Association between childhood cognitive skills & adult suicidal behavior: A systematic review and meta-analysis.

BACKGROUND: It is unclear whether cognitive skill deficits during childhood carry risk for suicide attempt or mortality later in adulthood at the population level. We conducted a systematic review and meta-analysis of population-based studies examining the association between childhood cognitive skills and adult suicidal behavior, namely attempt and mortality. METHOD: We systematically searched databases for articles then extracted study characteristics and estimates on the association between childhood cognitive skills (i.e., IQ or school performance at age ≤ 18 years) and later suicide attempt and mortality. Random-effect meta-analysis was used to quantify this association across all studies with available data. RESULTS: Twenty-three studies met the inclusion criteria and suggest an association between lower childhood cognitive skills and increased risk of suicidal behavior. Meta-analysis of the adjusted estimates from 11 studies (N = 2,830,191) found the association to be small but statistically significant. Heterogeneity was significant but moderate, and results were unlikely to be influenced by publication bias. In subgroup analyses, associations were significant only for males. No difference in effect size was found between suicide attempt and suicide mortality. LIMITATIONS: Cognitive skills were measured with different cognitive subtests. Heterogeneity in the age of cognitive skills assessment. Meta-regression and subgroup analyses were based on a relatively low number of studies. CONCLUSIONS: Individuals with lower cognitive skills in childhood have a greater risk of suicidal behavior in adulthood, especially males. Although the association was small, interventions improving cognitive skills may yield large effects on suicide prevention at the population level if the association is causal.

Associations between epigenetic aging and childhood peer victimization, depression, and suicidal ideation in adolescence and adulthood: A study of two population-based samples.

Background: Prior studies indicate that peer victimization (including bullying) is associated with higher risk for depression and suicidal ideation across the life course. However, molecular mechanisms underlying these associations remain unclear. This two-cohort study proposes to test whether epigenetic aging and pace of aging, as well as a DNA methylation marker of responsive to glucocorticoids, are associated to childhood peer victimization and later depressive symptoms, or suicidal ideation. Methods: Cohort 1: Epigenome-wide DNA methylation (EPIC array) was measured in saliva collected when participants were 10.47 years (standard deviation = 0.35) in a subsample of the Quebec Longitudinal Study of Child Development (QLSCD, n = 149 participants), with self-reported peer victimization at 6-8 years, depressive symptoms (mean symptoms, and dichotomized top 30% symptoms) and suicidal ideation at 15-17 years. Cohort 2: Epigenome-wide DNA methylation (EPIC array) was measured in blood collected from participants aged 45.13 years (standard deviation = 0.37) in a subsample of the 1958 British Birth cohort (1958BBC, n = 238 participants) with information on mother-reported peer victimization at 7-11 years, self-reported depressive symptoms at 50 years, and suicidal ideation at 45 years. Five epigenetic indices were derived: three indicators of epigenetic aging [Horvath's pan-tissue (Horvath1), Horvath's Skin-and-Blood (Horvath2), Pediatric-Buccal-Epigenetic age (PedBE)], pace of aging (DunedinPACE), and stress response reactivity (Epistress). Results: Peer victimization was not associated with the epigenetic indices in either cohort. In the QLSCD, higher PedBE epigenetic aging and a slower pace of aging as measured by DunedinPACE predicted higher depressive symptoms scores. In contrast, neither the Horvath1, or Horvath2 epigenetic age estimates, nor the Epistress score were associated with depressive symptoms in either cohort, and none of the epigenetic indices predicted suicidal ideation. Conclusion: The findings are consistent with epigenome-wide and candidate gene studies suggesting that these epigenetic indices did not relate to peer victimization, challenging the hypothesis that cumulative epigenetic aging indices could translate vulnerability to depressive symptoms and suicidal ideation following peer victimization. Since some indices of epigenetic aging and pace of aging signaled higher risk for depressive symptoms, future studies should pursue this investigation to further evaluate the robustness and generalization of these preliminary findings.

