MTHFR Ala222Val  polymorphism has repeatedly demonstrated to play a pathological role in numerous mental disorders, but none of these studies focused on the susceptibility of suicidal behavior in SCZ. In the present cross-sectional study, we recruited 957 chronic inpatients with SCZ and 576 healthy controls to assess the psychopathological symptoms of SCZ and compare the frequency of the MTHFR Ala222Val genotype in both suicide attempters and non-attempters. Our results demonstrated no significant differences in MTHFR Ala222Val genotype and allele distributions between the SCZ patients and controls (p > 0.05), but showed a statistical significance in the distribution of Ala/Val genotype between suicide attempters and non-attempters (p < 0.05). Further logistic regression analysis showed that MTHFR Ala222Val genotype, psychopathological symptoms, number of cigarettes smoked per day and drinking status were related to suicide attempts in SCZ (p < 0.05). Our study demonstrated that MTHFR Ala222Val polymorphism and some clinical characteristics might confer susceptibility to suicide in patients with SCZ.Patients with schizophrenia (SCZ) exhibit higher suicide rates than the general population. However, the molecular mechanism responsible for the high rate of suicidal behavior in SCZ remains poorly understood.  To date, suicide has been considered one of the most common causes of premature death in SCZ patients3. The average life expectancy of patients with SCZ is estimated to have decreased by approximately 14.6 years by suicide4. However, the pathophysiological mechanisms for the suicidality of SCZ patients are still unclear, particularly the biological pathological mechanism.Patients with schizophrenia (SCZ) are at high risk for attempting and completing suicide. Suicide is characterized by intentional termination of one’s life, which is an important sequelae of chronic schizophrenia5, which suggests that genetic risk factors may play an important role in suicidal behavior. The heritability of suicidal behavior is about 45%, while environmental components account for 57%6. Based on earlier genetic association studies focusing on several biological systems associated with suicidal attempts, a variety of potential candidate genes have been identified. For example, brain-derived neurotrophic factor (BDNF) and its receptor (NTRK2), which are highly expressed in the prefrontal cortex and hippocampus, have been shown as strong candidates for suicide; however, these results are controversial6. Also, another biological system involved in serotonergic transmission has been found to be candidate genes for suicidal behavior, including a tryptophan hydroxylase involved in synthesis, monoamine oxidase A involved in degradation, and a serotonin-1-A receptor involved in transmission7. Studies have demonstrated that nearly 90–98% of suicides are committed by people with some type of mental illness8. Thus, mental illness can be identified as the strongest risk factor for suicide. Among them, SCZ is one of the most common diseases associated with suicidal behavior9. Above evidences suggest that overlapping genetic risk factors are associated with suicidal behavior and psychopathology. However, genetic markers associated with the etiology and pathophysiology of suicidal behavior in psychiatric patients, especially SCZ patients, are still unknown.Evidence from twins, adoption, and family studies showed a significant increase of suicidal rates in suicidal probandsMTHFR gene, located on chromosome 1 encodes a 5-methylenetetrahydrofolate reductase that catalyzes the reduction of 5,10-methylenetetrahydrofolate (methylene THF). Impaired MTHFR metabolism leads to hypomethylation of DNA, which increases the mutation ratio and leads to chromosome instability10 in addition to increased concentrations of potentially toxic homocysteine (Hcy) and abnormal concentrations of neurotransmitters11. It was found that the substitution (C to T) of nucleotide 677 (rs1801133) in exon 4 of the MTHFR gene resulted in amino acid substitution  and a reduction of MTHFR activity, which was most frequently studied in relation to mental disorders including SCZ14, autism spectrum disorders17, affective disorders20, anxiety disorders22 and alcohol dependence23. To the best of our knowledge, only one study has investigated whether the MTHFR Ala222Val polymorphism was associated with suicide in 692 victims of completed suicide and 3257 healthy controls. No association was found between the MTHFR Ala222Val polymorphism and suicide24. In the study, only 16 of the 692 subjects were diagnosed with SCZ, and the diagnoses were based upon medical and forensic examinations of post-mortem rather than evaluations by psychiatrists. Recently, several striking meta-analyses have demonstrated that MTHFR Ala222Val polymorphism is significantly associated with SCZ28. In particular, three meta-analyses have shown a positive association between MTHFR Ala222Val polymorphism and SCZ in Asian population, but not in the European population29. Furthermore, the SCZ patients with the MTHFR Val/Val or Ala/Val genotype were found to have higher Hcy levels than those with the Ala/Ala genotypes, which might be associated with the increased risk of SCZ31. A recent meta-analysis also indicated that increased plasma total Hcy levels may be associated with a higher risk of SCZ27. Interestingly, one study showed that MTHFR Val/Val genotype affected gray matter density and memory impairment in SCZ patients in a Chinese population32. Taken together, these studies have suggested that the MTHFR gene polymorphisms, especially the MTHFR Val/Val genotype, may be involved in the psychopathology of suicidality in SCZ patients.The MTHFR gene, and the higher Hcy  level in SCZ, it would be interesting to investigate the relationship between the functional polymorphism of MTHFR Ala222Val and suicide attempt of SCZ patients in a genetically more homogeneous Han Chinese population. Based on previous literature, we hypothesized that the Val allele carriers of the MTHFR Ala222Val would be associated with increased risk for suicide attempt in SCZ and may interact with some relevant clinical risk factors. We then examined whether the interaction of genetic and environmental factors might affect the risk of attempted suicide in SCZ patients.Considering the high suicide rate of SCZ patients, the pathogenic role of the MTHFR Ala222Val genotypes were consistent with the Hardy-Weinberg equilibrium in both SCZ patients and healthy controls (both p > 0.05). Further, there were no significant differences in the Ala222Val allelic and genotypic distributions between SCZ patients and healthy controls (both p > 0.05). After adjusting for sex, smoking, BMI, and age, we still did not find any differences in  the Ala222Val allelic and genotypic frequencies between the two groups (both p > 0.05). In addition, we did not find any association between the MTHFR Ala222Val genotypes and the positive and negative syndrome scales33 (PANSS) total and its 3 subscale scores .The demographic and clinical characteristics are shown in Tables χ2 = 4.7, df = 2, p = 0.039), and the Ala/Val genotype was less frequent in attempters than in non-attempters (36.1% vs 46.6%) in the codominant models . The MTHFR Ala/Val genotype was associated with a lower suicide risk compared to the Val/Val genotype .The SCZ patients included 152 (15.9%) suicide attempters and 805 non-attempters. The Ala222Val allelic and genotypic frequencies of suicide attempters and non-attempters of patients are shown in Tables We further analyzed the characteristics related to suicide and found significant differences in drinking status (p < 0.001), onset age of smoking (p < 0.05), cigarettes smoked each day (p < 0.05), duration of illness (p < 0.01), positive factor of PANSS (p < 0.05), depression factor (p < 0.001), and excitement factor (p < 0.05) between attempter and non-attempter groups, as shown in Table χ2 = 5.6, p = 0.018), PANSS depression factor , number of cigarettes smoked per day , and drinking status  remained significant and MTHFR Ala222Val polymorphism  approached significance.In the binary logistic regression model for associations with suicide attempt, education . Moreover, the suicide attempt sample had 0.88–0.99 statistical power to detect this polymorphism to have relationship with suicide attempts as dominant, recessive, and log additive inheritance, with an OR of 2 .The whole sample in the current study had a power of 0.99 to detect the association of MTHFR Ala222Val polymorphism between the patients with SCZ and healthy controls, which was in accordance with a couple of previous studies36, suggesting that the Ala222Val polymorphism of the MTHFR gene did not contribute to increased risk of SCZ in our Chinese samples. Second, the MTHFR Ala222Val polymorphism appears to be weakly correlated with suicide attempts in SCZ. Moreover, compared to patients without lifetime suicide attempts, the Ala/Val genotype was significantly less frequent and the Val/Val genotype was more frequent in lifetime suicide attempts of SCZ patients . Third, in addition to MTHFR Ala222Val polymorphism, higher psychopathological symptoms and more severe depressive symptoms, more cigarettes smoked, and more drinking were significantly correlated to suicidal risk, suggesting that a multifactorial etiology involving both genetic heritability and environmental factors may contribute to suicidal behaviors among SCZ patients.There are three main findings in the present study. First, we did not find any significant difference in the MTHFR gene in the risk of pathogenesis of SCZ, but the results are inconsistent. We found that MTHFR Ala222Val polymorphism did not play a significant role in the pathogenesis of SCZ in our current study, which is consistent with several previous studies in Iranian, Korean, and Nordic populations37. However, the most recent meta-analysis reported a positive association between Ala222Val and SCZ in eastern Asia27, which is completely different from our results. A possible reason for the inconsistencies in these genetic association studies may be due to ethnic background. For example, the genotype frequency distribution of Ala222Val is significantly different between the Chinese and European populations. The frequency of Val/Val genotype in healthy controls was approximately 16% in the Chinese population, but only about 9% in the European population. Therefore, interethnic differences in the genotype frequencies of the MTHFR Ala222Val polymorphism may lead to inconsistent results across the different populations. Other factors may also