compared with the CCC-haploblock 4 (represented by rs12138897(C)). Secreted CA6 in saliva was weakly linked to CA6 gene variation. In conclusion, the results indicate that CA6 gene polymorphisms influence S. mutans colonization, tooth biofilm microbiota composition and risk of dental caries in Swedish adolescents.Carbonic anhydrase VI (CA6) catalyses the reversible hydration of carbon dioxide in saliva with possible pH regulation, taste perception, and tooth formation effects. This study assessed effects of variation in the  In mammalian cells, CAs are involved in several biological processes, including HCO3− dependent metabolic processes, secretion of electrolytes, respiration, pH regulation, bone resorption, biomineralization, and odontogenesis2. Today, 16 different CA isozymes have been identified in humans1, of which 13 have been shown to be enzymatically active. Some isozymes are expressed in most tissues, while others are tissue- or organ-specific. Five isozymes are cytosolic , five are membrane-bound , two are present in the mitochondria (VA and VB), and one  is a secreted isozyme4.Carbonic anhydrases (CAs) are zinc enzymes that catalyse the reversible hydration of carbon dioxide in the reaction CO5, and incorporated in the protein pellicle on tooth enamel and in dental biofilms8. CA6 supports neutralization of lactic and other bacteria-produced acids through conversion of saliva HCO3− to water and carbon dioxide and pH maintenance through the subsequent phase buffering9. CA6 is therefore suggested to be an important enzyme in oral physiology and tooth tissues integrity, i.e., resistance to caries and dental erosion10. However, data are conflicting as both high and low concentrations and activities of CA6 have been associated with caries12.CA6 is secreted into saliva with considerable individual variation in concentration and activityCA6 gene have been linked to CA6 concentration and activity in saliva and to caries status17, but findings are not consistent18. However, no study has confirmed the association between CA6 in saliva and S. mutans colonization in humans and no study has investigated CA6 polymorphism and levels of caries associated bacteria or overall oral microbiota.Single nucleotide polymorphisms (SNPs) and multisite haploblocks in the CA6 gene region on oral microbiota and specifically the acidophilic and caries-associated S. mutans in Swedish adolescents. Secondary aims were to evaluate associations between variation in the CA6 gene region and secreted CA6 protein and caries status.The primary aim of this study was to evaluate the effects of genetic variation in the S. mutans, with significantly higher proportions among the caries-affected than caries-free subjects , 75.3% of participants had detectable  p < 0.00; Table 1After quality control measures, 27 out of 30 Single Nucleotide Polymorphisms (SNPs) were retained for analyses. Two SNP (rs2274333 and rs17032942) were excluded due to sample call rate of 0%. An additional SNP (rs6697763) was excluded from analysis for deviation from Hardy-Weinberg equilibrium (p = 0.033), leaving 27 SNPs for further analyses.CA6 gene resulted in five haploblocks  covered the 2477 to 4512 bp region; haploblock 3 with three SNPs  covered the 12,141 to 15,160 bp region; and haploblock 4 with three SNPs  covered the 24,479 to 26,010 bp region of the CA6 gene.Evaluating potential multisite haploblocks within or around the cks Fig. . HaploblS. mutans in saliva or tooth biofilm by NGS , rs3737665 (T), rs12138897 (G), rs3765964 (A), and rs6680186 (A) were associated with increased odds of detectable S. mutans, whereas the GCAC-haploblock 2, and TCG and TTG haploblock 4 associated with increased odds  (p = 0.011), and if carrying the TTG allele versus the CCC haploblock 4 it was 3.3 (1.5–7.6) (p = 0.004).Further, the CCC-haploblock 4 and the AG-haploblock 5 associated with reduced odds to have S. mutans free , rs12138897 (G), rs6680186 (A), and rs7513804 (T), were associated with having mutans streptococci by saliva culturing (Table S. mutans status (as well as caries status as described in the next paragraph) led us to evaluate the association between the C and G allele of rs12138897 (C representing the CCC- and G- the TTG haploblock) and overall tooth biofilm microbiota by multivariate Partial Least Squares (PLS) analysis of NGS determined taxa. PLS modelling  displayed separation of participants carrying the G versus C allele based on their tooth biofilm microbiota  in univariate analysis.The association of the CCC-haploblock 4 and the TCG-haploblock 4 with ota Fig. . The sepbe1 Fig. . The C ae 2 Fig. . Five ofCA6 gene variation was associated with colonization of caries associated bacteria, such as S. mutans and S. wiggsiae, the association between the 27 SNPs and caries status (yes or no) was tested in an allelic association model. The rs12021597 (A), rs10864376 (T), rs3737665 (T), and rs12138897 (G) associated positively with having caries  were associated with caries status . The median  concentration of CA6 in saliva was 8.4  µg/mL and secreted CA6 11.0  µg/min Table .S. mutans or not, but secreted amounts (µg/min) was significantly higher in caries-free than caries-affected (p = 0.012) (Table between groups = 0.007) with an OR to be caries-free of 3.5 .CA6 in saliva (concentration or secreted amounts) was not associated with having d p = 0.02  as did the trend from the TCG to CCC variant (p < 0.001). The association with caries was less strong, but the proportions caries-affected participants tended to differ between the allele variant groups (p = 0.054) with a significant trend (p = 0.021), whereas caries prevalence (DeFS) did not differ between the groups though the trend did (p = 0.018).A summary of characteristics for the various allele variants in haploblock 4 is presented in Table CA6 gene is relevant to dental health and found that CA6 gene polymorphisms and haploblocks of CA6 were associated with S. mutans colonization, overall microbiota composition and dental caries in Swedish adolescents. Secreted CA6 in saliva tended to be linked to CA6 gene variation. Thereby, it is the first study to demonstrate effects of CA6 gene polymorphisms on the oral bacterial communities in a population-based setting.This study tested the hypothesis that variation in the CA6 polymorphisms in oral health, i.e., balancing less, against more, aciduric species, e.g., cariogenic S. mutans and S. wiggsiae, and de- and remineralization in the caries process. The results are in line with previous studies reporting a role of CA6 in caries development in children17. Though several of the physiological effects of CA6 are relevant for both oral microbiota ecology and the caries process, the detailed mechanisms for the effects remain to be elucidated. The underlying mechanisms for oral health of CA6 and CA6 gene variations may, alone or in concert, include tooth formation, local pH regulation, bacteria adhesion and metabolism and taste induced food preferences.Collectively, the present findings support a role of 2. Differences in enamel quality affects the initial tooth covering protein pellicle, and consequently the profile of attachment epitopes for bacteria19 and possibly oral microbiota composition. Differences in enamel quality also affects resistance of tooth tissue to destruction in an acidic environment.A tooth formation mechanism appears plausible because CA6 is expressed in the bud, cup, and bell stage of early tooth development, and by pre- and early differentiated odontoblasts and dental papilla mesenchyme cells, but most avidly in late tooth development with formation and maturation of the enamel20. The CA6 protein is suggested to regulate pH in secreted fluids1, and saliva buffer capacity was reported to differ by CA6 gene variation at rs227432716, and CA6 protein activity in some12, but not other5, studies. This aspect could not be tested in the present study as neither saliva buffer capacity nor pH were analyzed, but some gene variants were associated with presence of aciduric and acidogenic species, such as S. mutans and S. wiggisae, which are promoted at low pH. Since reported intake of sweets did not differ by CA6 gene variation this may reflect a less well functioning pH regulation by some CA6 gene variants. Impaired pH regulation may also increase or decrease the risk of tooth de- and remineralisation.Given the importance of pH homeostasis for the oral environment, local pH regulation by CA6 may be a contributory mechanism. Several of the most abundant bacteria in the mouth are glycolytic with avid acid formation, leading to pH fall and enrichment of aciduric, cariogenic species (dysbiosis)G and CCC associated SNPs were linked with mixed panels of oral bacterial such as S. mutans and S. wiggsiae, but also Treponema sp. HOT268, and Tannerella sp. HOT286 which thrive at a neutral to slightly basic pH. However, these suggested mechanisms are theoretical and need to be evaluated in future studies.Other important factors in oral biofilm ecology include access to attachment sites on host- and partner bacteria structures, and exposure to oxygen and nutrients. CA6 incorporated in pellicle and tooth biofilm may influence bacterial ecology and metabolism through effects unrelated to it’s enzymatic activity. Such effects may be two-fold; i.e., by proteolytic release of bioactive peptides or by providing a bacteria binding site. Here, the TTCA6 gene variation has been suggested to link to bitter taste and smell perception27. To date, reported associations with food selection are inconsistent and the present study did not find any effect of CA6 polymorphisms on sweet snack intake. This may reflect a “true” lack of association or the inherent error in measuring sugar intake in general and especially in the dental office. This aspect as well as the reported association between bitter taste receptor stimulation and innate immunity function28 should be addressed by a study design that is less prone to bias, such as using a biomarker or genetic marker reflecting sweet intake.Finally, CA6, allowing fine-mapping of both single genetic variants and recombination blocks. The phenotypes were also detailed, including