Adverse Childhood Experience, Genes, and PTSD Risk in Soldiers: A Methylation Study.

INTRODUCTION: Epigenetics can serve as a marker of susceptibility to many known psychiatric diseases. DNA methylation patterns of multiple genes have been studied in both civilian populations and military personnel with post-traumatic stress disorder (PTSD). Many of these genes serve various functions that span the hypothalamic-pituitary-adrenal axis, immune system, and central nervous system (CNS) growth factors and neurotransmission. It is thought that the methylation levels of such genes may be able to identify individuals who are at higher risk of developing PTSD. Our study seeks to establish whether previously reported PTSD genes possess a particular methylation pattern that is predictive of PTSD in active duty military members with combat exposure. MATERIALS AND METHODS: This is an institutional review board (IRB)-approved, cross-sectional, case control, gene-environment interaction study. About 170 active military members with and without PTSD were recruited. Patients with a history of structural brain damage, traumatic brain injury (TBI) resulting in loss of consciousness, predeployment diagnosis of PTSD or anxiety disorder, and predeployment prescription of an antidepressant or psychoactive medication were excluded. Validated measures of childhood trauma and adversity (adverse childhood experience [ACE] score), PTSD symptoms (PTSD check-list military version [PCL-M]), and combat exposure scales (CES) were measured via validated questionnaires for all subjects. After extracting DNA from peripheral blood provided by the 170 subjects, we determined methylation percentages, via pyrosequencing assays, for nine target areas within the following seven genes: BDNF, NR3C1, MAN2C1, TLR8, SLC6A4, IL-18, and SKA2. These genes are commonly reported in the literature as being highly correlated with PTSD and early-life traumatic experiences.Methylation levels were measured as a percentage at specific sites within the previously mentioned genes. Data were examined with SPSS v 22.0 Statistics and JMP v13.1 software using a general linear model for methylation × trauma (CES scores) split by diagnosis of PTSD or not, methylation versus childhood trauma (ACE scores), and methylation versus PTSD severity (PCL-M score). Two-way ANOVA was performed to control for antidepressant use. A two-tailed Student t-test was performed for PTSD analyses and was correlated with PTSD diagnosis, demographic information as well as ACE score, PCL-M score, and CES scores. RESULTS: Differentially methylated sites that were highly associated with PTSD diagnosis were found in three of seven candidate genes: BDNF, NR3C1, and MAN2C1. When compared to controls, patients with PTSD diagnosis had significantly lower levels of methylation, even after controlling for antidepressant use. PCL-M, ACE, and CES scores were significantly associated with PTSD diagnosis. CONCLUSION: Our study suggests that methylation of key genes involved in synaptic plasticity and the hypothalamic-pituitary-adrenal axis is associated with lower levels of methylation in military PTSD subjects exposed to combat when compared to their non-PTSD counterparts. Strengths of this study include controlling for antidepressant use and excluding TBI patients. Similar studies in an active duty population of this size are scarce. What is not clear is whether methylation changes are driving PTSD symptomology or whether they are merely a marker of disease. Future areas of research include prospective studies that measure methylation pre- and postcombat exposure in the same individual.

Temporal shifts in the collective dermatologic microbiome of military trainees.

BACKGROUND:  New military members undergo a highly-regimented 7-week training course during which trainees live and work within the same group of approximately 50 subjects for nearly 24 hours a day. This creates an optimal environment for assessing the impact of communal living on the collective skin microbiome. PURPOSE: The objective of this pilot study was to investigate dynamic changes of the skin microbiome in basic military trainees (BMT), in light of the unique environmental influences faced by this population. PATIENTS AND METHODS:  We evaluated collective changes in the skin microbiome of normal healthy adult basic trainees in response to communal living and universal Group A Strep prophylaxis with penicillin over the course of their initial 7-week training course. Samples from 10 flights of trainees were collected by swabbing upon arrival at Lackland AFB for their training (week 0) which is prior to prophylaxis with penicillin, at the 4 week point, and at the conclusion of their 7-week course of basic military training. Three separate high-throughput sequencing platforms and three bioinformatic pipeline analysis tools were utilized to assess the data. RESULTS: At all three time points we found that the top three bacterial genus identified were Propionibacterium, Staphylococcus, and Corynebacterium. We detected a community membership difference between the initial week 0 samples and the week 4 and 7 samples. A strong inverse correlation between Propionibacterium and Staphylococcus was noted with Propionibacterium being high at week 0 and much lower at weeks 4 and 7; conversely, Staphylococcus was low at week 0 and higher at weeks 4 and 7, this relationship was noted in both the individual and collective specimens. CONCLUSION: The collective dermatologic microbiome in the military trainee population examined exhibited a relative increase in Staphylococcus and Corynebacterium abundance coupled with a relative decrease in Propionibacterium abundance in this observational pilot study. Additional studies are needed to further assess the causal impact of communal living and widespread penicillin chemoprophylaxis.

A reduction in clot formation rate and strength assessed by thrombelastography is indicative of transfusion requirements in patients with penetrating injuries.

BACKGROUND: Bleeding is a major cause of death in patients with traumatic injuries. Recently, thrombelastography (TEG) has been suggested as an additional means of evaluating coagulation in trauma patients. We hypothesized that TEG data would aid in defining the coagulopathy of trauma in patients with penetrating traumatic injuries. METHODS: A retrospective study was performed of patients (n = 44) with penetrating injuries admitted to a combat support hospital during a 2-month period in 2004. Recorded data included standard laboratory data, TEG parameters, and blood product use in the first 24 hours after admission. Values were compared with clinically accepted ranges and those obtained from the Haemoscope Corporation. RESULTS: At admission, International Normalization Ratio, prothrombin time, and partial thromboplastin time were increased in 39% (>or=1.5), 31% (>16 seconds), and 37% (>40 seconds) of patients, respectively, suggesting hypocoagulation, but these variables did not correlate with the use of blood products (p > 0.05). TEG values obtained within 24 hours of admission (6 hours +/- 5.7 hours; median of 4.5 hours) demonstrated hypocoagulation based on delayed propagation of the clot (increased K time and reduced alpha-angle) and decreased clot strength (reduced maximal amplitude [MA]). MA correlated (r = 0.57, p < 0.01) with blood product use as well as platelet count (r = 0.61, p < 0.01). Patients with reduced MA (n = 23) used more blood products and had reduced platelet counts and hematocrit. CONCLUSION: Thrombelastography was a more accurate indicator of blood product requirements in our patient population than prothrombin time, partial thromboplastin time, and International Normalization Ratio. Thrombelastography enhanced by platelet count and hematocrit can guide blood transfusion requirements.

An improved arbitrary primed PCR method for rapid characterization of transposon insertion sites.

Modifications were made to published arbitrary primed polymerase chain reaction (AP-PCR) procedures that resulted in increased specificity and sensitivity. Several arbitrary primer sequences were also evaluated, resulting in recommendations for primer design.

Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis.

Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.