contribute to these controversial results, such as small or moderate genetic effects of Ala222Val, diet with increased folate consumption, relatively small sample sizes, heterogeneity in SCZ diagnosis, and gender stratification.Numerous studies have investigated the possible role of functional polymorphism of MTHFR Ala222Val polymorphism and suicidal  behavior in SCZ patients. Moreover, no significant association was found between the MTHFR Ala222Val genotypes and the PANSS total and its 3 subscale scores , suggesting that the MTHFR Ala222Val polymorphism appears not to be a general marker of a more severe disease but rather specific for suicide. Only one previous study investigated the association between suicide and Ala222Val polymorphism in postmortem and reported contradictory results24. A possible explanation for the inconsistency in results is that the subjects enrolled in the present study were suicide attempters and not suicide completers. In addition, different methods were used in the diagnosis of SCZ . Thus, the differences in the participants may account for the discordant results in these two studies. Our current study suggested that MTHFR Ala/Val genotype may lay a critical role in suicidal behaviors. The 222Val allele was considered to be a mutant allele. Ala222Val polymorphism was thought to be associated with alteration of MTHFR enzyme activity as the heterozygous genotype  and mutant homozygous genotype  have only 67% and 25% of the MTHFR enzyme activity of their Ala/Ala wild type counterparts, respectively22. In the present study, the Val/Val genotype was over-present and the Ala/Val genotype was less present among suicide attempters in patients with SCZ. We speculate that the association of MTHFR Ala/Val genotype with suicide attempt may be in relation to the reduced MTHFR enzyme activity. A reduction in MTHFR enzyme activity results in a decline in its ability to irreversibly reduce 5,10-methylene THF to 5-methyltetrahydrofolate (5-methyl THF) and produce methyl groups involved in epigenetic regulation22. The declined enzyme activity may either raise the concentration of potentially toxic Hcy in those with inadequate dietary folate intake, or lead to hypomethylation of DNA39. The studies in MTHFR gene knockout mice have shown that plasma total Hcy was 10-fold and 1.6-fold higher, respectively, in MTHFR -/- and MTHFR +/− mice than in wild-type controls41. Numerous studies have also demonstrated that elevated total Hcy levels in plasma are one of the most important risk factors for psychiatric diseases such as depression and SCZ45, although few studies have focused on the direct relationship between suicide attempt and Hcy in SCZ. However, it is worth mentioning that this explanation for the association between heterozygous genotypes and suicide attempts may stem from the enzymatic effects of genotypes, which is only our speculation. If our interpretation in this part was of major importance, there should be an even stronger difference between the wildtype (Ala/Ala) and the homozygous mutant genotype  than between Ala/Ala and Ala/Val genotypes, and the difference should also be obvious when alleles were compared. However, the fact is that the Ala/Ala and Val/Val genotypes were almost equally present and far from significantly different after clinical variables were controlled  , longer duration of illness, alcohol consumption, and more cigarettes smoked each day were associated with suicidal behavior in SCZ, suggesting important environmental factors in suicide behavior among SCZ patients.Both SCZ and suicide behavior have a multifactorial and complex etiopathogenesis, arising from a combination of genetic and environmental factorsMTHFR Val/Val genotype can be alleviated by increasing the consumption of folate and, to a degree, cobalamin60. However, we did not measure plasma folate and homocysteine levels in the subjects and did not have information on whether our subjects lacked folate food fortification or not. Second, there was a significant difference in the age between suicide attempters and non-attempters. Many studies have found that the common factors which may exacerbate folate deficiency are older age, male sex, and alcoholism63. Third, there were more female subjects in the SCZ group, which may have led to bias in the statistical analysis due to the imbalance in the number of subjects in each sex category in our current study. Fourth, it should be noted that we only found a weak association between the Ala222/Val genotype and suicide attempt in SCZ.There are several limitations that should be noted. First, the effect of MTHFR Ala222/Val polymorphism and  SCZ, but a weak association between this polymorphism and suicide attempt in SCZ. Moreover, psychological symptoms, drinking status, and smoking possibly interacted with the MTHFR gene variant and appeared to play an important role in suicide attempts among SCZ patients. However, considerable caution should be noted in the interpretation of the results. Since the sample size was small, the significant association between MTHFR Ala222/Val polymorphism and suicidal behavior may be due to multiple tests or small sample size. Further studies in a larger sample of various ethnic populations are unavoidable to investigate the interactions between these genes and the environment.To summarize, in the present study, we showed no relationship between 957 chronic inpatients with SCZ were recruited from two large psychiatric hospitals in Northern China. The inclusion criteria were as our prior studies: age 20–75 years; Han Chinese; and diagnosis of SCZ according to DSM-IV by the trained psychiatrists. All patients were of the chronic type with an illness duration of at least 5 years. Also, they had been taking stable doses of oral antipsychotic medication for at least one year prior to recruitment. Antipsychotic medication consisted mainly of prescribed drug monotherapy, including clozapine (n = 430), risperidone (n = 210), chlorpromazine (n = 69), sulpiride (n = 49), perphenazine (n = 45), quetiapine (n = 42), haloperidol (n = 34), aripirazole (n = 29), and others (n = 49). Average of antipsychotic dose  was 438 ± 407 mg/day.576 unrelated healthy controls were recruited through advertisement from local communities near the hospital during the same period as the patients. A research psychiatrist ruled out potential controls with Axis I disorders on DSM-IV at the present or in their lifetime after a clinical structured interview. All the healthy controls were Chinese Han population.We carried out a physical examination and laboratory tests from all subjects. Any subjects with abnormalities in physical health were excluded. All subjects gave informed consent to participate in the study, which was approved by the Institutional Review Board of Beijing HuiLongGuan hospital. In addition, all methods were performed in accordance with Declaration of Helsinki promulgated by the National Institute of Health.PANSS was used to assess symptoms of SCZ on the same day of blood drawing. Prior to the assessment, four psychiatrists received training sessions in  the use of PANSS and conducted repeated assessment tests. After training, inter-rater correlation coefficient was greater than 0.8 for the PANSS total score.64. The new positive factor was composed of P1, P3, P5, and G9, and the new negative factor was composed of N1, N2, N3, N4, N6, and G7. In addition, the three items P2, N5, and G11 of the PANSS scale constitute a cognitive factor, G2, G3, and G6 for a depressive factor, and P4, P7, G8, and G14 for an excitement factor.In this study, a five-factor model of SCZ symptoms was utilized, labeling symptoms as ‘positive’, ‘negative’, ‘cognitive’, ‘depression’, and ‘excitement’65. In this study, suicide attempts were defined as deliberate self-destructive behavior, with at least some intention to kill themselves, but not causing death65. During the study interview, the main question was “Have you tried to commit suicide in your lifetime?” If the answer was yes, then all subjects were asked about previous suicide attempts, including the following details: number of attempts, exact date and method of each suicide attempt. Totally, there were 152 patients who had a history of suicidal attempt (attempters) and 805 patients without history of suicidal attempt (non-attempters). The average number ± standard deviation of attempted suicide among attempters was 1.51 ± 0.94 times (ranging from 1 time to 5 times).History of suicide attempts were assessed by the same four psychiatrists based on record reviews and interview data according to a definition by WHO24, we selected one functional single nucleotide polymorphism Ala222Val in the promoter of MTHFR gene. The polymorphism was genotyped following the standard procedure as described in Sequenom Genotyping Protocol and the previous studies66. The primers and extent probe of MTHFR Ala222Val were: sense: 5′-ACGTTGGATGCTTGAAGGAGAAGGTGTCTG-3′, antisense: 5′-ACGTTGGATGCTTCACAAAGCGGAAGAATG-3′, and probe: AAGGTGTCTGCGGGAG. The sample genotyping success rate of this study averaged 98.9%. 5% of all DNA samples were repeated, showing 99.5% reproducibility for SNP results.Based on previous studiesχ2 test for goodness of fit. The differences in allele and genotypic frequencies of MTHFR Ala222Val polymorphism between groups were analyzed by using the χ2 test. Further, we used binary logistic regressions to calculate odds ratios (ORs) and their 95% confidence intervals (CIs) to evaluate the effects of different genotypes on SCZ or suicide after controlling for the confounding factors.Deviations from Hardy-Weinberg (HW) equilibrium were tested by using the Analysis of variance (ANOVA) for continuous variables and the chi-squared test for categorical variables were used to analyze the differences between groups . The relationship between the variables is evaluated using Pearson’s correlation coefficient. Bonferroni correction was used with each analysis to correct multiple tests. The variables that were significantly correlated with suicide attempt were included in the next logistic regression analysis, which was carried out to adjust for confounding factors. In the logistic regression model, the dependent variable was suicide, and the independent variables were those that were significantly different between suicide attempters and non-attempters.We used SPSS version 15.0 to perform all statistical analysis, with two-tailed p values of less than 0.05. The power of the sample was calculated using Quanto software, with the relative risk and known risk allele frequencies under dominant models, respectively.