a range of salivary, microbiome, and host characteristics which may affect oral microbiota ecology and the caries process directly or indirectly. Additional strengths were that (i) all participants were of the same age (17 years old), (ii) participants were recruited in connection with their regular dental survey which reduces the risk for selection bias, (iii) participants permanent teeth have been exposed to the oral environment for years allowing ecology stabilization and potential development caries, and (iv) have minimal confounding by diseases or medications. Further, the study design, i.e., consecutive inclusion of participants as they attended their regular visit for a dental examination, and that virtually all accepted to participate, limited the risk of a selection bias.Compared to previous investigations, the present study included detailed genotyping of a relatively large number of SNPs within and near S. mutans detection frequency between the different identification strategies. To reduce the impact of the sampling source, being S. mutans positive was based on presence in either saliva or tooth biofilm by NGS. In addition, self-reported information on lifestyle habits, here tooth brushing, tobacco use and intake of sweet snacks, is prone to bias, such as the well-known under-reporting of diet intake29.There are some limitations in the study that should be acknowledged. The relative low number of participants, including that only a subset was available for assessing CA6 amounts, limits the possibility of comparisons in subgroups. Further, identification of microorganisms by NGS is associated with potential errors at various steps, such as the PCR amplification, sequencing per se, and bioinformatic accuracy. It should also be noted that in contrast to culturing, DNA based identification by NGS identifies dead and alive bacteria, and that saliva detection, in contrast to tooth biofilm samples, reflects bacteria from epithelial and tooth surfaces. These differences contributed to the variation in 30, and in epithelial linings of the oesophagus, stomach, and large intestine. This has led to a wider search of its functions. Thereby, CA6 has been linked to acinic cell carcinoma31, colorectal cancer34, and Sjögrens syndrome37. Based on the findings from the current study it may be hypothesized that CA6 and CA6 gene variations contribute to the microbial environment in both the nasopharynx and gastro-intestinal tracts.CA6 was originally found in the salivary glands, but has now been found to be expressed by the lacrimal, tracheobronchial, nasal, and mammary glands , rs3737665 (T), rs12138897 (G) and haploblock TTG of CA6 are associated with S. mutans colonization, overall microbiota composition and dental caries in Swedish adolescents. Further studies need to elucidate the mechanisms.In conclusion, the results from the present study support that 38. Adolescents who had other chronic medical conditions, used medication regularly, required antibiotic treatment during the latest six months, who did not consent, or were unable to communicate in Swedish or English were excluded. All participants had answered a questionnaire with information on general health status, medication use, oral hygiene, dietary habits, and tobacco use, and samples of whole simulated saliva and supragingival biofilm were collected and stored at −80 °C until use.In this study 154 17-year-old adolescents were recruited from three public dental health care clinics in the city of Umeå, SwedenThe study was approved by the Regional Ethical Review Board in Umeå, Sweden  and abided by the Declaration of Helsinki and by the European General Data Protection Regulation (GDPR). Data collections and experiments were performed in accordance with relevant guidelines and regulations, including that all adolescents and their caregivers provided informed consent and data collection and handling followed the Helsinki declaration and the Swedish Law on personal data act (PuL).2 ≤ 0.8) and c) in the 10,000 bp up-stream to 10,000 bp down-stream region of the CA6 gene. Using the NIH snptag resource (https://snpinfo.niehs.nih.gov/snpinfo/snptag.html) 30 SNPs met these criteria. Genotyping was performed using a multiplexed primer extension chemistry of the iPLEX assay with detection of the incorporated allele by mass spectrometry using a MassARRAY analyzer . Raw data from the mass reader was converted to genotype data using the Typer software (Agena Bioscience)41. Two SNP markers had sample call rate 0%, rs2274333 and rs17032942, the reasons for the failure was low allele signals resulting in inaccurate genotype calls. An additional SNP (rs6697763) was excluded from analysis for deviation from Hardy-Weinberg equilibrium (p = 0.033), leaving 27 SNPs with valid genotype data, where the average call rate per sample was 99.4% and overall call rate was 99.8%  minor allele frequency (MAF) ≥ 5% in the CEU population b) not in complete linkage disequilibrium . All study participants refrained from oral hygiene on the morning of their visit to the dental clinic. All samples, except those for culturing, were stored at −80 °C until used.38. Briefly, samples were collected and lysed in buffer with lysozyme and mutanolysin (Sigma-Aldrich), treated with RNase and proteinase K, purified and washed. DNA quantity and quality were measured using NanoDrop 1000 Spectrophotometer . Multiplex 16S rDNA amplicon sequencing was performed with the Illumina MiSeq platform (http://www.illumina.com). 16S rDNA amplicon sequencing was conducted at the Forsyth Research Institute with the HOMINGS protocol42. Briefly, the V3-V4 hypervariable regions of the 16S rRNA gene were polymerase chain reaction (PCR) amplified with the 341 F (ACGGGAGGCAGCAG) forward primer and the 806 R (GGACTACHVGGGTWTCTAAT) reverse primer. Pair-end reads were fused using FLASH, and barcodes, primers, and ambiguous and chimeric sequences were removed within QIIME. Taxa were identified with the ProbeSeq customized BLAST program, which targeted recognition of 538 species (using 638 species-level probes) and 129 groups of closely-related species (using 129 genus-level probes)43. Taxa targeted by the genus probes (gp) are described in Table Genomic DNA was extracted from supragingival biofilm samples, saliva and from bacteria mock communities with the GenElute Bacterial Genomic DNA Kit  as previously described38. The numbers of tooth surfaces with caries in the enamel or into the dentine, or a filling, or were missing were recorded. The total numbers of decayed and filled tooth surfaces (DeFS) were calculated. Fissure sealants were not recorded. Missing surfaces were not considered to represent dental caries because tooth loss occurred for orthodontic reasons, tooth formation defects or aplasia in the study group.Visual and radiographic dental examinations were performed as described previouslyFor the first 90 participants’ saliva, samples were collected and stored at −80 °C until used for CA6 concentration measurements. CA6 protein concentration in saliva was measured by ELISA  following the manufacturer’s instructions. Briefly, 100 µL saliva, diluted 1:5 was used for analysis, CA6 protein was captured by a target specific antibody, then detected by a biotinylated secondary antibody, followed by an avidin-horseradish peroxidase (HRP) conjugate and tetramethylbenzidine (TMB) for colour development. The colour development reaction was terminated by sulfuric acid addition and the optical density (OD) measured at 450 nm using a MultiscanGO spectrophotometry . A standard curve using purified CA6 protein ranging from 0–1,000 pg/mL was run.Total saliva protein concentration was determined using Pierce Coomassie (Bradford) protein assay kit (ThermoFisher). Briefly, 5 µl diluted (1:1) saliva, was mixed with 250 µL protein dye solution and incubated for 30 min at room temperature. The optical density of each sample at 595 nm wavelength was assessed using a MultiscanGO spectrophotometry (Thermo Scientific) and compared to the optical density of a bovine serum albumin standard curve from known antigen concentrations to determine the sample concentration.NGS sequencing of saliva was performed in 139 tooth biofilm samples and 154 saliva samples where 2 samples failed. Caries information was missing for one participant who was excluded. All analyses with caries as outcome were restricted to the 129 subjects who reported brushing their teeth twice a day.2 or Fisher’s exact test. All tests were two-tailed. Correction for multiple comparison was done by the Benjamini Hochberg false discovery rate (FDR). P-values were considered significant at FDR < 0.25 and ≤0.06. The higher FDR was applied to avoid missing potential CA6 SNPs of interest as described earlier45.SPSS software version 24.0 was used for descriptive statistics, including medians , frequencies (n), proportions (%) and odds ratios (OR) with 95% confidence intervals (CI). Group comparisons of continuous variables were done using Mann-Whitney U test and for categorical variables Chiet al.46. Haploblocks with frequencies <1% were not included in the analyses. Association between genetic variation (SNPs and haploblocks) and phenotypes was evaluated using Haploview and Chi2 tests. The tests were two-tailed tests and corrected for multiple comparison by FDR as described above. Phenotype penetrance was assessed in common allele, recessive and dominant models47.Haploview software (version 4.2) was used to evaluate characteristics of SNPs , as well as evaluation of potential haploblocks using the algorithm described by Gabriel Clustering of participants by presence (0/1) of bacterial taxa in saliva and tooth biofilm, respectively, was modelled using partial least square (PLS) regression. Saliva taxa did not yield a significant model and therefore PLS analyses employed tooth biofilm taxa. Variables with a Variable Importance in the Projection (VIP) > 1.5 were considered influential. SIMCA P+ version 14.1 (Umetrics AB) was used for these analyses.Carbonic Anhydrase 6 Gene Variation influences Oral Microbiota Composition and Caries Risk in Swedish adolescentsTable S2