Perceived friend support buffers against symptoms of depression in peer victimized adolescents: Evidence from a population-based cohort in South Korea.

BACKGROUND: Peer victimization is associated with an increased risk for depression, but there is less evidence on how certain factors such as friend support can buffer this association. This study investigated the associations between friend support and depressive symptoms among victimized and non-victimized adolescent girls and boys from South Korea. METHODS: Participants includes 2258 students from the Korean Children and Youth Panel Survey, a nationally representative sample of middle school students in South Korea. Self-reported perceived friend support, depressive symptoms and peer victimization were measured using validated scales during middle school year 3 (mean age= 15.7 years). RESULTS: The association between peer victimization and depressive symptoms varied by sex (p for sex by peer victimization interaction<0.05). Peer victimization was more strongly associated with same year depressive symptoms in girls (ß=0.55) than boys (ß=0.24). After controlling for key confounders, including prior year mental health symptoms, higher levels of friend support were found to attenuate the association between peer victimization and depressive symptoms (p for friend support by peer victimization interaction <0.05). Peer victimization was associated with more depressive symptoms for adolescents with low and moderate friend support, but not those with high friend support. LIMITATIONS: Peer victimization, depressive symptoms, and friend support, were self-reported and measured the same year. CONCLUSIONS: Friend support protects victimized South Korean adolescents from the negative effect of peer victimization on depressive symptoms, hence contributes to closing the gap in depression between victimized and non-victimized adolescents.

Are early-life antecedents of suicide mortality associated with psychiatric disorders and suicidal ideation in midlife?

OBJECTIVE: To establish whether previously identified early-life antecedents of suicide mortality (i.e. low birthweight, younger maternal age, higher birth order, externalizing problems and adversities) are associated with proximal psychiatric disorders and suicidal ideation, which are themselves associated with an increased risk of suicide. METHODS: Participants were from the 1958 British birth-cohort (N = 8905) with information on prenatal/childhood experiences and the Clinical Interview Schedule-Revised at age 45 years. Outcomes were as follows: any internalizing disorder (anxiety disorder/depressive episode), depressive episode, alcohol use disorder and suicidal ideation. RESULTS: After adjustment, higher birth order (Ptrend = 0.043), younger maternal age (Ptrend = 0.017) and increased number of childhood adversities (Ptrend = 0.026) were associated with an increased risk of internalizing disorders. For example, the OR (95% CI) in fourth- or later-born children was 1.48 (1.06-2.07) and for young maternal age (<19 years) was 1.31 (0.89-1.91). Effect sizes were similar in magnitude for depressive episode and suicidal ideation, although associations did not reach conventional significance levels. No associations were found for low birthweight and externalizing problems (in males) and investigated outcomes. CONCLUSION: Associations for younger maternal age, higher birth order and adversities with adult internalizing disorders suggest that psychiatric disorders may be on the pathway linking some early-life factors and suicide.

Depressive symptoms and body mass index: co-morbidity and direction of association in a British birth cohort followed over 50 years.