Environmental factors such as stress drive the development of drug addiction in genetically vulnerable individuals; the genes underlying this vulnerability are unknown. One strategy for uncovering these genes is to study the impact of environmental manipulation on high-throughput phenotypes that predict drug use and addiction-like behaviors. In the present study, we assessed the viability of this approach by evaluating the relative effects of environmental enrichment and isolation housing on three high-throughput phenotypes known to predict variation on distinct aspects of intravenous drug self-administration. Prior to behavioral testing, male and female C57BL/6J and DBA/2J mice (BXD founders) were housed in enrichment or isolation for ten weeks beginning at weaning. Enrichment significantly reduced novelty reactivity; this effect was significantly more robust in C57BL/6J mice relative to DBA/2J mice. Enrichment significantly reduced novelty preference; this effect was significantly dependent on novel environment characteristics and was significantly more robust in DBA/2J mice relative to C57BL/6J mice. Enrichment significantly increased anxiety; this effect was not strain-dependent. Collectively, these data indicate that (1) environmental enrichment influences novelty reactivity, novelty preference, and anxiety via distinct genetic mechanisms in mice, and (2) the BXD panel can be used to discover the genetic and epigenetic mechanisms underlying this phenomenon. Intravenous drug self-administration in mice2 provides a direct but technically challenging approach for discovery of genetic and environmental factors driving drug use and addiction-like behaviors. A complementary strategy for uncovering these mechanisms is the study of easily quantifiable behavioral phenotypes  that predict drug use and addiction-like behaviors8. A distinct advantage of using these predictive phenotypes as a proxy for intravenous drug self-administration is that they can be quantified in mice using high throughput, low stress protocols that require no drug exposure. These characteristics allow quantification of multiple predictive phenotypes in the same mouse and same study using identical experimental conditions; this allows direct comparison of genotype, environment, and sex effects across phenotypes. In summary, this strategy provides an easily accessible window onto genes that have a high probability of influencing drug use and addiction-like behaviors.Drug addiction is a heritable disease driven by unknown genetic and environmental factors9; the genes underlying this vulnerability are largely unknown. In experimental animals, isolation housing10 and environmental enrichment11 provide rodent models of high-stress and low-stress environments, respectively13. Studies in mice and rats using these models reveal that isolation housing potentiates, and environmental enrichment attenuates, both drug use and phenotypes that predict drug use15. Together with observations of correlated drug use phenotypes and predictive phenotypes8, these data suggest that the biological mechanisms influencing both drug use and predictive phenotypes are themselves influenced by housing manipulation. Consequently, a strategy of housing manipulation  followed by quantification of predictive phenotypes could be used to identify mouse strains with alleles that confer vulnerability or resistance to addiction following stress exposure. Employing this strategy using a recombinant inbred mouse panel  in the context of a systems genetics approach would enable the discovery of the precise genetic mechanisms underlying these strain differences in stress-induced addiction vulnerability.Epidemiological studies reveal that environmental stress drives the development of drug addiction in genetically vulnerable individuals6. To index these phenotypes, we used exploration of a novel open field, preference for a novel chamber in a conditioned place preference apparatus, and preference for the closed arms in an elevated zero maze, respectively. We assessed these effects in the two founder strains of the BXD recombinant inbred panel (C57BL/6J and DBA/2J) to determine if a larger systems genetics study using the BXD panel18 would be feasible. Specifically, our goal was to determine if C57BL/6J alleles and DBA/2J alleles differentially influence the effect of housing manipulation on the assessed predictive phenotypes.In the present study, we assessed the effects of housing condition, strain, sex, and the interaction among these factors on three phenotypes that have previously been shown to predict substance use variation in mice, rats, or both: novelty reactivity, novelty preference, and anxietyMale and female C57BL/6J and DBA/2J mice were weaned at four weeks and housed in enriched or isolated housing for ten weeks prior to behavioral testing. Enriched mice were housed in large cages with conspecifics, exercise wheels, nesting materials, and toys. Isolated mice were singly housed in standard shoebox cages without enrichment items. To control for variation of environmental variables that were not explicitly manipulated, each isolation-housed mouse was matched to an enrichment-housed littermate; these littermates were housed side-by-side on the same shelf throughout the experiment and tested in the same apparatus. Following the experiment, data were analyzed to assess the effects of the three independent variables on the addiction-predictive behavioral endpoints.Experiments were conducted in The Department of Psychology at The University of Memphis and approved by the Institutional Animal Care and Use Committee at the University of Memphis. Experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and with the ARRIVE guidelines. Efforts were made to reduce the number of animals used and to minimize animal pain and discomfort.Male and female C57BL/6J mice (stock number: 000664) and DBA/2J mice (stock number: 000671) were purchased from The Jackson Laboratory . A single male and a single female of the same strain were housed together in standard cages on ventilated racks. Offspring from these breeder pairs were weaned at four weeks of age and were used as experimental subjects. Assignment to experimental conditions occurred as follows: Two mice of the same sex were randomly selected from a litter and those mice were randomly assigned to the isolation housing condition or environmental enrichment condition. Mice were housed in these conditions for 10 weeks at which point testing began. Mice remained housed in isolation or environmental enrichment conditions on the days that they were tested apart from the brief time that they were in the testing apparatus. Mice were maintained in a temperature-controlled environment (21 ± 1 °C) on a 12:12 light:dark cycle (lights on at 0800). Mice had free access to food and water throughout the experiment with the exception of the brief time in the testing apparatus.Mice in the isolation housing condition were housed individually in clear polycarbonate standard size mouse pens (11.5″ L × 7.5″ W × 5″ H). Aspen wood bedding was used to line the bottom of the pen. No other items were placed in the pen. Mice in the environmental enrichment condition were housed in same-sex groups in clear polycarbonate standard size rat pens (19.5″ L × 10″ W × 8″ H) which were significantly larger than a standard size mouse pen. Three C57BL/6J mice and three DBA/2J mice were housed together in each environmental enrichment pen. In addition to bedding, each environmental enrichment pen contained the following items: both a vertical and a horizontal running wheel for exercise; an opaque PVC tube and one half of a glove box for shelter; three Nestlets for nest building. Isolation housed mice and their enrichment housed littermates were part of the same experimental cohort. Enrichment housed and isolation housed mice from the same cohort were maintained side-by-side on the same shelf in the mouse housing room to ensure they were exposed to the same microenvironment with the exception of experimentally-manipulated housing variables.Novelty reactivity testing was performed using six standard Med Associates  mouse open field arenas (ENV-510) with dimensions of 10.75″ L × 10.75″ W × 8″ H. Each arena was constructed of clear acrylic walls and a white opaque base. A white opaque cover was placed over the top of each arena during testing to prevent mice from jumping out of the apparatus. Integrated IR controllers (ENV-520) in combination with associated hardware and software allowed for automated data collection. Each open field apparatus was housed within a sound attenuating cubicle (ENV-022V) containing a ventilation fan to reduce ambient noise.Novelty preference testing was performed using six standard Med Associates conditioned place preference apparatus for mice (MED-CPP-MS) with overall interior dimensions of 18.4″ L × 5″ W × 5″ H. Each apparatus consisted of a center compartment (4.6″ L × 5″ W × 5″ H) with a neutral gray finish and a smooth PVC floor, (2) a left compartment (6.9″ L × 5″ W × 5″ H) with white walls and a stainless steel wire mesh floor, and (3) a right compartment (6.9″ L × 5″ W × 5″ H) with black walls and a stainless-steel bar floor. The center compartment was connected to the left and right compartments via manually operated guillotine doors. A hinged clear polycarbonate lid over each compartment allowed for loading and unloading of mice and prevented escape from the apparatus during testing. Activity level and percentage of time in the different compartments were collected by means of an integrated array of infrared detectors and associated hardware and software.Anxiety testing was performed using three standard AccuScan Instruments Inc.  elevated zero mazes separated by opaque partitions. Each maze was elevated 43″ from the floor and consisted of a black circular platform (2″ W with 16″ outer diameter) evenly divided into 2 closed and 2 open quadrants. The closed quadrants had clear acrylic walls (11″ H) on the inside and outside of the maze platform. The open quadrants did not have walls. During testing, each maze was illuminated by a 15 W red light bulb suspended 49″ above the maze platform. Activity level and percentage of time in the open and closed quadrants of each maze were collected using integrated IR detectors and associated hardware and software.Mice were tested in 10 cohorts. For each cohort, behavioral testing was conducted across three days. Order of testing was novelty reactivity on day one, novelty preference on day two, and anxiety on day three. Littermates were tested on the same day using the same apparatus. Testing was conducted between 1000 and 1700. For all assays, mice were acclimated to the testing room for 10 min. Mice were picked up by the base of the tail to place them in the apparatus. Open field and novelty place preference testing were conducted in darkness. Elevated zero maze testing was conducted with illumination from a 15 W red light bulb.Mice were placed in the center of and facing the rear of the open field. The open field was covered to prevent mice from jumping out of the apparatus. The doors to the sound attenuating cubicle were closed during testing. Mice could explore the open field for 120 min after which they were removed and placed back in the home cage. Total distance traveled during testing was used as the index of novelty reactivity.This test consisted of an exposure phase and a testing phase. During the exposure phase, mice were placed in the center compartment of the apparatus with both doors closed. The door to the black or white side was opened  and mice could explore for 15 min. Following this, mice were placed back in the center compartment with both doors closed. The testing phase immediately followed this. During the testing phase, both doors were open, and mice were allowed to explore both the familiar compartment (the one they explored during the exposure phase) and the novel compartment. Percentage of time in the novel compartment during the test phase was used as the index of novelty preference.20. At the beginning of the test, mice were placed in and facing the center of the closed quadrant on the left half of the zero maze. Mice could explore the maze for five minutes after which they were removed and placed back in the home cage. Percentage of time in the closed quadrants was used as the index of anxiety.Testing procedures have been described in detail previouslyAnalysis of variance (ANOVA) was