Responding to an unprecedented increase in the suicide rate among soldiers, in 2008 the US Army and US National Institute of Mental Health funded the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS), a multicomponent epidemiological and neurobiological study of risk and resilience factors for suicidal thoughts and behaviors, and their psychopathological correlates among Army personnel. Using a combination of administrative records, representative surveys, computerized neurocognitive tests, and blood samples, Army STARRS and its longitudinal follow-up study (STARRS-LS) are designed to identify potentially actionable findings to inform the Army’s suicide prevention efforts. The current report presents a broad overview of Army STARRS and its findings to date on suicide deaths, attempts, and ideation, as well as other important outcomes that may increase suicide risk . The findings highlight the complexity of environmental and genetic risk and protective factors in different settings and contexts, and the importance of life and career history in understanding suicidal thoughts and behaviors. Length A soldier’s military occupation, which relates to an array of expected stressors and protective training, is related to risk. The adjusted odds of attempting suicide were higher in combat arms soldiers (OR = 1.2 [95% CI = 1.1–1.2]) and combat medics , compared to all other occupations . Combat The extensive use of improvised explosive devices (IEDs) by enemy forces in the Iraq and Afghanistan wars was unprecedented . ControlAdditional findings suggest that Army units with a history of suicide attempts may be important targets for preventive interventions. In models adjusted for sociodemographics, service-related characteristics, prior mental health diagnosis, unit size, and unit deaths from suicide, combat, and accidents, enlisted soldiers were more likely to attempt suicide if 1 or more suicide attempts occurred in their unit during the past year, with odds increasing as the number of unit attempts increased to 5 or more (ORs = 1.4–2.3) . The assViolence within a soldier’s family  was also associated with risk of suicide attempt. Adjusted odds of suicide attempt were higher in soldiers with a history of family violence documented in legal, Family Services, and medical records (OR = 2.9 [95% CI = 2.6–3.2]) . Risk inThe HADS contains performance data from a computerized neurocognitive testing battery that is completed by all soldiers before deployment and provides baseline data in the event of TBI. Poorer neurocognitive test performance was prospectively associated with suicide attempt, even after controlling for sociodemographics and prior mental health diagnosis . AdditioUnlike the HADS, which focuses on medically documented STB, the AAS provides self-report data from a representative sample of the Army population. In early analysis of the first 5428 AAS respondents (representative of non-deployed soldiers), lifetime prevalence estimates of self-reported suicide ideation, plans, and attempts were 13.9%, 5.3%, and 2.4%, respectively . Most reN = 29,982), which includes the full AAS sample and respondents from the pre-deployment survey of the PPDS), found that prevalence estimates for lifetime suicide ideation were 12.7% among men and 20.1% among women, and for lifetime suicide attempts were 2.5% and 5.1%, respectively ). MRAP2 is expressed in brain and adrenal cortex and is a plausible susceptibility gene for STB. Polygenic risk score (PRS) analyses showed some association of suicide attempt with bipolar disorder ), PTSD treatment (OR = 6.3 [95% CI = 5.7–6.9]), suicide attempt (OR = 3.0 [95% CI = 2.5–3.6]), demotion (OR = 2.1 [95% CI = 1.9–2.3]), and attrition (OR = 1.2 [95% CI = 1.1–1.2]). Findings indicate that sexual assault victimization is associated with considerable distress and likely decreases force readiness [Among all female Regular Army soldiers with administratively recorded sexual assault victimization from 2004–2009  or discontinued treatment in the past year (N = 145) [Mental health service utilization and barriers to care are significant concerns in the Army’s efforts to prevent suicide and improve force health and readiness. Among 5428 AAS respondents, only 21.3% of soldiers who reported a past 30-day mental disorder indicated they were currently receiving mental health treatment . ReporteN = 145) . More thN = 145) .In support of precision medicine approaches and cost-effective intervention targeting, Army STARRS investigators were among the first to demonstrate the potential utility of machine learning to assist clinical decision-making through enhanced risk prediction for adverse mental health and other outcomes. For example, the days, weeks, and months following discharge from psychiatric hospitalization are known to be a particularly high-risk period for suicide. During 2004–2009, 68 soldiers died by suicide within 12 months of hospital discharge , equivalent to 263.9/100,000 person-years vs. the Army-wide rate of 18.5/100,000 person-years . A totalMachine learning models have also been developed to predict the following: suicides after outpatient mental health specialty visits , sexual Army STARRS, called by some the Framingham of mental health studies , continuThe work that began in Army STARRS continues in STARRS-LS (2015–2020), the longitudinal follow-up study funded by the DoD, which adds historical administrative data from 2010 to 2015 and reassessment of the original Army STARRS survey respondents via web- and telephone-based interviews. These follow-up surveys will capture soldiers who are still serving and those who have separated from the Army. The number of soldiers leaving the Army and returning to civilian life each year will increase as the Army downsizes with the end of the Afghanistan war. The transition from military to civilian life is known to be stressful , 87 and


Takotsubo cardiomyopathy (TCM), also known as broken heart syndrome or stress-induced cardiomyopathy, is a rare condition with an estimated incidence of 0.02% of all hospitalizations in United States and 2% of all acute coronary syndrome presentations. TCM predominately presents as a transient wall motion abnormality of the left ventricular apex due to emotional or physical stress. Cardiac rupture in the setting of TCM is an extremely rare phenomenon with limited published case reports. We present a case of a 75-year-old female who had cardiac rupture secondary to TCM and performed a literature review using Ovid MEDLINE for published cases showing this association. After the literature review, we found 20 cases showing this association, which are listed in a tabular fashion. Takotsubo cardiomyopathy (TCM), also known as stress-induced cardiomyopathy, was first described in Japan in 1990 . Balloonμl), white count (14.4 k/μl), INR (1.1), creatinine (0.66 mg/dl), and AST (87 μ/l). Electrocardiogram (ECG) on presentation showed sinus rhythm at 74 beats/min, left axis deviation, Q waves in V1 to V3, 1 mm ST segment elevation of V2-V3 (present in prior ECG), and poor R wave progression . The patient presented to the local emergency department with substernal chest pain radiating to the left jaw and arm and was found to have an elevated troponin (0.26 ng/ml). She was appropriately given aspirin, heparin, and nitroglycerine and was transferred to our medical centre for further care. On presentation, she was alert and oriented, her blood pressure was 200/100 mmHg, nontachycardic, nontachypneic, and sating at 97-98% on 2 litres per minute of oxygen. Pertinent physical exam findings include bibasilar crackles, no murmurs/rubs, normal heart sounds, and no pedal edema. Pertinent laboratory values include haemoglobin (14.2 gm/dl), platelet count . While in the elevator, en route to the cardiac catheterization laboratory, the patient became unresponsive. Resuscitation was immediately started, labs at the time of code blue were significant for acute drop in haemoglobin from 14.2 to 6.2, hypokalaemia of 2.5 mmol/l, and arterial blood gas showed metabolic acidosis . The catheterization team was unable to establish arterial access hence coronary catheterization was not performed. Unfortunately, despite after aggressive resuscitative efforts for 50 minutes, the patient died.Autopsy was performed and revealed 1000 ml of blood in chest wall cavity. Gross pathology revealed a slit-like rupture of 1 cm × 0.8 cm, transmural and located anteriorly 1.5 cm inferior to the base of the heart. The pericardial surface was smooth and tan red in colour . MicroscTCM is generally classified by an acute and profound but reversible left ventricular dysfunction in the absence of significant coronary artery disease. The revised Mayo criteria are most commonly used currently to establish diagnosis of TCM which includes combination of clinical presentation, ECG, transthoracic echo, and angiographic finding . It is oAs per Kumar et al., the characteristics of patients with cardiac rupture in TCM when compared to patients with TCM who did not had cardiac rupture include the following: female gender, older age group, higher systolic and diastolic blood pressure, higher frequency of ST elevations in inferior lead, low ejection fraction (EF), and higher left ventricular peak systolic fraction . Our patAs histopathological slides from our patient showed no thrombus and minimal atherosclerosis of coronary arteries, our case falls into the newly diagnosed category of MINOCA . TCM is one of the categories of MINOCA; other categories include coronary spasm, coronary dissection, plaque disruption, spontaneous coronary emboli, myocarditis, and coronary microvasculature dysfunction . Our patA thorough literature review of 20 previous cases of TCM complicated with cardiac rupture is listed in The prognosis of TCM is usually favourable, and left ventricular function improves in the majority of cases with conservative management. Currently, there is no standardised treatment protocol for the patients with TCM . AlthougDue to devastating complications of TCM, our case highlights the need for close monitoring of patients with TCM for the first few days. Special consideration should be paid to older female patients as they have higher rates of cardiac rupture.