BACKGROUND: An unhealthy body mass index (BMI) has been associated with depression but the direction of association is uncertain. Our aim was to estimate the co-morbidity and direction of association between BMI and depressive symptoms at several ages, from childhood to mid-adulthood. METHOD: The data were from 18,558 individuals born in 1 week in March 1958, in England, Scotland and Wales, with follow-up at ages 7, 11, 16, 23, 33, 42, 45 and 50 years. Depression (scores>or=90th percentile) was identified from child/adolescent (teacher questionnaires) and adult (self-complete questionnaires and clinical interview) measures. BMI (kg/m2) measured in child/adolescence and adulthood was classified as underweight, normal, overweight or obese. RESULTS: In cross-sectional analyses, obesity and underweight (not overweight) from 11 to 45 years were associated respectively with 1.3-2.1 and 1.5-2.3 times the risk of depression compared with normal weight. Using the time-lagged generalized estimating equation (GEE) approach, we tested (a) whether underweight or obesity at prior ages (7 to 45 years) predicted subsequent risk of depression (11 to 50 years), adjusting for baseline depression; and (b) whether depression at prior ages (7 to 42 years) predicted subsequent risk of underweight or obesity (11 to 45 years), adjusting for baseline BMI. In longitudinal analyses, underweight predicted subsequent depression in both sexes [odds ratio (OR) 1.25, 95% confidence interval (CI) 1.11-1.40] and depression predicted subsequent underweight in males only (OR 1.84, 95% CI 1.52-2.23). Obesity predicted subsequent depressive symptoms in females only (OR 1.34, 95% CI 1.14-1.56), but depression did not predict obesity. CONCLUSIONS: Clinicians should consider screening routinely for depression patients with unhealthy BMI, namely underweight and obesity, and vice versa.

Prenatal and childhood antecedents of suicide: 50-year follow-up of the 1958 British Birth Cohort study.

BACKGROUND: We aimed to elucidate early antecedents of suicide including possible mediation by early child development. METHOD: Using the 1958 birth cohort, based on British births in March 1958, individuals were followed up to adulthood. We used data collected at birth and at age 7 years from various informants. Suicides occurring up to 31 May 2009 were identified from linked national death certificates. Multivariable Cox proportional hazard models were used to investigate risk factors. RESULTS: Altogether 12399 participants (n = 44 suicides) had complete data. The strongest prenatal risk factors for suicide were: birth order, with risk increasing in later-born children [p trend = 0.063, adjusted hazard ratio (HR)], e.g. for fourth- or later-born children [HR = 2.27, 95% confidence interval (CI) 0.90-5.75]; young maternal age (HR = 1.18, 95% CI 0.34-4.13 for ⩽19 years and HR = 0.41, 95% CI 0.19-0.91 for >29 years, p trend = 0.034); and low (<2.5 kg) birth weight (HR = 2.48, 95% CI 1.03-5.95). The strongest risk factors at 7 years were externalizing problems in males (HR = 2.96, 95% CI 1.03-8.47, p trend = 0.050) and number of emotional adversities (i.e. parental death, neglected appearance, domestic tension, institutional care, contact with social services, parental divorce/separation and bullying) for which there was a graded association with risk of suicide (p trend = 0.033); the highest (HR = 3.12, 95% CI 1.01-9.62) was for persons with three or more adversities. CONCLUSIONS: Risk factors recorded at birth and at 7 years may influence an individual's long-term risk of suicide, suggesting that trajectories leading to suicide have roots in early life. Some factors are amenable to intervention, but for others a better understanding of causal mechanisms may provide new insights for intervention to reduce suicide risk.

Morning salivary cortisol and cognitive function in mid-life: evidence from a population-based birth cohort.

BACKGROUND: The hormone 'cortisol' has been associated with cognitive deficits in older ages, and also with childhood cognition. The extent to which the associations of cortisol with cognitive deficits in later life reflect associations with childhood cognition ability is unclear. This study aimed to assess associations between adult cortisol levels and subsequent cognitive functions, while considering childhood cognition and other lifetime covariates. METHOD: Data are from the 1958 British Birth Cohort. Two morning salivary cortisol samples were obtained at 45 years: 45 min after waking (t1) and 3 h later (t2). Standardized tests assessing immediate and delayed verbal memory, verbal fluency and speed of processing were administered at 50 years. Information on cortisol, cognitive outcomes and covariates [e.g., birthweight, lifetime socio-economic position (SEP), education, smoking and drinking habits, body mass index (BMI), menopausal status, and depression/anxiety] was obtained for 4655 participants. RESULTS: Worse immediate and delayed verbal memory and verbal fluency at 50 years were predicted by elevated t2 cortisol at 45 years. For instance, for 1 standard deviation (s.d.) increase in t2 cortisol, individuals scored -0.05 s.d. lower on verbal memory and fluency tests. Childhood cognition explained about 30% of these associations, but associations with adult cognition remained. CONCLUSIONS: This study suggests that higher cortisol levels in late morning at 45 years are associated with poorer verbal memory and fluency at 50 years, with a contribution from childhood cognition to these associations.