used to assess the effects of independent variables on behavioral endpoints. We performed factorial ANOVAs to examine the effects of housing condition, strain, and sex on novelty reactivity, novelty preference, and anxiety. Strain  and sex were between-subjects factors in all ANOVAs. Housing condition  was a between-subjects factor in all ANOVAs except for those in which the difference between enriched and isolated littermates was used as the dependent variable. Normality of all measures was assessed by inspecting normal probability plots. The assumption of homogeneity of variance across groups and timepoints was assessed using Mauchly’s test of sphericity. The Huynh–Feldt correction was used when this assumption was violated. When performing multiple comparisons, Fisher's Least Significant Difference procedure was used. When analyzing novelty preference data, novel side  was a between-subjects factor. Block 1–8) was a within-subjects factor when analyzing novelty reactivity data. Dependent variables for novelty reactivity, novelty preference, and anxiety were distance traveled in the open field, percentage of time spent in the novel compartment of the conditioned place preference apparatus, and percentage of time spent in the closed quadrants of the zero maze, respectively. To directly assess and visualize the effects of strain and sex on environmentally induced changes in novelty reactivity, novelty preference, and anxiety, we performed ANOVAs using the difference between enriched and isolated littermates on these behavioral phenotypes as the dependent variable. Table –8 was a We assessed phenotypic relationships within each of the experimental groups by calculating Pearson correlation coefficients Table . Out of p < 0.0001], strain , sex , and the three-way interaction of housing condition, strain, and block . Post hoc tests indicated that, on all blocks, environmentally enriched mice explored the open field significantly less than isolated mice; this was true for both C57BL/6J and DBA/2J strains and for males and females within these strains   irrespective of housing condition and strain. DBA/2J mice explored significantly less than C57BL/6J mice in both the isolation and enrichment housing conditions (p < 0.05); the magnitude of this strain difference was significantly greater in the isolation housing condition relative to the enrichment housing condition .Novelty reactivity was significantly influenced by housing condition  and the two-way interaction of strain and block . Post hoc tests indicated that the reduction in novelty reactivity following environmental enrichment was significantly greater (p < 0.05) in the C57BL/6J strain than in the DBA/2J strain in both male and female mice  = 22.06, , 98 = 22p < 0.0001], novel side , the two-way interaction of sex and novel side , and the three-way interaction of housing condition, strain, and novel side . The three-way interaction of housing condition, strain, and sex approached significance . Post hoc tests indicated that environmentally enriched mice preferred the novel side significantly less than isolated mice; this relationship was true for both C57BL/6J and DBA/2J strains and for males and females within these strains . However, this relationship was strongly dependent on the characteristics of the novel side   = 58.29, p < 0.01]. Post hoc tests indicated that reduction in novel side preference following environmental enrichment was significantly greater in DBA/2J mice (p < 0.01), but only when the novel side consisted of black walls with a bar floor  = 9.69, p < 0.05], strain , and the two-way interaction of strain and sex . Post hoc tests indicated that enriched mice spent significantly more time in the closed quadrants of the zero maze then isolated mice, irrespective of strain (p < 0.05)  ; time spent in the open quadrants of the zero maze by male and female C57BL/6J mice did not differ significantly.Anxiety in a novel environment  was significantly influenced by housing condition [F  = 5.93, In the present study, we assessed the effects of housing condition , mouse strain , and sex on novelty reactivity, novelty preference, and anxiety. Littermates of the same sex were weaned at four weeks and housed in environmental enrichment or isolation for 10 weeks prior to testing. To control for variation in environmental variables that were not explicitly manipulated, each isolation-housed mouse was matched to an enrichment-housed littermate; these littermates were housed side-by-side on the same shelf throughout the experiment and tested in the same apparatus. Environmental enrichment significantly reduced exploration of a novel open field in males and females of both strains  a significantly greater effect on novelty reactivity in C57BL/6J mice, (2) a significantly greater effect on novelty preference in DBA/2J mice, and (3) an effect on anxiety that was statistically equivalent in C57BL/6J and DBA/2J mice. Collectively, these data indicate that environmental enrichment influences novelty reactivity, novelty preference, and anxiety via distinct genetic mechanisms in mice. Moreover, because the C57BL/6J and DBA/2J strains are the founders of the BXD recombinant inbred panel, these data indicate that the BXD panel can be used to identify the biological mechanism underlying these distinct gene x environment interactions.In the present study, environmental enrichment significantly reduced locomotion in a novel open field (i.e. novelty reactivity) in male and female C57BL/6J and DBA/2J mice Fig. a–d, and 15. Indeed, this effect may be the most frequently observed behavioral consequence of home cage environmental manipulation. The influence of genetic background on this effect has been much less frequently investigated, but several studies in rats have found that strain significantly influences the attenuating effect of environmental enrichment on novelty reactivity23. In a study that is quite similar to the one reported here, Võikar and colleagues24 assessed novelty reactivity and other behavioral endpoints in mice from substrains of C57BL/6 and DBA/2 following either isolation housing or group housing . These authors reported that, as in the present study, isolation housed mice displayed significantly higher novelty reactivity relative to enriched mice. However, these authors did not observe a significant strain by environment interaction. Several methodological differences between the study reported here and the Võikar et al. study may explain this:The attenuating effect of environmental enrichment on novelty reactivity has been widely observed in both rats and miceFirst, in the present study, we used multiple environmental enrichment components including access to conspecifics, exercise wheels, nesting materials, and toys. In contrast, the enrichment in Võikar et al. was limited to the presence of conspecifics. This suggests that social enrichment by itself is sufficient to attenuate novelty reactivity in these two mouse strains. Moreover, these findings suggest that (1) nonsocial and social components of environmental enrichment have distinct effects on novelty reactivity, and (2) nonsocial enrichment or the combination of social and nonsocial enrichment, but not social enrichment alone, interacts with strain to influence novelty reactivity in C57BL/6J and DBA/2J mice.25 have found differences in novelty reactivity between enriched and isolated mice in dim or dark testing conditions but not lighted testing conditions. Importantly, one of these studies identified a housing × strain interaction on novelty reactivity under dim light but not bright light conditions23. This suggest that other phenomena, such as anxiety, may influence the expression of novelty reactivity in a strain- and environment-dependent manner.Second, in both the present study and the Võikar et al. study, mice were tested during the light component of the light:dark cycle. However, in the study reported here, novelty reactivity was quantified in darkness, whereas Võikar et al. quantified novelty reactivity in a well-illuminated cage (approximately 700 lux). These differences are relevant because at least two studies26 since the separation of these substrains may account for some of the behavioral differences observed in these two studies. Collectively, these data suggest that the strain dependent effect of environmental enrichment on novelty reactivity observed in C57BL/6J and DBA/2J mice in the present study was dependent on nonsocial aspects of environmental enrichment, qualities of the testing environment that influence phenotypes other than novelty reactivity , the precise polymorphisms present in the JAX maintained substrains of C57BL/6 and DBA/2 mice, or some combination of these factors.Finally, the mice used in Võikar et al. were C57BL/6JOlaHsd and DBA/2OlaHsd which are distinct substrains from the C57BL/6J and DBA/2J mice used in the present study. Thus, genetic polymorphisms which have accumulated27.In the present study, environmental enrichment significantly reduced preference for a novel chamber in a conditioned place preference apparatus (i.e. novelty preference) in male and female C57BL/6J and DBA/2J mice. In contrast to the effect of environmental enrichment on novelty reactivity, the effect of environmental enrichment on novelty preference has been infrequently studied. Studies that have investigated this phenomenon have found, as in the present study, that environmental enrichment attenuates novelty preferencenovelty preference in DBA/2J mice, it had the greatest effect on novelty reactivity in C57BL/6J mice. These distinct strain dependent effects suggest that novelty reactivity and novelty preference are driven by distinct genetic mechanisms; this hypothesis is consistent with previous research30. More importantly, these findings reveal that environmental enrichment influences these mechanisms differentially as a function of genetic background. This suggests that individuals are uniquely genetically vulnerable or resistant to the effects of environmental factors on novelty reactivity and, distinctly, on novelty preference.A striking finding from the present study is that the effect of environmental enrichment on novelty preference was significantly more robust in DBA/2J mice relative to C57BL/6J mice  Fig. . SpecifiIn the present study, environmental enrichment significantly increased time in the closed quadrants of a zero-maze (anxiety). This effect was equivalent in C57BL/6J mice and DBA/2J mice Fig. . Althoug35. Unlike the consistent findings that environmental enrichment attenuates novelty reactivity, the effects of environment enrichment on anxiety have been less clear. For example, zero maze studies using male mice, female mice, or both from C57BL/6 substrains have found increased anxiety (present study), decreased anxiety34, or no difference in anxiety33 relative to controls following varying types and degrees of environmental enrichment. The lack of clarity offered by these studies mirrors the variation in findings across the literature regarding the effects of environmental enrichment on anxiety15. Collectively, these studies suggest that variables including quality and duration of environmental enrichment, sex, testing apparatus, light intensity during testing, or a combination of these factors may influence anxiety and, consequently, the ability to detect strain dependent effects of environmental enrichment on anxiety.Multiple studies have investigated the effects of environmental enrichment on anxiety using the elevated zero maze in mice36. Neither of those strategies was used in this study. The addition of these enrichment components could reveal strain dependent effects of environmental enrichment on anxiety in C57BL/6J and DBA/2J mice that were not detected in this study. Moreover, the use of these enrichment components could enhance or diminish the strain dependent effects on novelty reactivity and novelty preference that were observed in this study.It should be noted that some investigators add daily handling and weekly introduction of novel objects to their environmental enrichment protocol8. The most important finding from the present study is that environmental enrichment had a unique strain-dependent effect on each predictive phenotype: novelty reactivity was reduced significantly more in C57BL/6J mice, novelty preference was reduced significantly more in DBA/2J mice, and anxiety was significantly increased independent of strain. Collectively, this