Median estimated glomerular filtration rate (eGFR) was 17 mL/min/1.73 m2. Older age (r = −0.46), male sex (r = −0.34), lower eGFR (r = 0.27), lower serum-corrected calcium (r = 0.27), higher PTH (r = −0.36), and higher ALP level (r = −0.20) were identified as determinants of lower number of RTE. In contrast, serum concentrations of FGF23 and phosphorus were not correlated with RTE. Multivariate non-linear regression analysis indicated a negative association between serum PTH and log-transformed RTE . However, the serum levels of FGF23 and ALP were not associated with RTE. In patients with CKD, serum PTH concentrations were related to thymic atrophy which contributes to immune abnormality.Premature immune ageing, including thymic atrophy, is observed in patients with chronic kidney disease (CKD). Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23), which are mineral and bone disorder (MBD)-related factors, affect immune cells and possibly cause thymic atrophy. We examined the cross-sectional association between thymic atrophy, evaluated as the number of CD3 The thymus generates T cells, and upon atrophy, the supply of naïve T cells and the diversity of the cell repertoire diminish. The loss of such diversity results in susceptibility to infection due to insufficient responses to new pathogens. Patients with DiGeorge syndrome, in which the thymus is congenitally hypoplastic, become infected easily due to T-cell deficiency4. When the number of naïve T cells decreases and the diversity of the repertoire is lost, effector T-cell population increases. This condition is related to chronic inflammation and arteriosclerosis5.Chronic kidney disease (CKD) promotes premature immune ageing, including thymic atrophy, which might be associated with high mortality due to infection or cardiovascular disease (CVD)14.However, the specific CKD pathology that is associated with thymic atrophy remains unknown. CKD is a disease in which the function of the kidneys gradually decreases. In patients with CKD, mineral and bone disease (MBD) can cause abnormal serum concentration of calcium, phosphorus, parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and alkaline phosphatase (ALP), resulting in bone-related abnormalities and vascular calcification. This complication is associated with increased mortality by CVD or infectious disease15, and left ventricular hypertrophy is mediated by FGF receptor 4 by the action of FGF2316 through direct organ involvement. Additionally, PTH and FGF23 act on immune cells18 and it exhibits thymic atrophy in klotho-hypomorphic mice and FGF23 knockout mice20. These facts indicate that MBD might likewise affect the thymus in humans.In recent years, MBD has been found to act on organs other than bone and vascular calcification. Cachexia is mediated by the PTH receptorBased on this, we hypothesised that MBD is involved in thymic atrophy in human. We examined this hypothesis by investigating the relationship between thymic function and MBD markers  in patients with non-dialysis dependent CKD.2) ≥60 (CKD stage 1 or 2), 15–60 (CKD stage 3 or 4), and <15 (CKD stage 5), respectively. The median (interquartile range) age was 71  years, and 63% of the participants were men. Pre-existing CVD was evident in 21% of patients. The aetiologies of kidney disease comprised diabetic nephropathy , glomerulonephritis , nephrosclerosis , and others . Glomerulonephritis was frequent (83%) in the group with eGFR ≥60 mL/min/1.73 m2. A phosphate binder was used only when eGFR was <15 mL/min/1.73 m2 (43% of patients). Active vitamin D was used when eGFR was 15–60 mL/min/1.73 m2 (9% of patients) or <15 mL/min/1.73 m2 (59% of patients). The serum-corrected calcium level did not change even when eGFR decreased. The median serum phosphorus level was 5.1 mg/dL with eGFR <15 mL/min/1.73 m2. Serum levels of both PTH and FGF23 increased as eGFR decreased, but serum ALP level did not change despite the decrease in eGFR.Table +CD4+CD45RA+CD31+ cells [Recent thymic emigrants (RTEs)/μL] (RTE) and the proportions of CD45RA+CD31+ cells in the CD3+CD4+ cell subset (RTE%), as previously reported 21. Thymic function is reportedly altered in patients on dialysis1. Therefore, we examined correlations between thymic function and eGFR and MBD-related factors and found that eGFR was correlated positively with RTE . Furthermore, among the variables associated with MBD and thymic atrophy, serum level of PTH and corrected calcium correlated negatively and positively, respectively, with both RTE and RTE% , whereas serum ALP level was correlated negatively with RTE . Serum level of FGF23 and phosphorus correlated with neither RTE nor RTE%. That is, thymic function correlated with eGFR as well as the serum concentration of PTH, ALP, and corrected calcium, which are factors related to secondary hyperparathyroidism, in addition to age and male sex, but not with the serum concentration of FGF23 or phosphorus, which are associated with phosphorus metabolism.Table We next performed multivariate, non-linear regression analyses to detect variables explaining LnRTE . Figure Figure Then, we examined whether active vitamin D supplementation was related to RTE or RTE% as active vitamin D might affect both thymic function and serum PTH level. As shown in Supplemental Table. 22. For example, the effect of PTH on fat and muscle cells causes cachexia in patients with CKD15. PTH receptors are also expressed on T cells, and mediate the secretion of Wnt10b, which plays an important role in the osteogenic action of PTH18. The action of PTH in bone is mediated through cAMP/PKA and Wnt-independent activation of β-catenin/T-cell factor (TCF) signalling23. Continuous infusion of PTH also acts on GαS on naïve T cells and promote their differentiation to Th1724. As a result, the output from the thymus might be depleted through the loss of naïve T cells. In addition, β-catenin/TCF signalling in T cells is involved in differentiation regulation in the thymus and peripheral tissue25. Increased Wnt signalling by PTH in thymic epithelial cells might lead to decreased thymic function26. An additional mechanism associated with thymic atrophy caused by PTH is the effect of haematopoietic stem cells. Lymphoid progenitor cells differentiate from haematopoietic stem cells and become thymocytes upon reaching the thymus. Therefore, a decrease in the number of haematopoietic stem cells results in downstream thymic atrophy27. Thymic atrophy might also occur via a reduction in the number of haematopoietic stem cells caused by PTH, because patients with CKD have fewer CD34-positive cells, including haematopoietic stem cells than healthy individuals28, and serum PTH is associated with a decreased number of CD34-positive cells29. Although intermittent PTH (1–34) administration expands haematopoietic stem cells32, persistent PTH elevation in CKD patients might decrease haematopoietic stem cells due to exhaustion in a manner similar to the sulfonylurea-induced exhaustion of insulin secretion that occurs during diabetes treatment33. To understand the role of PTH in thymic atrophy, thymic epithelial cell-specific knockout of PTH receptors in mice may be performed.This study showed that thymic atrophy was associated with higher PTH level, but not with FGF23 or ALP level, after adjustment for eGFR, active vitamin D supplementation, and other variables. One mechanism that could explain the association between serum PTH level and thymic atrophy is the effect of PTH activity on organs because PTH receptors are found throughout the body, and thus this hormone can act on organs as well as bones19 and klotho-hypomorphic mice20. Notably, hyperphosphatemia is also associated with thymic atrophy in mice, because crossing klotho-hypomorphic mice with Npt2a knockout mice improves thymic atrophy34. Although neither serum level of FGF23 nor phosphorus were found to be associated with thymic atrophy in the present study, there is a possibility that it was not related to phosphorus metabolism because of the differences between humans and mice.Thymic atrophy was not found to be associated with serum FGF23 level in this study, whereas it has been shown to develop in FGF23 knockout35 to evaluate thymic atrophy, as this is an established method that is used to assess thymic function36. The number of naïve T cells derived from the thymus before an antigen encounter are usually maintained by the peripheral division. However, RTEs are naïve T cells that divide at a lower frequency, and these reflect the production of T cells by the thymus35. Thymic atrophy can also be evaluated by measuring T-cell receptor excision circles. This method closely correlates with measuring RTEs by flow cytometry, but it includes PBMC separation and PCR35, and its application is subject to considerable variation35. The size of the thymus can be measured using chest CT, but this is not quantitative and depends on observer subjectivity37. Therefore, quantitative flow cytometry is the most practical method for evaluating thymic atrophy.We measured RTEs by flow cytometry38. This suggests that RTE is thought to reflect more thymic atrophy than RTE%.PTH was associated with RTE in multivariate analysis in this study. However, RTE% is not significantly associated with PTH in multivariate analysis although RTE% correlated with PTH in univariate analysis and tended to be associated with PTH in multivariate analysis. RTE has a higher correlation coefficient with T-cell receptor excitation circles, which is another indicator of RTEs, than RTE%2. In addition, one study has found an association between thymic atrophy and CVD39. In another study, anti-thymocyte globulin increases the incidence of CVD when administered to patients receiving renal transplantation40. Since atherosclerosis comprises vascular wall inflammation that is affected by the immune system, thymic atrophy in association with immune ageing might cause CVD. Serum PTH level is associated with the onset of CVD, infectious disease, and mortality in an epidemiological study14. For these reasons, PTH may be involved in the pathogenesis of these complications by inducing immune abnormality through thymic atrophy in addition to regulating bone metabolism.In this study, it was demonstrated that thymic atrophy in CKD may be promoted by PTH. Because lower serum calcium and higher ALP are also correlated with thymic atrophy, it can be said that secondary hyperparathyroidism is associated with thymic atrophy. Thymic atrophy reduces repertoire diversity and increases susceptibility to infection2D that was not evaluated might have been confounders. However, multivariate regression analysis did not associate oral active vitamin D supplementation with thymic function D or 1,25(OH)In conclusion, we showed that serum PTH concentrations are associated with thymic atrophy. Although this is a cross-sectional study, PTH may cause thymus atrophy and contribute to immune abnormality, which may also partially explain the increased mortality due to elevated PTH level. Further investigation is required to explain the relationship between PTH and thymic atrophy.The Ethics Committee at Osaka Minami Medical Center approved this study  and the study was conducted according to the ethical principles of the Declaration of Helsinki. Informed consent was obtained from all the participants. We enrolled 125 patients with non-dialysis dependent CKD who were hospitalised between June 2013 and October 2017. The effect of an acute decline in kidney function and inflammation was avoided by applying the following exclusion criteria: age <20 years old, acute kidney injury, hepatitis B or C virus infection, use of immunosuppressive agents, active infection, and inflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, and vasculitis.2)], the presence or absence of hypertension and diabetes, haemoglobin, serum levels of calcium, phosphorus, intact PTH, ALP, albumin, C reactive protein, urinary protein, medications (phosphate binder and active vitamin D), prior immunosuppression, and smoking status. Estimated glomerular filtration rate (eGFR) (mL/min/1.73 m2) was calculated using the following equation41:Demographic and clinical data at the time of study initiation were collected from the medical records of Osaka Minami Medical Center, and included age, sex, body mass index +CD4+CD45RA+CD31+ T cells were defined as RTEs as described in a previous report35, and they are expressed as RTE and RTE%. The absolute number of RTE in the peripheral blood was calculated based on the number of peripheral blood lymphocytes at the time of sampling. Figure We used PBMCs or peripheral blood to detect T cells by flow cytometry. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS  and stored in liquid nitrogen. Serum aliquots from each patient were stored at −20 °C in our biorepository. Cryopreserved PBMCs were thawed and washed twice in RPMI  containing 10% foetal calf serum (complete medium). Peripheral blood and PBMCs were stained with the following conjugated antibodies: CD3-PE-Cy7 , CD4-FITC , CD8-PerCP-Cy5.5 , CD31-PE , and CD45RA-ECD . Samples were acquired by using Navios (Beckman), and data were analysed by FlowJo software . CD32), differences in the clinical characteristics of patients were compared using Kruskal-Wallis tests for continuous variables and chi-squared tests for categorical variables. Correlations were assessed by calculating Spearman’s correlation coefficients. To examine the effect of serum PTH level on RTE and RTE%, multivariate non-linear regression analysis that included RTE and RTE% as dependent variables were conducted separately with adjustments for age, sex, eGFR, diabetes, serum-corrected calcium, serum phosphorus, active vitamin D supplementation, and the use of phosphonate binder. To satisfy the assumption of linear regression models that the residuals are normally distributed, RTE was log-transformed. A similar analysis with the above non-linear regression model was performed to assess the impacts of serum FGF23 and ALP level on RTE and RTE% separately. We also performed a multivariable non-linear regression analysis including log-transformed RTE and RTE% separately as a function of active vitamin D supplementation and other covariates described above to assess the association between active vitamin D supplementation and RTE or RTE%. All statistical analyses were used to determine the two-sided significance level at P < 0.05 using R software version 3.3.2 (https://www.r-project.org/foundation/) using the “rms” package43.Data are presented as medians (interquartile ranges). Categorical variables are presented as numbers (%). After classification based on eGFR (<15, 15–60, or ≥60 mL/min/1.73 mSupplementary Table


Alzheimer’s disease (AD) dementia, a progressive neurodegenerative disease, exerts significant burden upon patients, caregivers, and healthcare systems globally. The current study investigated the associations between AD dementia patient disease severity and health-related quality of life (HRQoL) of both patients (proxy report) and their caregivers living in Japan, as well as caregiving-related comorbidities such as depression.This cross-sectional study used self-reported data from caregivers of people diagnosed with AD dementia by a healthcare provider in Japan. Caregivers were identified via online panels and invited to participate in an online survey between 2014 and 2015. Caregivers completed survey items for themselves, in addition to providing proxy measures for patients with AD dementia for whom they were caring. Patient and caregiver HRQoL was measured using the EuroQoL 5-Dimension (EQ-5D). Additional outcomes for caregivers of AD dementia patients included the Patient Health Questionnaire (PHQ-9) of depressive symptomology, as well as comorbidities experienced since initiating caregiving for their AD dementia patients. These outcomes were examined as a function of AD dementia severity, as measured by long-term care insurance (LTCI) categories. Bivariate analyses between LTCI and outcomes were conducted using independent t-tests and chi-square tests. Multivariable analyses, controlling for potential confounders, were conducted using generalized linear models (GLMs) specifying a normal distribution.Across 300 caregiver respondents, multivariable results revealed that increasing AD dementia severity was significantly associated with poorer patient and caregiver EQ-5D scores and a high proportion of caregivers (30.0%) reported PHQ-9 scores indicative of major depressive disorder (MDD). The most frequent comorbidities experienced after becoming caregivers of AD dementia patients included hypertension (12.7%) and insomnia (11.0%). Depression and other comorbidities did not differ significantly by patient severity.The current study provides unique insight into the specific degree of incremental burden associated with increasing AD dementia severity among patients and caregivers alike. Importantly, greater disease severity was associated with poorer quality of life among both patients and caregivers. These results suggest that earlier detection and treatment of AD dementia may provide an opportunity to reduce the burden of disease for patients, caregivers, and society at large. Alzheimer’s disease (AD) dementia is a progressive neurodegenerative disease that poses a significant burden to patients, caregivers, healthcare systems, and society. Disease symptomatology includes impaired cognition  and is thus associated with difficulty in performing daily activities and consequent functional dependence on others for help. These symptoms and the progressive nature of AD dementia can result in increasing degrees of care required from formal  and informal (family) caregivers . The cauCritically, the health, societal, and economic burden associated with AD dementia may increase as the severity of the disease increases –5, with The high cost of informal care in AD dementia highlights the unique burden and responsibility placed on families of persons with AD dementia. Many AD dementia patients depend on care from family members due to the associated impairments in functioning and cognition that are hallmarks of this progressive disease. Notably, the care needs of patients with AD dementia can increase significantly as disease severity progresses , and stuGlobally, the impact on caregivers has shown a range of negative outcomes in the context of AD dementia care , 18, incJapan is ranked among the countries with the highest prevalence, with a 2010 study estimating 2.5 million adults suffering from dementia, a number predicted to reach 7.3 million by 2025 , 38. To This cross-sectional study collected new data from caregivers of people with a clinical diagnosis of AD dementia who were previously identified in the 2012/2013 Japan National Health and Wellness Surveys , with additional caregivers supplemented from Lightspeed Research (LSR) opt-in ailment panels. Convenience sampling, stratified by sex and age, was implemented to achieve demographic characteristics that match the adult population in Japan. Further details about the NHWS and respondent recruitment are provided in Laks et al.  and GoreSociodemographic variables included sex and age for both patients and caregivers. An additional background variable for patients included LTCI levels reported by their caregivers. The implementation of LTCI in Japan required detailed assessments of patients’ needs to effectively assign care resources. The Government-certified Disability Index (GCDI) is an 85-item measure developed to serve this purpose , providiAdditional background variables for caregivers included marital status, number of children in the household, change in employment status due to caregiving for the AD dementia patient, relationship with patient, and level of involvement with caregiving.Patient disease severity was reported by caregivers based on patients’ LTCI scores. Previous research has demonstrated that LTCI is associated with other measures of disease severity , supportCharlson comorbidity index (CCI)  scores sTwo forms of the EuroQoL-5Dimension (EQ-5D) with 3 levels , a widely used measure of health status, were used: one to assess the caregiver’s HRQoL, and a proxy version for the caregiver to rate the patient’s HRQoL from the patient’s perspective , 48. TheThe Patient Health Questionnaire (PHQ)-9 , 52 was ns and %s for categorical variables and means, medians, standard deviations (SDs), and minimum and maximum values for continuous variables. Bivariate associations were examined between the primary independent variable, LTCI, and several HRQoL and comorbidity dependent measures using chi-square tests for categorical variables  and t-tests for continuous variables , with two-tailed p-values indicating statistical significance. Results of these bivariate analyses helped inform variables that were included as covariates in multivariable analyses. Covariates were identified according to statistically significant differences in bivariate comparisons (p < .05) across the AD dementia severity categories. Some core variables of conceptual interest  were included regardless of their association with the severity categories.Descriptive analyses were conducted on all study variables of interest; these included Multivariable analyses were used to assess patient and caregiver outcomes as a function of patient severity . The final list of identified covariates included: caregiver gender, caregiver age, caregiver employment status, caregiver marital status, percentage average daily care for which caregiver is responsible, percentage average daily care from visiting nurse/home-helper, total average hours of care required per day, years providing care for AD dementia patient, relationship between caregiver and patient, patient age, and patient gender. Separate generalized linear models (GLMs) specifying appropriate distributions  were chosen for each outcome of interest that was associated significantly with AD dementia severity in bivariate analyses. Multivariable models included exploratory interaction terms between age categories and employment status of caregivers on both caregiver and patient outcomes. These interaction terms were included to test whether the relationship between employment status and outcomes depended upon age of caregivers. These interaction terms were expected to account for cultural expectations surrounding age and work status .with a caregiver-reported patient age of 4 years), the mean age was 83.7 years , with minimum and maximum values of 38 and 106 years, and 78.7% of patients were female.Across 299 patients (excluding 1 outlier n = 37 in this “low severity” group) indicating that they did not know their patients’ LTCI (n = 3) or that their patients had no LTCI (n = 15) or support level 1 (n = 7) or 2 (n = 12); 63.7% (n = 191 in this “medium severity” group) indicating that patients had nursing care level 1 (n = 52), 2 (n = 75), or 3 (n = 64); and 24.0% (n = 72 in this “high severity” group) indicating that patients had nursing care level 4 (n = 43) or 5 (n = 29) , with minimum and maximum values of 20 and 96 years. A total of 61.0% were between the ages of 50 and 64 and 10.7% were 65 years of age or older. Of the caregiver sample, 55.0% were male, 62.3% were married or living with a partner, 51.0% had at least one dependent adult in the household, 17.7% had at least one dependent child in the household, and 26.7% reported changing employment status due to caregiving.Across 300 caregivers (see Table n = 276), 55.8% had provided at least 4 years of care; 13.0% had provided at least 10 years of care. Among all caregivers, 39.7% were secondary caregivers, 30.0% were primary caregivers but shared responsibilities, and 30.3% were the only primary caregivers. Caregivers were personally responsible for an average of 13.0 h (SD = 8.01) of care per day, with 48.0% of respondents responsible for 13 h of care per day or greater. Data were collected regarding the caregiver’s relationship to the patient . Age and relationship data revealed that caregivers responded inconsistently regarding the direction of the parent vs. offspring relationship; therefore, responses were recoded into more logically defensible categories: offspring of patient , spouse/sibling/parent of patient , or in-law/extended family/not related to patient .Among those who