Mucosal immune responses and protection against tetanus toxin after intranasal immunization with recombinant Lactobacillus plantarum.

The use of live microorganisms as an antigen delivery system is an effective means to elicit local immune responses and thus represents a promising strategy for mucosal vaccination. In this respect, lactic acid bacteria represent an original and attractive approach, as they are safe organisms that are used as food starters and probiotics. To determine whether an immune response could be elicited by intranasal delivery of recombinant lactobacilli, a Lactobacillus plantarum strain of human origin (NCIMB8826) was selected as the expression host. Cytoplasmic production of the 47-kDa fragment C of tetanus toxin (TTFC) was achieved at different levels depending on the plasmid construct. All recombinant strains proved to be immunogenic by the intranasal route in mice and able to elicit very high systemic immunoglobulin G (IgG1, IgG2b, and IgG2a) responses which correlated to the antigen dose. No significant differences in enzyme-linked immunosorbent assay IgG titers were observed when mice were immunized with live or mitomycin C-treated recombinant lactobacilli. Nevertheless, protection against the lethal effect of tetanus toxin was obtained only with the strains producing the highest dose of antigen and was greater following immunization with live bacteria. Significant TTFC-specific mucosal IgA responses were measured in bronchoalveolar lavage fluids, and antigen-specific T-cell responses were detected in cervical lymph nodes, both responses being higher in mice receiving a double dose of bacteria (at a 24-h interval) at each administration. These results demonstrate that recombinant lactobacilli can induce specific humoral (protective) and mucosal antibodies and cellular immune response against protective antigens upon nasal administration.

Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects.

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.

Overexpression of glutathione peroxidase increases the resistance of neuronal cells to Abeta-mediated neurotoxicity.

Senile plaques are neuropathological manifestations in Alzheimer's disease (AD) and are composed mainly of extracellular deposits of amyloid beta-peptide (Abeta). Various data suggest that the accumulation of Abeta may contribute to neuronal degeneration and that Abeta neurotoxicity could be mediated by oxygen free radicals. Removal of free radicals by antioxidant scavengers or enzymes was found to protect neuronal cells in culture from Abeta toxicity. However, the nature of the free radicals involved is still unclear. In this study, we investigated whether the neuronal overexpression of glutathione peroxidase (GPx), the major hydrogen peroxide (H2O2)-de-grading enzyme in neurons, could increase their survival in a cellular model of Abeta-induced neurotoxicity. We infected pheochromocytoma (PC12) cells and rat embryonic cultured cortical neurons with an adenoviral vector encoding GPx (Ad-GPx) prior to exposure to toxic concentrations of Abeta(25-35) or (1-40). Both PC12 and cortical Ad-GPx-infected cells were significantly more resistant to Abeta-induced injury. These data strengthen the hypothesis of a role of H2O2 in the mechanism of Abeta toxicity and highlight the potential of Ad-GPx to reduce Abeta-induced damage to neurons. These findings may have applications in gene therapy for AD.

Use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors.

The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e., Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying the gfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescent L. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.

Efficient gene transfer and long-term expression in neurons using a recombinant adenovirus with a neuron-specific promoter.