pattern of evidence reveals that environmental enrichment influences novelty reactivity, novelty preference, and anxiety via distinct genetic mechanisms in mice.In the present study, we directly compared the effects of environmental enrichment on three high-throughput, noninvasive phenotypes: novelty reactivity, novelty preference, and anxiety. We selected these phenotypes because each explains unique variance on the gold standard intravenous drug self-administration paradigm37. To accomplish this, littermates from multiple BXD strains would be, as in the present study, housed in isolation or enrichment conditions. Using these mice, novelty phenotypes, anxiety phenotypes, and gene expression in addiction relevant brain regions would be quantified. Using these data, systems genetics analyses would enable identification of genetic loci associated with enrichment-induced variation in these endpoints. In the context of the housing manipulation used in the present study, genes identified using this approach would be strong candidates for validation on novelty phenotypes, anxiety phenotypes, and intravenous drug self-administration phenotypes. Moreover, BXD strains exhibiting extreme effects of environmental enrichment on multiple novelty and anxiety phenotypes could prove to be valuable models of environmentally induced addiction risk. The addition of ATAC-seq38 or MeDIP-seq39 to the systems genetics approach would enable discovery of underlying epigenetic mechanisms  driving the gene-by-environment interactions observed in the present study; in this regard, others have described epigenetic mechanisms underlying some effects of environmental enrichment41, the use of ATAC-seq in a systems genetics context42, and approaches for perturbation of the epigenome43. Collectively, these techniques offer a direct approach for discovering and validating genetic and epigenetic mechanisms through which environmental enrichment affects drug use and phenotypes which predict drug use.The findings presented here indicate that a systems genetics approach using the BXD recombinant inbred panel would enable discovery of the unknown genetic mechanisms driving the observed strain-dependent effects of environmental enrichment on intravenous drug self-administrationSupplementary Information


The vitamin D receptor (VDR), coded by the VDR gene, plays a pivotal role in executing cellular functions when bound by the active form of vitamin D. Gene polymorphisms in this receptor have been increasingly associated with a heightened state of vulnerability to certain diseases. However, limited data is available concerning the role of VDR gene polymorphisms in preterm infant complications. In 114 premature infants (< 32 weeks gestation) we analyze four single nucleotide VDR polymorphisms (rs2228570 (FokI), rs1544410 (BsmI), rs797532 (ApaI), rs731236 (TaqI)) for their association with respiratory distress syndrome (RDS), intraventricular hemorrhage (IVH), bronchopulmonary dysplasia (BPD), necrotizing enterocolitis (NEC) and retinopathy of prematurity (ROP). The results show that BPD was almost four times more likely in infants with the genotype CC of ApaI (rs7975232) . While both BPD and NEC were 2.1 times more likely to occur in preterm infants with the allele C of ApaI (rs7975232) . The ApaI VDR polymorphism appears to influence incidence of BPD and NEC in preterm infants. Considering VDR polymorphisms in future genetic investigations, in preterm complications, may prove clinically relevant. VDR is expressed extensively in immune system cells, thymus, placenta, alveolar type-II cells, osteoblasts, chondrocytes, and skin epithelium3. In contrast to the later, erythrocytes, mature striated muscle cells, and highly differentiated neural cells of the cerebral cortex present considerably less vitamin D receptors4.Vitamin D activity is regulated by the vitamin D receptor (VDR), also known as the calcitriol receptor or Nr1I1. It is a nuclear transcription factor that, when activated by a ligand, modulates gene expression5. The resulting interactions trigger the tissue-specific responses and their molecularly correlated cascading effects.In the human body, most of the utilized vitamin D is sourced by endogenous cutaneous production. Pre-vitamin D is provided by photoisomerization from 7-dehydrocholesterol (ergosterol) due to ultraviolet radiation (UV-B). Then, it is transformed to cholecalciferol, and after hydroxylation in the liver and kidneys—to calcitriol. This active hormonal form binds to the vitamin D binding protein (VDBP) in the blood and reaches the target cell. Once it enters the cell, it attaches to VDR in the cytoplasm and constructs a combined complex. This fusion enters the nucleus, where it heterodimerizes with retinoic acid X receptor and enhances vitamin D-dependent gene transcription6. To date, the most explored VDR polymorphisms are rs2228570 (FokI), rs1544410 (BsmI), rs797532 (ApaI), rs731236 (TaqI)7. TaqI and FokI consist of a single nucleotide change—A to G and G to A in exons 9 and 2, respectively. Whilst BsmI and ApaI are located in intron 8 and result in a change from G to A and A to C, respectively. Possible associations are being explored between VDR gene variation and the development of different types of cancers 8, autoimmune diseases (risk of developing multiple sclerosis is still being investigated)9, diabetes11, asthma12, cardiovascular diseases13, osteoporosis14, and susceptibility to infection15.As with many types of receptors, the genes responsible for coding the VDR present with subtle allelic variants located on chromosome 12The following work is centred to investigate VDR polymorphisms  and their association with risk for neonatal complications such as respiratory distress syndrome, intraventricular hemorrhage, bronchopulmonary dysplasia, necrotizing enterocolitis and retinopathy of prematurity.The study comprises a population of 114 premature infants born within 22 + 6 to 32 + 0 weeks of gestation, between the years 2014 and 2019. All infants were hospitalized at the Department of Neonatology  located in the Clinical Hospital of Gynecology and Obstetrics at Poznan University of Medical Sciences in Poznan, Poland. Homogenic consistency was maintained as a result of all infants being of Caucasian origin. Excluded were neonates born after 32 + 0 weeks of pregnancy, multiple pregnancy births, infants with chromosomal abnormalities, TORCH infections  and infants without antenatal steroid therapy.Clinical factors were observed for association with VDR gene polymorphism and its influence on neonatal development and preterm complications. The clinical features include: gender, gestational age , birth weight , mode of delivery , APGAR score in 1st and 5th minute, and pH and blood base excess (BE) in cord blood. The five preterm complications evaluated were respiratory distress syndrome (RDS), intraventricular hemorrhage (IVH), bronchopulmonary dysplasia (BPD), necrotizing enterocolitis (NEC) and retinopathy of prematurity (ROP). Infants who were delivered outside third-level hospitals were also analyzed for developing complications of preterm birth.16. This is manifested by breathing disorders and an increase in the oxygen requirement, in conjunction with a characteristic chest radiograph or ultrasound . Criteria for the classification of RDS was obtained from European Consensus Guidelines on the Management of Respiratory Distress Syndrome17.The diagnosis of RDS is based on a clinical picture of a preterm infant with the onset of progressive respiratory failure shortly after birth18.Intraventricular hemorrhage was diagnosed with the use of cranial ultrasound . Routine cranial ultrasound screenings were performed on the 1st, 3rd and 7th days post birth in accordance with the local hospital standards. Classification of IVH was obtained using criteria based on the Papile IVH Severity Scale19. Notably, this definition does not take into account the severity of respiratory disease or extent of prematurity22.Bronchopulmonary dysplasia was defined as the need for at least one form of oxygen supplementation either at 28 postnatal days or 36 weeks postmenstrual age (PMA)23. Severity of NEC can vary in degree in preterm neonates and can manifest in variable stages. Diagnosis of NEC was based of the Modified Bell’s clinical criteria assessment of NEC stages using an abdomen ultrasound  and an X-Ray of the abdomen24. Within the Modified Bell’s clinical criteria, stages were divided into three specific components for the most accurate recognition and diagnosis of NEC25.Necrotizing enterocolitis is characterized by ischemic necrosis of the intestinal mucosa, which is associated with severe inflammation, invasion of enteric gas forming organisms, and dissection of gas into the bowel wall and portal venous system26. Patients were examined with binocular indirect ophthalmoscopy by a team of three trained paediatric ophthalmologists specializing in ROP. The initial fundus examination was performed 4 weeks postnatally, with further follow-up every 7–10 days until full vascularization of the retina, stabilization of the changes or the necessity for treatment. Findings were classified according to the Revisited International Classification of Retinopathy of Prematurity (ICROP)28.Retinopathy of prematurity is classified as a developmental vascular proliferative disorder that occurs in the retina of preterm infants with incomplete retinal vascularization. The screening examination followed the guidelines of Polish Ophthalmological Society for ROP screening The criteria for selections of candidate genes in the present study were their potential involvement in the pathogenesis of RDS, IVH, NEC, BPD or ROP. We studied 4 single nucleotide polymorphisms of vitamin D receptor: rs2228570 (FokI), rs1544410 (BsmI), rs797532 (ApaI), rs731236 (TaqI).: CC (FF) 267 bp; CT (Ff) 267, 197, 70 bp and TT (ff) 197, 70 bp. For detection of the rs1544410 mutation, PCR was amplified with starters: 5′-CAACCAAGACTACAAGTACCGCGTCAGTGA-3′, 5′-AACCAGCGGGAAGAGGTCAAGGG-3′ (PCR product 821 bp long) and hydrolyzed at 37 °C with Mva12691 (BsmI) restriction enzyme . The following genotypes were obtained: GG (bb) 646, 175 bp; GA (Bb) 821, 646,175 bp; AA (BB) 821 bp. The rs7975232 and rs731236 polymorphic sites of the VDR gene were detected using starters: 5′-CAGAGCATGGACAGGGAGCAA-3′, 5′-GCAACTCCTCATGGCTGAGGTCTC-3′. PCR product (745 bp long) was hydrolyzed with ApaI restriction enzyme at 25 °C overnight  and found the following genotypes: AA (AA) 745 bp; AC (Aa) 745, 528, 217 bp and CC (aa) 528 bp, 217 bp. For detection of the rs731236 polymorphism, 745 bp PCR product was hydrolyzed overnight at 65 °C with TaqI restriction enzyme . TaqI has two binding sites on the PCR product and produces a following fragments: TT (TT) 494, 251 bp; TC (Tt) 494, 293, 251, 201 bp and CC (tt) 293, 251, 201 bp. DNA fragments obtained after respective restrictive enzyme digestion and the DNA size marker were electrophoresed on a 2.5% agarose gel with Midori Green Advanced DNA Stain .A sample of blood was taken following birth, after admission to the intensive care unit. Genomic DNA was extracted from blood leukocytes using QIAamp DNA Blood Mini Kit . Genotyping was performed using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) procedures. For detection of rs2228570 mutation PCR was amplified with starters: 5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′, 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′ (PCR product 267 bp long) and hydrolyzed at 37 °C with FokI restriction enzyme . The following genotypes were obtainedDescription of the studied polymorphism genes is presented Table All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the Bioethics Committee of Poznan University of Medical Sciences (no. 66/14 and 799/16). Informed consent was obtained from all parents.p value of less than 0.05 indicates statistical significance. Categorical variables were presented as a percentage. Non-normally distributed continuous variables were tested by the Shapiro–Wilk test and showed as median and ranges. To evaluate the association between prematurity complications and categorical variables the following test were performed: the Fisher exact probability test, the Chi-square test, Fisher Freeman Halton, and Chi-squared test with Yates correction. U Mann–Whitney test was used to analyze differences in non-normally distributed continuous variables.Statistical analysis was performed using