recalled the total duration of caregiving (SD = 0.224) and median of 0.595. EQ-5D caregiver index values ranged from − 0.110 to 1.000, with a mean score of 0.809 (SD = 0.216) and median of 0.785  and hypertension (12.7%). Most caregivers (92.0%) reported no diagnosed comorbidities since becoming caregivers, however. A total of 90 re-contact caregivers 30.0%) experienced MDD as indicated via PHQ-9 scores ≥10 (see Table 0.0% expeM = 0.69 SD = 0.16) than did those AD dementia patients at the medium severity LTCI level  as well as patients at the high severity LTCI level ; p’s < .05. AD dementia severity significantly predicted EQ-5D scores for patients after adjusting for covariates in the multivariable model  than did caregivers of AD dementia patients at the low severity LTCI level , p < .05. EQ-5D scores did not significantly differ between caregivers of patients at medium severity and low severity LTCI levels, however. After controlling for covariates, AD dementia severity remained a significant predictor of caregiver EQ-5D scores in the multivariable model  to be developed to support caregiving responsibilities and ultimately reduce burden and improve outcomes among patients and caregivers alike. For example, there is a cluster-randomized study that assessed psychosocial behavior management programme for home-dwelling people living with dementia . This prImportantly, a higher proportion of caregivers of AD dementia patients in this study reported depressive symptomology suggestive of MDD, compared with previous research that included caregivers of patients with dementia defined more broadly . A totalAlthough significant interactions were found between age  and employment status, they did not reveal a consistent pattern across age groups. Speculatively, there was a non-significant trend toward poorer HRQoL with employment, suggesting that younger caregivers who were also employed  may have tended to have an especially difficult time coping with the multiple demands of caregiving and maintaining a job, which may also have had an effect on patients’ HRQoL. This effect was reduced among 50–64-year-olds, perhaps implying that those with more established positions at work or without dependent children in the household were in relatively better shape to cope with the multiple demands.The online survey format of the current study provides certain sampling advantages but may under-represent caregivers without access to or sufficient comfort with online technology. This may result in a biased population from which surveys are drawn, such as respondents from higher socioeconomic backgrounds as well as respondents who are relatively young; however, it is important to note that if caregivers with access to and comfort with technology report burden associated with caregiving, the current study results may actually be an underestimation of burden among AD dementia caregivers in Japan.There were a number of differences in demographic characteristics between the current study sample and the general population in Japan, with respondents in the current study being more likely to be younger in age (10.7% vs. 26.7% over age 65), male (55% vs. 48.7%), and socioeconomically advantaged (69% vs. 58.6% employed) . These dA further notable limitation of the current study is the cross-sectional, contemporaneous nature of the data, which precludes causal assessment . For example, greater caregiver burden may lead to the implementation of increasing levels of nursing care, thus resulting in a positive correlation between these two factors. Further research is needed to examine these relationships over time and to best guide future policy and supportive care initiatives.The current study also utilized a proxy measure of patient HRQoL completed by their caregivers. Whereas evidence suggests this to be appropriate , previouThe current findings provide specific quantification of burden among AD dementia patients and caregivers alike, and suggest that greater AD dementia severity is associated with a higher degree of burden, most notably related to HRQoL. This study also provides important and novel insight into the experience of those with mild disease symptoms, a group that has received little attention in previous research. In the context of an aging global population and the progressive nature of AD dementia, these results further highlight the need for efforts in promoting early detection and treatment, an issue that has received increasing attention through national organizations in Japan and globally , 66. New


Globally, 13–20% of women experience a common mental disorder (CMD) postnatally. Unfortunately, the burden of CMDs is disproportionally substantial in women from low-income countries. Nevertheless, there is a growing recognition of the buffering effect of social support (SS) on psychiatric morbidity and the need for mental well-being support services/interventions. This study evaluated the relationship between psychiatric morbidity and SS levels, and factors influencing the mental health functioning of Zimbabwean women postnatally. Data were collected from 340 mothers and were analysed through structural equation modelling.The mothers’ mean age was 26.6 (SD 5.6) years. The mean Multidimensional Scale of Perceived Social Support score was 42.7 (SD 10.8), denoting high levels of SS. Additionally, 29.1% of the population reported excessive psychiatric morbidity, the median Shona Symptoms Questionnaire score was 5 (IQR: 2–8). The structural equation model demonstrated the buffering effects of SS on psychiatric morbidity , and accounted for 70% of the variance. Being unmarried, increased maternal age, lower educational and income levels were associated with poorer maternal mental health. There is a need for routine; surveillance and treatment of CMDs in women in the postnatal period, including integration of low-cost, evidenced-based and task-shifting SS interventions.The online version of this article (10.1186/s13104-019-4151-1) contains supplementary material, which is available to authorized users. Globally, between 13 and 20% of women who have just given birth experience a mental disorder . For exaDespite the significant burden of CMDs among women in Sub-Saharan Africa, in-depth information on mental health issues in the postnatal period is limited , 12. NevWe conducted a cross-sectional study at Harare City Council primary health centres. The clinics offer a variety of health services including: curative, maternity and postnatal care. Six clinics were purposively selected to ensure recruitment of participants across the socio-economic continuum. Two of the six clinics were in low to medium density catchment areas with four clinics being located in high-density suburbs . AssuminA purpose-built questionnaire was used to capture the participants’ age, gender, marital status, educational level, employment status and perceived level(s) of income. The Shona Symptom Questionnaire (SSQ), an indigenous generic screen, was used to evaluate the report of CMDs in the past 7 days. The SSQ is a binary outcome i.e. “yes” and “no” responses are scored as one and zero respectively. The score range is 0–14 and scores ≥ 8 indicate risk of CMDs. The SSQ is especially sensitive in screening for depression and anxiety and has been extensively validated in the research setting , 19. TheAfter receiving ethical and institutional approvals, the principal investigator (TK) approached prospective participants in the treatment waiting area(s). The researcher explained the study rationale, applied the selection criteria in recruiting participants and afterwards issued a detailed information sheet to mothers meeting the inclusion criteria. Mothers were obliged to provide written consent to participate in the study. All outcomes were primarily self-administered, however, the principal investigator aided participants where necessary.χms2)—criterial value: p > 0.05, root mean square error of approximation (RMSEA)—criterial value: ≤ 0.06, Comparative Fit Index (CFI)—criterial value: ≥ 0.90, Tucker–Lewis Index (TLI)—criterial value: ≥ 0.90 and the standardized root mean square residual (SRMR)—criterial value: ≤ 0.06 [Data were entered into Microsoft Excel and analysed using STATA (Version 15). Normality was checked using the Shapiro–Wilk Test. Descriptive statistics (frequencies and means) were used to describe participants’ sociodemographics and responses on the SSQ and MSPSS. Thereafter, univariate analysis  was applied to determine factors influencing mothers’ mental health. Contextual factors (patients characteristics) and study primary outcomes (SSQ and MSPSS sub-scores) were then entered into the structural equation model as endogenous and exogenous variables respectively. The following parameters were set as a minimum criterion for model fit; Likelihood Ratio Chi squared Test , attained secondary education (83.4%), unemployed (65%) and reported of medium levels of income (55.3%). Their children were mostly males (50.9%), with an average age of 22.6 SD 13) weeks. Mothers received the least and greatest amount of social support from friends and family respectively, and the mean MSPSS score was 42.7 (SD 10.8), denoting high levels of SS. Additionally, 29.1% of the mothers showed excessive psychiatric morbidity and the median SSQ score was 5 (IQR: 2–8)  and the associated contextual factors. The model accounted for 70% of the variance  was relatively higher compared to the global lifetime prevalence of 18% , and a 1.1% was rIn our study, having fewer resources (lower education and lower income), small social network (being unmarried) and maternal characteristics  negatively influenced maternal mental health. Married and cohabiting mothers showed the least risk of psychiatric morbidity. Traumatic experiences such as the death of a loved one, losing a job and relationship breakdown or divorce are associated with poor mental health functioning –36. ThesCurrent evidence also suggests that increased maternal age is a risk factor for CMDs and this is in contradiction to previous studies , 32, 33.Collectively, our study outcomes point out the need for the provision of support services such as professional counselling for the improvement of the mental health of mothers in the postnatal period. However, the lack of human resources is a massive threat towards the closure of the huge mental health treatment gap in low-resource settings . This thThe prevalence of CMDs was 29.1% and mothers who received an adequate amount of SS showed optimal mental health. Being unmarried, lower education status, lower income level, and increased maternal age were associated with poorer maternal mental health. There is need for routine surveillance and treatment of CMDs in women in the postnatal period. More importantly, there is also need for integration of low-cost, evidenced-based and task-shifting interventions such as the Friendship Bench  in mitigCausality cannot be inferred as data were collected cross-sectionally.Purposively selection of study sites and participants may have introduced selection bias.Clinical data used in applying the selection criterion were self-reported.Institution-based participant recruitment may have precluded selection of community-dwelling mothers at risk of poor mental health.Additional file 1. Frequencies of responses on the MSPSS, N = 340. Table denotes frequencies of responses on the MSPSS, a 12-item social support outcome measure. Responses are rated on a five-point Likert scale, ranging from “strongly disagree = 1” to “strongly agree = 5”.Additional file 2. Frequencies of responses on the SSQ, N = 340. Table denotes frequencies of responses on the SSQ, a 14-item, binary common mental disorders (CMDs) screen. Respondents indicate if they had experienced any of the enlisted symptoms in the last seven days. A yes response is scored as “one” and no as “zero”, a score ≥ 8 is indicative of risk of CMD.Additional file 3. Variance explained by the model. Table denotes the variance accounted by the variables and the total model expressing the relationship between contextual factors, levels of perceived social support and report of common mental disorders.


Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.The Eurasian grapevine ( Vitis vinifera) is one of the most extensively cultivated crops with worldwide economic importance, being the only grapevine species used for the production of quality wine. V. vinifera is characterized by high genetic and phenotypic variability, but selection carried out by humans has limited cultivation to a reduced number of varieties selected for their yielding capacity and traits related to quality, such as berry composition, phenology and resistance to stresses, capturing only part of the diversity of the species1. The V. vinifera varieties that are prevalently cultivated globally are susceptible to various pathogens responsible for severe crop losses, including the Oomycete Plasmopara viticola, the causal agent of downy mildew. This pathogen has a polycyclic behaviour and infects all the green parts of the host leading to quantitative yield losses, due to direct infection of the berries, as well as qualitative damage2. The threat posed by this fungal pathogen, combined with the ineffectiveness of agronomic practices in halting its diffusion, makes frequent spraying of fungicides unavoidable in areas experiencing high disease pressure. This contributes to the status of viticulture as the agricultural activity which makes the most intensive use of plant protection products . This leads not only to negative impacts on farmers’ economic situations, human health and the environment, but also to a potential reduction of future disease control due to the development of fungicide resistance3. For these reasons, the cultivation of pathogen-resistant grapevine varieties is one of the most straightforward strategies to reduce the impact of plant protection against P. viticola while maintaining the quantity and quality of yield. Nonetheless, this process is complicated by genetic factors: Vitis accessions that co-evolved with the pathogen and show partial or total resistance to P. viticola, originating from North America  and Asia (e.g. V. amurensis), can be used as sources of resistance genes in breeding programs2. However, the use of these non-vinifera parents, which are less suitable for winemaking, often introduces unwanted traits in the offspring during breeding, resulting in a serious reduction of wine quality. This important limitation of breeding programs could be avoided by employing resistance genes of V. vinifera origin, but until recently no indigenous varieties have been shown to exhibit these traits. In fact, previous studies suggested that V. vinifera does not have a specific response to infection compared to the American grapevines5.The Eurasian grapevine  of leaf tissues with the pathogen and compared with the same parameters measured for susceptible cultivar Pinot noir.To characterize the interaction of P. viticola followed an analogous pattern in the two cultivars until two dai: the asexual spores of the pathogen penetrated through the stomatal pore and differentiated the substomatal vesicle , being 3.4 times lower than on Pinot noir. Analogously, sporulation per leaf unit showed a significant nine-fold reduction , indicating that the plant employs defense reactions to reduce pathogen infection and sporulation in comparison with Pinot noir  confocal microscopy analyses were carried out following aniline blue staining to establish the growth of pathogen within leaf tissues of Mgaloblishvili and Pinot noir. The analyses showed that the colonization of leaf tissues by cle Fig.  from whicle Fig. . Startinoir Fig.  showed tted Fig.  and ill ted Fig. , and shoted Fig. . At the oir Fig. . These rV. vinifera and non-vinifera samples confirmed the suitability of PN40024 12X v2 grape reference transcriptome for read mapping of non-vinifera genotypes, such as the Vitis interspecific crossing Bianca and other interspecific hybrids19.Whole transcriptome analysis was performed on inoculated and non-inoculated leaves of Mgaloblishvili, Pinot noir and Bianca collected at three time points . The trimmed reads were mapped on the PN40024 12X v2 grape reference transcriptome. The average number of unique mapping reads per sample was ~52 million, ranging from 48 to 55 million reads, and a percentage of 78% successfully mapped reads (from 73 to 82% of reads). The similar percentages of mapped reads overall the The overview of count table has been represented by heatmap and hierarchical analysis Fig. . The samP. viticola.Differentially expressed genes (DEGs) obtained by comparing inoculated and non-inoculated samples of each cultivar are listed in the Table At 1 dai, the three genotypes shared a core of 181 DEGs, and the highest number of DEGs (774) was shared between Bianca and Pinot noir, followed by Mgaloblishvili and Pinot noir (600 DEGs), and Bianca and Mgaloblishvili (550 DEGs). At 2 dai, Bianca and Pinot noir shared two DEGs, while at 3 dai the three cultivars shared seven DEGs Fig. .Moreover, at each time point, cultivar-specific DEGs were found: for example, in Mgaloblishvili 28%  and 64%  of total DEGs were not shared with Pinot noir nor Bianca Fig. . FinallyV. vinifera, the gene expression patterns of Mgaloblishvili and Pinot noir induced by P. viticola inoculation were analyzed and compared. The highest number of DEGs was detected in the two varieties following inoculation with P. viticola at 1 dai. Subsequently, the number of DEGs strongly decreased, reaching the lowest value at 2 dai and increasing a little at 3 dai  that identify pathogen-associated molecular patterns (PAMPs), molecules which are essential for microbes to establish an infection22. P. viticola PAMPs include β-glucans and cell wall components, and recognition occurs via the invading haustorium23. Laminarins in particular have been shown to elicit a defense reaction in V. vinifera24.Oomycetes undergo a series of developmental stages throughout a successful infection cycle, including the formation of sporangia, release of motile zoospores, their encystment and germination to form hyphae, haustoria and, finally, sporangiophores25 were strongly overexpressed in Mgaloblishvili upon P. viticola inoculation 28. An increasing number of effectors has been characterized for Oomycetes28. Based on homology sequences, the presence of a wide array of effectors, supposedly interfering with the response of the V. vinifera defense mechanism, has also been detected in the P. viticola genome, with extensive differences between European and Chinese strains29. To counteract the action of the pathogen, plants have evolved specific receptors called NBS-LRR (nucleotide-binding site leucine-rich repeat), proteins encoded by resistance R genes, able to recognize the effectors and ultimately activating defense mechanisms leading to effector-triggered immunity (ETI) through hypersensitive response (HR)30. Among the known NBS-LRRs is the rust resistance Lr10 locus, encoding for CC-NBS-LRR receptors involved in wheat leaf rust resistance31. Surprisingly, in the present study several isoforms of the Lr10 gene were found to be overexpressed in Mgaloblishvili, indicating that this cultivar could be capable of specifically recognizing fungal effectors. In contrast, a strong downregulation of these gene isoforms was found in Pinot noir, highlighting the uniqueness of Mgaloblishvili in this respect. Since Mgaloblishvili exploits genes encoding for PAMPs receptors and effectors upon P. viticola inoculation, it seems likely that the plant activates PAMP-triggered immunity (PTI) and an incomplete ETI response. In fact, the wide array of effectors present in the P. viticola genome, along with the upregulation of isoforms of a single NBS-LRR receptor, could explain why the resistant phenotype is not related to HR in Mgaloblishvili.Successful pathogens use effector molecules, encoded by 32. Several RING H2-type E3 ligases, involved in ubiquitination process, are activated in response to biotic and abiotic stresses. The upregulation of genes encoding for RING-H2 ATL proteins is related to P. viticola resistance in non-vinifera grapevine species. In contrast, these genes are downregulated in susceptible V. vinifera cultivars33. This is confirmed by the results obtained in the present study for Pinot noir, where two genes encoding for RING-H2 ATL proteins (ATL39 and ATL20) were downregulated. For the first time in a V. vinifera cultivar, Mgaloblishvili has been demonstrated to exhibit a gene encoding for a RING-H2 finger protein-like, namely ATL22, which was strongly upregulated on P. viticola inoculation  to release elicitors that induce plant defense responses, and by a direct effect derived from digestion of glucan fibers and the resulting weakening of fungal cell walls40. The modulation of β-glucanases has previously been detected in response to artificial inoculation with P. viticola in V. riparia and V. vinifera27. Interestingly, the analysis of promoter regions for NAC, WRKY and β-glucanase genes revealed the presence of stress-responsive cis-elements, such as GCC-box (data not shown), known as recognition sites for ethylene-responsive transcription factors41.The plant defense mechanism is regulated by a complex network of signaling transduction pathwaysne Table , involvene Table  and GDSLne Table , indicatAC Table , WRKY anAC Table , were diGenes related to the IAA signaling system were also upregulated, but later than the ethylene pathway. Indeed, tryptophan aminotransferase-related protein gene Table , leading43. Interestingly, genes implicated in the synthesis of terpenoids, i.e. several cytochrome P450s . PCA explained almost all variability among samples (90%) and clearly distinguished the two varieties on the principal component 1 (PC1 = 66%). PC2 (24%) partially identifies a shift between inoculated and non-inoculated samples, but this difference was also partly explained by PC1  was validated through Real-time RT-PCR Table  on MgaloPC1 Fig. . These rP. viticola was compared to that of a reference resistant variety, Bianca, to explore the possible existence of shared or disparate mechanisms underpinning resistance. Bianca is an interspecific hybrid selected and cultivated in Hungary for wine production and obtained through a long and complex hybridization process. The parentage of Bianca is approximately 80% V. vinifera and 20% of non-vinifera, with a genetic background originating from other resistant Vitis species, such as the American V. labrusca, V. rupestris, V. berlandieri and V. lincecumii54. Resistance to P. viticola in grapevine Bianca is controlled by a major dominant gene, associated with localized HR in leaf tissues immediately after pathogen infection, preventing mycelial growth and sporulation within host tissues55. Resistance based on single resistance genes  generally induces a strong selection pressure on pathogen populations and, as a consequence, a rapid increase in the frequency of strains that are able to overcome the host defence reactions. Indeed, resistance breaking isolates of P. viticola have been found in Bianca55.Finally, Mgaloblishvili response to P. viticola growth was found in Mgaloblishvili , synthesis of antimicrobial compounds (cytochrome P450 78A4 element) and structural defenses (cellulose synthase-like protein G3)57, and a BTB/POZ domain-containing protein involved in the indirect activation of disease resistance protein PR1, associated with Systemic Acquired Resistance58; enzymes involved in the synthesis of antimicrobial compounds, such as a cytochrome P450 (CYP72A219 element), and a valencene synthase, involved in the biosynthesis of valencene59, a sesquiterpene acting in the inhibition of hyphal