Adenoviruses are highly efficient vectors for gene transfer into brain cells. Restricting transgene expression to specific cell types and maintaining long-term expression are major goals for gene therapy in the central nervous system. We targeted gene expression to neurons by constructing an adenoviral vector that expressed the E. coli LacZ reporter gene under the control of the rat neuron-specific enolase promoter (Ad-NSE). Expression from Ad-NSE was compared with that from an adenoviral vector encoding the same reporter gene under the control of the Rous sarcoma virus LTR promoter (Ad-RSV). Both recombinant adenoviruses were injected stereotactically into rat hippocampus, cerebellum and striatum. Anatomical and immunohistochemical analyses of the Ad-NSE-stained cells showed that neurons were preferentially transduced. More neurons were stained in the hippocampus following infection with Ad-NSE than with Ad-RSV. Cytotoxicity from Ad-NSE was lower than from Ad-RSV. beta-Galactosidase gene expression after Ad-NSE infection remained stable for 3(1/2) months, and was detectable for 6 months. Thus, the NSE-adenoviral vector can be used to transfer potentially therapeutic genes into neuronal cells. The use of a cell-specific promoter also resulted in high in vivo efficiency and long-term transgene expression.

Neuron-restrictive silencer elements mediate neuron specificity of adenoviral gene expression.

Neuron-restrictive silencer elements (NRSEs) were used to target the gene expression of adenoviral vectors specifically to neuron cells in the central nervous system. By generating adenoviral constructs in which NRSE sequences were placed upstream from the ubiquitous phosphoglycerate kinase promoter, the specificity of expression of a luciferase reporter gene was tested in both cell lines and primary cultures. Whereas transgene expression was negligible in nonneuronal cells following infection with an adenovirus containing 12 NRSEs, neuronal cells strongly expressed luciferase when infected with the same adenovirus. The NRSEs restricted expression of the luciferase gene to neuronal cells in vivo when adenoviruses were injected both intramuscularly into mice and intracerebrally into rats. This NRSE strategy may avoid side effects resulting from the ectopic expression of therapeutic genes in the treatment of neurological diseases. In particular, it may allow the direct transfection of motor neurons without promoting transgene expression within inoculated muscles or the secretion of transgene products into the bloodstream.

Ki 67 expression in 35 borderline ovarian tumours: relations with clinicopathologic parameters and ploidy.

OBJECTIVE: To test the diagnostic and prognostic interest of Ki 67 expression and DNA ploidy in ovarian tumours. METHODS: The reactivity of tumour cells with the monoclonal antibody MIB 1 against Ki 67, was assessed by an indirect immunoperoxidase staining technique applied to 25 benign, 35 borderline and 20 malignant tumours of the ovary. In each cases, ploidy was analyzed by DNA flow cytometry. RESULTS: Ki 67 immunopositivity was detected in three benign tumours (12%), 14 borderline tumours (40%) and 14 ovarian carcinomas (70%). The mean and standard deviation of Ki 67 positive cells in benign, borderline and malignant tumours were: 1% +/- 8, 5% +/- 9.6 and 44.5% +/- 31 respectively. A difference in Ki 67 immunostaining was found between carcinomas and benign tumours (P < 0.0001), and between borderline and carcinomas (P = 0.001) but not between benign and borderline tumours. Evaluable DNA histograms from flow cytometry were generated in 77 cases (96.2%) of ovarian tumours. All 25 benign, 97.1% of 35 borderline and 44.5% of 20 malignant ovarian tumours were diploid. A difference was found between borderline and carcinomas (P = 0.001), but not between borderline and benign tumours. CONCLUSION: Ki 67 labelling was correlated with neither histologic and prognostic parameters, nor with survival, nor with DNA ploidy in borderline ovarian tumours.

An adenoviral vector-based system to study neuronal gene expression: analysis of the rat tyrosine hydroxylase promoter in cultured neurons.