CytelStudio version 11.1.0  and Statistica version 10 . A Table RDS developed in 107/114 (93.9%) infants. No significant differences were observed in the clinical characteristics between the RDS and non-RDS infants, as seen in Table Furthermore, forty of the 114 infants (35.1%) suffered from BPD. Frequency of BPD was higher in children born before 29 weeks of gestation . BPD incidence was also more pronounced in newborns with birth weight less than 1000 g , or had lower Apgar scores in first  and fifth minute of life .Almost a quarter of the included infants developed NEC . The incidence of NEC was observed to be higher in children born before 29 weeks of gestation .In the 47/114 (41.2%) infants who developed ROP, incidence was significantly higher in those with earlier gestation . Higher rate of ROP was also recorded in infants with birth weight less than 1000 g , and with lower Apgar score in the first minute .Analysis showed higher prevalence of BPD in premature infants with the genotype CC ApaI rs7975232) gene polymorphism . Furthermore, the study revealed that BPD and NEC were approximately two times more likely to occur in infants with the allele C of ApaI (rs7975232) . No other significant associations were found with the rest of the polymorphisms. Genotype and allele distribution in infants with complications of prematurity is presented in Table 2 gene po29. A physiological response to vitamin D, is regulated by nuclear VDR and its resulting transcription cascade. The overall mechanism of the VDR protein revolves around its binding to the active form of vitamin D, producing a protein interaction with the retinoid X receptor, resulting in the regulation of transcription factors30. Such regulation mainly involves the process of mineral metabolism but has also been associated in the regulation of other metabolic pathways pertaining to embryological and immunological responses and development31.Abnormalities in vitamin D levels have consistently been reported to produce adverse effects on human health and disease development32. Polymorphisms in the VDR gene have also been found to influence neonate development, and is being increasingly explored for its role and prognostic value in certain neonatal complications. Maternal and fetal VDR genotype variants may increase the risk for preterm births33. As prematurity carries risk for multiple complications34, understanding the role of VDR polymorphisms in such complications become essential for elaborate prognosis. To date, few preterm infant complications have been linked to such polymorphic changes. However, a majority of the preterm complications remain unassessed for their association with VDR polymorphisms.The subtle variations in the genetic makeup of VDR, also known as polymorphisms, have been linked to a wide array of diseases including metabolic, immunological, and multiple cancers35. Glasgow and colleagues argued for the significance of vitamin D playing a role as a prophylactic for respiratory distress in preterm babies36. An evaluation of RDS in preterm infants found that high levels of vitamin D decreased the probability of RDS by a ratio of 3.34 times37. Both Vitamin D and the VDR receptor influence embryological and perinatal development. VDR TaqI (rs731236) polymorphism, CC and CT genotype has been associated with increased risk of developing RDS in preterm neonates38. The authors found that within a TaqI polymorphism, a CC genotype variance was equated with a 5.22 times increased risk in developing RDS, while a TaqI CT genotype variance was 3.26 times more likely present in preterm infants with RDS.RDS is a common complication in preterm children, as a result of underdeveloped lungs, which interferes with the absorption of appropriate levels of oxygen38. Our study does replicate the lack of Fok1 VDR polymorphism influence on the incidence of RDS but disputes that of the TaqI VDR polymorphism. These antithetical results reveal the importance of further investigation into the influence of VDR polymorphisms on RDS in order to deduce possible genetic risk factors.The mechanisms involving the VDR polymorphism with RDS, to date, are not well understood. Within the results of our study, the genetic variability of the VDR gene did not alter the VDR receptor significantly enough in order to influence the incidence of RDS in preterm neonates. A most recent publication by Ustun et al. argue that VDR TaqI polymorphism did influence the incidence of RDS but the Fok1 polymorphism did not40. Crucial components observed in the pathological variations of IVH include alterations in venous and arterial cerebral blood flow, which have been associated with the fragility of the germinal matrix vasculature43. To date, the influence of Vitamin D on IVH in preterm infants is up for debate with little to no apparent investigation on its influence of the VDR gene. Elimination of the VDR receptor in knockout mice has revealed alterations in the vascular function of vascular endothelial cells44. With the absence of endothelial VDR receptors, Ni and colleagues found a reduction of endothelial nitric oxide synthase (eNOS) expression. Polymorphic changes in endothelial nitric oxide synthase, have been associated with a 3.4-fold increased risk of developing IVH in preterm infants45.The pathogenesis of intraventricular hemorrhage, involves a multifactorial etiology, influenced by both environmental and genetic elements46. Considering the multifactorial nature of IVH, further investigation is required to deduce potential hallmarks of VDR polymorphisms and IVH interactions.According to the results of our study, VDR polymorphisms do not affect the incidence of IVH. The slight variations in the VDR genetic makeup do not appear to influence IVH incidence rates. To the authors' knowledge, no previous study analyzing a potential association between VDR polymorphisms and IVH was identified. It is worth noting, that observations of preterm infants in the German Neonatal Network, showed no detectable differences in incidence frequency of IVH in preterm neonates with high and low Vitamin D levels48. Hypovitaminosis D has been established as a potential risk factor for improper lung regulation and respiratory morbidities, such as RDS and BPD49. As such, VDR gene polymorphisms may have slight, but meaningful, variations that mimic vitamin D abnormalities and lead to its consequent effect32. In the performed analysis, the VDR gene polymorphism rs7975232 (ApaI) was significantly associated with a higher incidence of BPD. Preterm infants with the receptor genotype CC ApaI (rs7975232) were roughly 4 times more likely to develop BPD, while the infants with the allele C of ApaI (rs7975232) were about twice as likely. This is in contrast to a mirror study performed in 2014 by Koroglu et al.50. The study analyzed the same VDR polymorphisms  in 109 preterm infants (47 of which developed BPD), and concluded that only the FokI receptor polymorphism was associated with an increased frequency of the dysplasia. Such contrasting findings highlight the need for further research concerning VDR polymorphism association to BPD.Both animal and human studies have placed emphasis on the role of vitamin D in proper lung maturation51. Abnormalities within this signalling pathway in the intestines leads to an unregulated activation of nuclear factor-kappa B (NF-κB), that in turn increases the production of proinflammatory cytokines53. This sequence of events leads to tissue damage and eventual NEC. Vitamin D plays a protective role in the intestines by regulating TLR4, and a deficiency in this vitamin has been reported as a feature of preterm NEC infants55. Nonetheless, to the authors’ knowledge, no previous studies have explored the potential role of VDR polymorphisms in NEC. The results of our analysis show that preterm infants with the VDR allele C of ApaI (rs7975232) were twice as likely to develop NEC. Such a finding highlights the possible role of VDR genetic variation on the overall interaction between vitamin D and the TLR4 cascade. An increasing number of reports have placed emphasis on the genetic predisposition of preterm infants to NEC61. However, due to lack of extensive research and coherence of results, validating studies remain necessary to elucidate the relationship between gene variation and NEC62. Including VDR polymorphisms in future genetic studies may prove beneficial in understanding certain infants vulnerability to NEC.In the pathogenesis of NEC, the transmembrane protein, toll-like receptor 4 (TLR4), and its cascading effect has been established as a possible key component63. Eventually, the resulting hypoxic stress from improper vascularization of the retina, stimulates the production of excess angiogenic factors  that lead to new abnormal blood vessel formation (second phase)64. In a study evaluating the relationship between vitamin D levels and ROP, Kabatas EU et al. found that infants with lower serum 25-hydroxy vitamin D were 1.14 times more likely to develop ROP65. They hypothesized that vitamin D deficiency may facilitate angiogenic conditions in the retina, reinforcing the development of ROP. Such a link is yet to be extensively explored.Two phases for ROP pathogenesis are generally recognized. The first phase involves suppressed insulin-like growth factor-1 (IGF-1) and VEGF, due to a hyperoxic neonatal state, that leads to a disruption of retinal vascularization68. However, according to the results of our study, VDR polymorphisms remain inconsequential to ROP development, and require further investigation.To the authors' knowledge, this is the first study analyzing a potential association between VDR polymorphism and ROP. The mild variations in the genetic makeup of the receptor did not appear to influence ROP incidence. Polymorphisms, particularly those relating to VEGF, have previously been highlighted as risk factors in ROP, underlining a possible genetic predispositionIn the presented study, the vitamin D levels in the preterm infants were not analysed for a possible correlation to the prematurity complications. The assessment protocols focused solely on the genetic background of these complications. Including such a variable in further analysis can assist in deconstructing the relationship between receptor polymorphisms and development of preterm complications.71. Understanding the role of this novel pathway and its similar, yet distinct, vitamin D hydroxyderivatives (and its receptors) may further unveil some clinical relevance to preterm infants and their associated complications. In addition to varying pathways of vitamin D activation, different receptors for the active metabolite should also be taken into consideration for further analysis72. Receptors such as ROR (both α and γ) and AhR have shown interactions with varying vitamin D derivatives, underlining the misconception that VDR is the sole receptor for the vitamin75. Exploring the downstream influence of ROR and AhR gene polymorphisms, alongside VDR, allows for more elaborate understanding of this pivotal and versatile vitamin.Notably, in recent years, alternative pathways of vitamin D activation have also been identified. Slominski AT and colleagues defined a novel pathway for metabolizing vitamin D3, revolving around the mitochondrial enzyme CYP11A1, and found predominantly in the adrenal glands, placenta, and keratinocytes of the epidermisDespite providing fundamental insight into the relationship between VDR polymorphisms and preterm infant complications, the above-mentioned genetic analysis acts as preliminary and requires further investigation. Larger sample sizes in non-homogenous populations can provide valuable information concerning patterns of pathogenesis and genetic susceptibility of certain infants. Allocating efforts and resources into the role of polymorphisms, including that of VDR, in preterm complications carry promising potential and can allow for future construction of more personalized care.Exploring genetic variations in the vitamin D physiological pathway of functioning may broaden the current understanding of genetic susceptibility to preterm complications. The genotype CC of ApaI (rs7975232) VDR gene polymorphism was associated with a 3.8 times increase in likelihood of developing BPD in preterm infants. Furthermore, infants with the allele C of Apa I (rs7975232) were twice as likely to develop BPD and NEC. Including VDR polymorphisms in future genetic assessments of preterm complications is advised.