growth and zoospore viability in Oomycetes60.Remarkable differences in the defense mechanism, associated with unique characteristics of Mgaloblishvili, were found in genes related to pathogen recognition, signaling and antimicrobial compound synthesis Table . The mosV. vinifera germplasm of Georgia, previously explored for its resistance or tolerance to other important pathogens, such as phytoplasmas62. The putative defense response observed in Mgaloblishvili, schematically represented in Fig. V. vinifera cultivar and exhibiting unique features compared to those of an American grapevine, which relies on HR. The response to P. viticola is associated not only with PAMP and DAMP recognition but also with a weak effector recognition, mediated by Lr10 locus, not leading to HR. This could be compatible with the absence of co-evolution between P. viticola and Mgaloblishvili, resulting in an inability to recognize all the effectors of the pathogen. P. viticola was in fact introduced into Europe from Northern America in 1878, reaching South Caucasus in the last decade of the century63. The historic time period since pathogen introduction has apparently been too brief to allow co-evolution between the pathogen and plant, an effect that is undoubtedly exacerbated by the asexual vegetative propagation of this crop plant. Overall, the defense mechanism is mainly mediated by the ethylene signaling pathway and consists of the limitation of P. viticola growth and sporulation through the expression of genes involved in the synthesis of antimicrobial compounds, mainly terpenoids and flavonoids but also glucanases affecting pathogen cell wall integrity, and in the deposition of structural barriers that both thicken the host cell wall and encapsulate hyphae in a callose-like material.The results reported in this work show for the first time the existence of resistance pathways against one of the most important grapevine biotic stresses, downy mildew, within the V. vinifera gene pool is an unique precedent, opening new and promising perspectives for sustainable disease management of grapevine because it can lead to: (i) a more durable resistance through the exploitation of new target genes for V. vinifera genetic engineering (ii) changes in the strategies adopted for disease resistance breeding, which can now concentrate on the Eurasian grapevine as an easier method than hybridization with American grapevines; (iii) screening of other V. vinifera varieties to detect novel resistant genotypes; (iv) increased efforts for biodiversity conservation, to preserve Georgian and other local grapevine genetic resources as a sources of useful traits to be exploited in additional branches of viticulture.The identification of resistance traits within the 63 and phytoplasmas61 in previous studies, demonstrates that the Eurasian grapevine V. vinifera is more capable of responding to biotic stresses than was previously known or believed. This confirms yet again the importance of V. vinifera gene pool from the more ancient area of grapevine cultivation, extending from South Caucasus to Central Asia.Generally speaking, the detection of resistance to downy mildew in our study, and to powdery mildewV. vinifera variety Mgaloblishvili, the international V. vinifera variety Pinot noir, and the Vitis interspecific hybrid variety Bianca were grown in greenhouse  at the Department of Agricultural and Environmental Sciences  in 5 L pots filled with sand-peat mixture (7:3 v/v), regularly watered via a drip system and fertilized twice a year with Osmocote Topdress fertilizer .Two-year-old plants of the Georgian P. viticola prepared as described by Toffolatti and coworkers9, on the underside of the 2nd–5th leaves starting from the apex of the shoots. 2.5 × 104P. viticola sporangia were sprayed per leaf. Shoots were covered with transparent plastic bags to keep humidity high and to favor the infection process where needed.Four plants per variety were used for the experiment. For each plant, two shoots were used in the experimental procedure: the first one was spray-inoculated with the pathogen and the second one was sprayed with sterile distilled water. Experimental inoculations were carried out by spraying a sporangia suspension of P. viticola) were randomly collected at 0, 1, 2 and 3 days after inoculation (dai). A leaf disc (2 cm diameter) was cut from each sample for confocal microscopy analysis, and the rest of the leaf tissues was frozen at −80 °C for RNA extraction. The leaf discs were fixed in 0.1 M phosphate buffer (pH 7.2) containing 2% paraformaldehyde and 3% glutaraldehyde and kept at 4 °C. All microscopy reagents were purchased from Sigma-Aldrich .Three leaves  per variety  and treatment .Furthermore, three inoculated leaves were sampled at 6 dai for assessing disease severity (I%I), evaluating the number of sporangia formed by the pathogen2HPO4 (pH 9) for 24 hours21. This dye was selected as its chemical properties allow it to bind with β-1,3-glucans present both in the cell wall of the pathogen and in the callose deposition produced by plant cells in response to infection65. Confocal microscopy analyses were performed using an inverted microscope, Leica DMIRE2 with a HCX APO L U-V-I 63.0 × 0.90 water immersion objective, equipped with a Leica TCS SP2 laser scanning device . To detect aniline blue fluorescence, leaves were excited by the 405 nm UV laser and the emission was collected between 450/530 nm. For chlorophyll detection, samples were excited at 514 nm line of the Argon laser and the emission was collected between 650/750 nm. Images were captured as z-series (50–60 stacks spaced every 0.5 µm) of optical sections (3-D), keeping the same parameter settings in all acquisitions, and displayed as single maximum-intensity projections (MIPs) obtained by Fiji, an open-source platform for biological-image analysis66. MIPs were obtained for the fluorescence of both targets and then merged to obtain the images shown in Figs The pathogen development was observed on samples prepared from leaves collected at 1, 2, 3, and 6 dai on Mgaloblishvili, Pinot noir and Bianca. Leaf discs were stained with 0.05% aniline blue in 0.067 M KRNA was extracted from 100 mg of sampled tissues. Samples wer ground with liquid nitrogen and RNA was extracted using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich), according to the manufacturer’s instructions.67.RNA was quantified using Qubit® RNA HS Assay Kit by Qubit® 3.0 Fluorometer ; its integrity and purity (260/230 and 260/280 ratios) were assessed using respectively Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer , and NanoDrop Spectrophotometer . The samples showing a 260/230 ratio lower than 1.8 were subjected to lithium-chloride purificationStarting from 1 μg of high quality total RNA per sample, 72 cDNA libraries were constructed according to the KAPA Stranded mRNA-Seq Kit . Each library was barcoded using the SeqCap Adapter kit A and B  and the final size of 250–280 bps was confirmed on High Sensitivity D1000 ScreenTape by Tapestation 2200 (Agilent). All the libraries were quantified by KAPA Library Quantification kit – Illumina (Kapa Biosystems) using the LightCycler 480 , and randomly multiplexed in seven pools in equimolar way. Each pooled library was sequenced on an Illumina HiSeq2500 platform  with paired end runs of 2 × 50 bps. Base calling and quality control were performed on the Illumina RTA v1.13 (Illumina) sequence analysis pipeline. The original sequencing datasets have been deposited in the European Nucleotide Archive (ENA) with the accession number PRJEB24540.68 and then processed to remove low quality bases and contaminants (Illumina adapters). Cleaning phase was then performed with Trimmomatic software version 0.3669 using an average quality cut-off of 30 (Phred score) and a minimum read length of 40 bp. Cleaned reads were then mapped against predicted mRNAs (PN40024 12X v2 grape reference transcriptome) obtained from the gene prediction version 2.0 of the National Centre for Biotechnology Information. Mapping was performed using Bowtie270 tool with default parameters. Alignments were first converted in a binary alignment map (BAM), a binary representation of the Sequence Alignment/MAP (SAM), and then sorted and indexed for the count of reads per mRNA, using SAMtools71 software package. The number of reads aligning to each transcript were counted using an ad hoc Python script.Raw reads quality was visually inspected by means of FastQC software72. Transcripts with less than 5 reads were not included in the analysis. To determine the differentially expressed genes (DEGs) between the different treatments (inoculated vs non-inoculated samples) of each variety at each time point, a multifactor designs method has been performed with DESeq2 R package. For each transcript, the log2 fold change (FC), p-value and adjusted p-value were evaluated, and only RefSeq IDs with false discovery rate (FDR)-adjusted p-value < 0.05 were retained. The protein sequence and functional information of DEGs were obtained from UniProt database73 or InterProScan tool for functional domain identification74.To have an overview of similarities and dissimilarities among samples, the count data were used to perform heatmap analysis with hierarchical clustering and Principal Component Analysis (PCA) with DESeq2 R package75, using DEGs as input data. The graphical representation of heatmap, hierarchical clustering and Principal Component Analysis (PCA) were performed using the function heatmap.2 implemented in gplots R package76.Two and three-way Venn diagrams showing the overlaps among different time points per genotype were calculated and built using the jVenn web-server77. Enrichment test was run on DEGs showing a FDR-adjusted p-value < 0.05 and gene2GO annotation file was retrieved by the Grape genome browser of CRIBI Center78. A classical enrichment analysis was performed by testing the over-representation of GO terms within the group of DEGs, using the statistical Fisher’s exact test. The top-50 significantly enriched GO IDs were listed and recorded based on the terms of biological process ontology.Gene ontology (GO) enrichment analyses were performed with R package topGO version 2.26.0P. viticola at 1 dai, was investigated through semi-quantitative real-time reverse transcriptase (RT)-PCR analyses , according to the manufacturer’s instructions. The cDNA was diluted 1:1 with sterile water and amplification reactions were carried out in three technical replicates per sample.79. Ubiquitin80 and actin81 genes were used as references for data normalization. Real-time RT-PCRs were performed using a SYBR green method on a StepOne Plus Real-Time PCR System (Thermo Scientific) thermal cycler. Each 10 μl PCR reaction contained 0.6 μl of each primer (10 μM), 1 μl of diluted cDNA, 1X SYBR Green Real-Time PCR Master Mix (Thermo Scientific) and sterile water. Thermal cycling conditions were: 95 °C for 10 min followed by 40 cycles of 94 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s, and a melt cycle with 1 °C increments from 55 to 96 °C. The expression of each gene in different varieties and treatments was calculated by comparing their 2−ΔΔCt values82. Such values were employed in a PCA analysis (SPSS) revealing the collective differential expression patterns of the five genes. Linear correlation between the differential expression levels of the five genes obtained by RNA-seq and real-time RT-PCR analyses was tested in SPSS. Goodness of fit was evaluated through significance and R2 values.Gene specific primers Table  were desSupplementary informationTable S1Table S2Table S3