We validated an adenoviral vector-based system as a move toward the characterization of regulatory sequences that are involved in the control of cell-type specificity and ligand regulation of neuronal gene expression in cultured neurons. We constructed recombinant adenoviruses, incorporating the luciferase gene under the control of different fragments of the rat tyrosine hydroxylase (TH) promoter. Similar results for luciferase expression were obtained in immortalized cells either by infection using adenoviral constructs or by transfection using conventional plasmid vectors. Taking advantage of adenoviral vectors, we extended our experiments to various primary cell cultures. The first 800 bp of the TH promoter were found to be sufficient to confer a cell-type preferential activity in noradrenergic neurons of the rat superior cervical ganglia. Furthermore, using this neuronal culture model, we showed that the same promoter region carries leukemia-inhibitory factor (LIF)-responsive element(s). Our results demonstrate that the first 800 bp of the rat TH promoter contains a functionally important core region for constitutive and LIF-regulated expression of TH in peripheral noradrenergic neurons. Moreover, the study validates the adenoviral vector-based system as a new strategy for studying the regulation of neuronal gene expression.

Adenovirus-mediated transfer of a functional GAD gene into nerve cells: potential for the treatment of neurological diseases.

The recent development of efficient virus-mediated gene transfer into nerve cells allows the prospect of new strategies for the treatment of drug-resistant neurological diseases. Some forms of epilepsy may be amenable to gene therapy. Although there is no obvious candidate gene, the consensual GABA hypothesis of epilepsy suggests that the GAD gene may be beneficial. GAD gene expression may be useful in supplying the inhibitory neurotransmitter GABA to particular critical brain territories. We show herein that a nonreplicative recombinant adenovirus carrying the GAD67 gene under the control of Rous sarcoma virus long terminal repeat promoter is able to express the transgene in primary cultures of neurons and glial cells. Expression of the GAD67 gene was assessed by immunoblotting and immunohistochemical analysis. We demonstrated the functionality of the transgene, the expression of which resulted in production of large amounts of GABA in neuronal and glial cell cultures. Substantial production of the enzyme was also detected for several weeks in infected organotypic slices cultured from new-born rat hippocampal tissues. The virally encoded GAD67 was also expressed in vivo in various brain areas involved in various neurological disorders and thus may be of value for the development of gene therapies.

An adenovirus encoding CuZnSOD protects cultured striatal neurones against glutamate toxicity.

Superoxide dismutase (SOD), a key enzyme in the detoxification of free radicals, catalyses the dismutation of superoxide O2.- to oxygen and hydrogen peroxide (H2O2). It is therefore a promising candidate for gene transfer therapy of neurological diseases in which free radicals are thought to be involved. We have constructed a recombinant adenoviral vector containing the human copper-zinc SOD cDNA. Using this vector we were able to drive the production of an active human copper-zinc SOD protein (hCuZnSOD) in various cell lines and primary cultures. Infection of striatal cells with a recombinant adenovirus expressing hCuZnSOD protected these cells from glutamate-induced cell death.

Common accessory genes for the Bordetella pertussis filamentous hemagglutinin and fimbriae share sequence similarities with the papC and papD gene families.

The Bordetella pertussis filamentous hemagglutinin (FHA) is a major virulence factor responsible for attachment, one of the early events in bacterial pathogenesis. Deletion of its structural gene, fhaB, or a Tn5 insertion in fhaA, downstream of fhaB, resulted in a FHA- and fimbriae- phenotype, although fhaB and the fim genes are not linked. The fhaB downstream region therefore most likely encodes accessory proteins required for the biosynthesis of FHA and fimbriae, despite the lack of sequence similarities between these two proteins. The nucleotide sequence of this area contains the open reading frames fhaD and fhaA, whose products share sequence similarities with the papD and papC gene products, respectively. PapD is a periplasmic chaperone protein able to bind to the Escherichia coli P pilin subunits and to transport them towards the outer membrane protein PapC which is responsible for pilus membrane translocation. An additional open reading frame, fhaE, is located downstream of fhaA. Its amino acid sequence shares similarities with those of the fimbrial subunits. Deletion analyses suggest that fhaB and the downstream genes can be transcribed as a polycistronic operon, and primer extension analysis revealed the presence of a second promoter between fhaB and fhaD.