Pseudomonas. Regarding biochemical characterization, all strains displayed in vitro PGP capabilities, but these were in different proportions that grouped them according to their site of origin. This study contributes with microbial isolates from natural extreme environments with biotechnological potentials in improving plant growth under cold stress.The isolation of soil bacteria from extreme environments represents a major challenge, but also an opportunity to characterize the metabolic potential of soil bacteria that could promote the growth of plants inhabiting these harsh conditions. The aim of this study was to isolate and identify bacteria from two Chilean desert environments and characterize the beneficial traits for plants through a biochemical approach. By means of different culture strategies, we obtained 39 bacterial soil isolates from the Coppermine Peninsula (Antarctica) and 32 from Lejía Lake shore soil (Atacama Desert). The results obtained from the taxonomic classification and phylogenetic analysis based on 16S rDNA sequences indicated that the isolates belonged to four phyla , and that the most represented genus at both sites was  Deschampsia antarctica) and the Antarctic carnation (Colobanthus quitensis) have been able to germinate and develop despite the extreme low temperature [Calamagrostis crispa, Nassella nardoides, and Jarava frigida [By definition, extreme environments meet conditions that represent a difficult life opportunity for any species . The geoperature . We beliperature , but it perature , which a frigida ,16 at an frigida . Studyin frigida .Bacteria with the capacity to contribute to plant tolerance to different environmental vicissitudes have been described in extreme environments . These bTwo extreme Chilean environments were selected for this study. The first site corresponds to Lejía Lake (23°30′ S 67°41′24″ W) in the Atacama Desert, and the second site corresponds to the Coppermine Peninsula on Robert Island, Antarctic Peninsula (62°22′43″ S 59°42′21.9″ W). From both sites, approximately 100 g of bulk soil samples were taken in quadruplicate at 10 cm depth to avoid contamination with other surfaces. Each sample was placed in sterile plastic bags and immediately stored in cold (IcePack). Samples were used for the preparation of Soil Extract Medium (SEM) as a matrix for bacterial isolation and for physical–chemical analysis. Soil samples were taken under similar environmental conditions (during the summer after the rainy season) but in different years. Sampling in the Atacama Desert was conducted in April 2014 and that in the Antarctic Peninsula was performed in January 2019 by the ECA-55 (LV Antarctic Scientific Expedition) of INACH.p/v) and measurement was obtained using a pH-Meter . The elemental composition of the soil was measured by Total Reflection X-ray Fluorescence Spectrometry (TXRF), using Bruker S2 PICOFOX equipment [Temperature was recorded using a digital thermometer  and geolocation was obtained using a Global Positioning System (GPS) equipment . Soil pH was achieved by mixing soil:distilled water . This fraction was obtained using 80 g of soil mixed with 80 mL of distilled water. This mix was agitated for 1 hour and then decanted overnight. The soluble fraction was used to hydrate the agar (1.5%), and finally, the mixture was autoclaved and plated. LB was prepared using 10 g of yeast extract, 5 g of peptone, 10 g of sodium chloride, 10 g of agar, and 1 L of distilled water. To obtain bacterial isolations, the protocol described by Mandakovic et al. [After field sampling, the isolation of bacteria from the collected samples was performed. Two culture media were used in both sites to obtain bacterial isolates: SEM and Luria–Bertani (LB) . Brieflyc et al.  previousw/v) agarose gel electrophoresis and sequenced . Taxonomy was defined using the EzBioCloud (16S database), and sequences were deposited in the NCBI Genbank database. Accession numbers can be found in The DNA of each strain was extracted using a DNeasy Blood & Tissue Kit . The 16S rDNA region was amplified using the 27F and 1492R primers. The PCR mix contained 12.5 µL of Mastermix Go Taq Promega, 8.5 µL of nuclease-free water, 1 µL of each primer, and 2 µL of DNA. Thermal cycling followed the following steps: 10 min at 95 °C, 30 cycles of 95 °C for 60 s, 58 °C for 30 s, and 72 °C for 60 s, and a final extension at 72 °C for 10 min. PCR products were kept at 4 °C until use. PCR products were visualized in 1% . Multiple cluster alignment and phylogenetic analysis were performed on MEGA software (v. 7.2) based on the neighbor binding method using a 1000 repetition bootstrap to evaluate statistical support .To biochemically classify bacteria with beneficial plant traits, five capacities were evaluated for each strain: siderophore production, auxin (AIA) production, nitrogen fixation, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and phosphate solubilization, as described in Maza et al. . The proThe similarity evaluation was performed using Past software (v. 4.03) through a multivariate analysis with a matrix that included each isolate, genus, and origin site. The algorithm used was the unweighted pair group method with arithmetic mean (UPGMA) and Jaccard similarity index.Two Chilean desert sites were selected for this study: Lejía Lake, located in the north of Chile, specifically in the highlands of the Atacama Desert, and Coppermine Peninsula, located in the extreme-south of the country, which is part of Robert Island in the Antarctic Peninsula. To characterize the physical–chemical features of these two Chilean deserts, we performed environmental measurements during sampling and subsequently identified soluble elements in the soil through TXRF. We observed that despite being obtained from distant environments, the environmental conditions of both soil sampling sites were not significantly different in temperature, pH, and humidity at the time of sampling . We expeTwo culture media (SEM and LB) were used to isolate strains from Coppermine Peninsula and Lejía Lake soils . A total of 39 bacteria were isolated from Coppermine Peninsula and 32 were isolated from Lejía Lake . To taxoWhen compared to other studies carried out that isolated bacteria from soils under extreme environmental conditions in different parts of the world, our results were consistent with the isolation of mainly Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria ,41,42. TPseudomonas was the most represented genus in our study, with 22 isolates from Coppermine Peninsula (56.4%) and 11 isolates from Lejía Lake (34.4%). We also observed the presence of less represented but specific genera in Lejía Lake, such as Stenotrophomonas (DD1), Halomonas (N5), Arthrobacter (AF3), Microbacterium (M1-B and M2-A), Rhodococcus (D4), Streptomyces (M1-A), Paenibacillus (5B and AF2), Oceanobacillus (E4), Alkalihalobacillus (E3-18 and E4-18), Bacillus , Peribacillus (B1), and Mesobacillus . Regarding the Coppermine Peninsula, exclusive genera found in the isolates from this site were Pusillimonas (L3D), Janthinobacterium (S6), Pseudarthrobacter , Paeniglutamicibacter (L1D and L2D), Frondihabitans (R8), Cryobacterium (S5), Enterococcus (S1), Sporosarcina (R6D and R7D), Filibacter (R9), and Flavobacterium (R4 and S4). Romaniuk et al. [Pseudomonas was one of the most abundant genera isolated from the soil from King George Island; however, another abundant genus was Psychrobacter, from which we obtained no isolates. This may be due to the difference in culture media used in each study. As for the rest of the studies related to microorganisms isolated from Antarctic soils, they focused more on the search for fungi [Regarding genera isolated, k et al.  similarlor fungi ,48,49 oror fungi , and notPseudomonas (isolate C3) and Peribacillus (isolate B1) were isolated from the Lejía Lake, which had four positive PGP capacities, while in the Coppermine Peninsula, the soil isolate with the highest number of PGP capacities corresponded to the genus Paeniglutamicibacter (isolate L1D) with three PGP capacities out of the five evaluated. In general, Antarctic strains had less PGP traits than Lejía Lake isolates. Most of the Coppermine Peninsula strains possessed ACC deaminase activity, while the strains from Lejía Lake were more heterogeneous in terms of their PGP capabilities. The fact that a high percentage of bacteria present in the soil had at least one PGP capacity despite being isolated from bulk soil is consistent with a study carried out in Namibia Desert, in which a higher percentage of bacteria with PGP activities was isolated from bulk soil rather than from rhizospheres, proposing these strains as potential biofertilizers for arid areas [Sporosarcine was only represented in the Coppermine Peninsula, presenting two of the five PGP capacities evaluated. In this context, Yadav et al. [Sporosarcine to be a potent PGP candidate to use in low-temperature crops. Additionally, Halomonas genus was part of the set of bacteria isolated and characterized as plant beneficial traits from Lejía Lake. This genus was also isolated and characterized in the desert of southern Tunisia, and the genus was proposed as a potential plant growth promoter under salinity and drought conditions [Two bacteria of the genus id areas . The genv et al.  describenditions . Other anditions ,55 have nditions . To evaluate the similarity among all the isolates, we calculated the Jaccard distance considering the origin of the sample, their taxonomic identification, and their PGP capacities. We expected that the bacteria isolated in the two sampling sites would have equivalent PGP capabilities, considering that both deserts have similar environmental conditions and also share taxonomic groups. Therefore, we estimated that when performing a multivariate test, the main factors that would cluster together would be taxonomy and plant beneficial traits, regardless of the origin of the sample. Nevertheless, at the time of conducting the similarity test using the Jaccard index, taxonomy was irrelevant, and isolates were grouped by their PGP capabilities, which also coincided with their isolation site. This could be explained by the fact that despite having similar environmental conditions during sampling, sites at other times of the year are completely different, which could impact site-specific microbiological adaptations and PGP characteristics. For example, for most of the year, the Coppermine Peninsula is covered with snow , while iAlthough further investigations should be done, including the addition of more isolates, we think that these differences could be due to nutritional factors  , suggestFinally, Jorquera et al.  declare Extreme environments have been studied in various settings due to the interesting capacities developed by the different species that inhabit them . In partArthrobacter, Bacillus, Janthinobacterium, Pseudomonas, and Sporosarcine, among others, have been described as having different mechanisms associated with cold stress tolerance, such as cold acclimation proteins (Caps) and antifreezing proteins (AFPs) that protect the specific cytoplasmic and extracellular components involved in cold adaptation [Bacillus genus presents mechanisms of response to cold stress through the formation of biofilms, the production of Reactive Oxygen Species (ROS), and also through the expression of the acdS gene, which is related to the ACC deaminase activity committed to the PGP capacity. The ability of bacteria to adapt and survive in cold environments attracts many researchers interested in solving this adaptive attribute and also in developing new or better biotechnological tools. Bacteria of the genus aptation . In addiaptation  showed t


Background and Objectives: Hemodynamic stress during resistance training is often a reason why this training method is not used in cardiac patients. A lifting protocol that imposes rests between repetitions (IRRT) may provide less hemodynamic stress compared to traditional resistance training (TT). The aim of this study was to verify differences between set configurations on hemodynamic stress responses in resistance training. Materials and Methods: We compared hemodynamic (heart rate (HR), systolic blood pressure (SBP), and rate pressure product (RPP)) responses assessed with the auscultatory method in elderly (age = 75.3 ± 7.3 years) coronary male patients who were participating in a cardiac rehabilitation program allocated to either TT or IRRT with the same load (kg) and total number of repetitions (24) in the bilateral leg extension exercise. Results: IRRT resulted in significant lower values than TT for RPP at repetitions 8  and 16 . Conclusions: IRRT appears to be a viable method of reducing the hemodynamic response  to resistance training and, thus, may contribute to the safety of cardiac rehabilitation programs. Further studies with more cardiac patients and other measurement techniques should be conducted to confirm these important findings. Resistance training (RT) has been traditionally considered less suitable for cardiac patients than aerobic training. The excessive pressure response observed in classical studies ,2 was thThe traditional approach for organizing RT session is to complete single or various sets with a determined number of repetitions and the targeted intensity in each exercise. More recently, a different set configuration called cluster or inter-repetition rest training (IRRT), which consists of including rest intervals after each single repetition, has been recommended and has been demonstrated to be very effective for maintaining mechanical performance  during RT sessions with less fatigue than traditional set configurations ,14. PrevTherefore, the objective of the current investigation was to describe and compare the changes in HR, SBP, and RPP during a RT session with TT and IRRT configurations in a sample of elderly coronary patients undergoing a cardiac rehabilitation program. Based on previous studies, our hypothesis was that mean SBP and RPP during the IRRT session would be lower than during the TT session.Fifteen elderly (≥65 years old), stable coronary male patients with a minimum of one year of experience in RT (3 days a week) who regularly participated in a cardiac rehabilitation program were invited to participate in this study. All were informed about the procedures and the risks associated with the protocols and provided written informed consent. Inclusion criteria included completing all testing sessions, not using a pacemaker, no evidence of ischemia or arrhythmias, not presenting discomfort the days of evaluation, and maintaining medications prescribed during the study. One patient used a pacemaker, and another did not complete the evaluations. Thus, a final sample of 13 coronary male patients was included in this study. Their characteristics are included in Patients presented different diagnosis before participation in the rehabilitation program including: hypertension (8), coronary insufficiency (2), acute myocardial infarction (5), cerebrovascular accident (1), myocardial revascularization surgery (2), dyslipidemia (5), diabetes mellitus type II (1), and coronary angioplasty (4). During the time of the study, the patients were taking beta-blockers (6), statins (8), cholesterol lowering drugs (5), antidiabetics (8), and antihypertensives (10).On a first day, the patients were evaluated for body composition and completed the physical activity readiness questionnaire (PAR-Q)  and the Forty-eight hours after the first evaluation, the patients performed one of the experimental sessions in randomized order: TT, which consisted of 3 sets of 8 repetitions with the load (kg) determined in the 8-RM test of the first day with a passive recovery between sets of 3 min, and IRRT, which consisted of 1 set with the same load (kg) and number of repetitions (24) but with the total recovery time divided between each repetition . Both protocols were performed with the maximal intended velocity and with a normal respiration avoiding the Valsalva maneuver. In a third day, the patients completed the other experimental session. Experimental sessions were separated by at least 48 h. During these sessions, HR was monitored with a HR monitor  and SBP was recorded by means of the auscultatory method by an experienced clinician before and after the 8th, 16th, and 24th repetitions and 10 min after the last repetition completed. Of note, given the short time interval for BP measurements, the cuff was inflated by ~10 s before the start of the recovery interval to ensure enough time for recording SBP in both conditions . Subsequt-tests with Bonferroni’s correction. Effect sizes for the pairwise comparisons are reported as Hedge’s g (G) with their corresponding 95% confidence intervals (CI). Additionally, a post hoc power analysis and a sensitivity analysis were performed. The statistical power for the interaction of the 2 × 5 repeated measures ANOVA, with a sample size of 13, a correlation among repeated measures of 0.7, and a medium effect size (f = 0.25) is 0.81. Additionally, the sensitivity of the tests for an alpha level of 0.05 and a power of 0.90 was sufficient for detecting a medium effect size (f = 0.28) for the interaction. All the analyses were carried out by using the statistical package SPSS version 20.0  and the G Power software . The level of statistical significance was set at 0.05.Descriptive values are reported as mean ± SD. Normality assumption was verified by using the Shapiro–Wilk test. In order to contrast the evolution of HR, SBP, and RPP throughout the sessions, a two-way repeated measures ANOVA was performed. The factors considered for each ANOVA were session  and time . When a significant session × time interaction was detected, pairwise comparisons were performed using paired p = 0.024; G = 0.329; 95% CI: 0.061, 0.598) and 16 .The results of ANOVA for HR, SBP, and RPP are show in The results of the current study confirm our hypothesis of a smaller hemodynamic  stress during IRRT while performing bilateral leg extension exercise compared to TT with the same load (kg) and total number of repetitions. Specifically, there were significant time × session interactions only for RPP after the 8th and 16th repetitions. To the best of our knowledge, this is the first evidence in the literature reporting a reduced hemodynamic load during IRRT in a clinical population which agrees with previous reports with young healthy individuals ,16. ThesThis study is not without limitations. First, recordings of SBP were with the auscultatory method and after lifting loads. Therefore, it would be expected that our SBP recordings were ~31% lower in both conditions than those measured with direct methods during lifting . Future In summary, this study suggests that a cluster set configuration may induce a lower cardiovascular stress  in elderly coronary male patients when compared to a matched workload (kg × number of repetitions) with a traditional set configuration.