Influence of correlation between HLA-G polymorphism and Interleukin-6 (IL6) gene expression on the risk of schizophrenia.

Converging evidence suggests important implications of immuno-inflammatory pathway in the risk and progression of schizophrenia. Prenatal infection resulting in maternal immune activation and developmental neuroinflammation reportedly increases the risk of schizophrenia in the offspring by generating pro-inflammatory cytokines including IL-6. However, it is not known how prenatal infection can induce immuno-inflammatory responses despite the presence of immuno-inhibitory Human Leukocyte Antigen-G (HLA-G) molecules. To address this, the present study was aimed at examining the correlation between 14 bp Insertion/Deletion (INDEL) polymorphism of HLA-G and IL-6 gene expression in schizophrenia patients. The 14 bp INDEL polymorphism was studied by PCR amplification/direct sequencing and IL-6 gene expression was quantified by using real-time RT-PCR in 56 schizophrenia patients and 99 healthy controls. We observed significantly low IL6 gene expression in the peripheral mononuclear cells (PBMCs) of schizophrenia patients (t = 3.8, p = .004) compared to the controls. In addition, schizophrenia patients carrying Del/Del genotype of HLA-G 14 bp INDEL exhibited significantly lower IL6 gene expression (t = 3.1; p = .004) than the Del/Ins as well as Ins/Ins carriers. Our findings suggest that presence of "high-expressor" HLA-G 14 bp Del/Del genotype in schizophrenia patients could attenuate IL-6 mediated inflammation in schizophrenia. Based on these findings it can be assumed that HLA-G and cytokine interactions might play an important role in the immunological underpinnings of schizophrenia.

CHRFAM7A gene expression in schizophrenia: clinical correlates and the effect of antipsychotic treatment.

Earlier studies have implicated CHRNA7, coding α-7 nicotinic acetylcholine receptor (α7 nAChR), and its partially duplicated chimeric gene CHRFAM7A in schizophrenia. However, the relationship between the alterations in peripheral gene expression of CHRFAM7A and severity of clinical symptoms has not been examined. Furthermore, potential influence of the antipsychotic medication on CHRFAM7A expression in drug-naive or drug-free schizophrenia is an unexplored area. CHRFAM7A gene expression in lymphocytes was analyzed in 90 antipsychotic-naïve or free schizophrenia patients using TaqMan-based quantitative RT-PCR. Psychotic symptoms were assessed using Scale for Assessment of Positive Symptoms and Scale for Assessment of Negative Symptoms (SANS). The relationship between psychopathology and CHRFAM7A expression was examined. In addition, measurement of CHRFAM7A gene expression was repeated during follow-up after short-term antipsychotic treatment in 38 patients. There was significant inverse correlation between CHRFAM7A expression and total negative psychopathology score-SANS, and this relationship persisted after accounting for possible confounders such as age, sex and smoking. On exploration of the factor structure of psychopathology using principal component analysis, all the negative symptoms-affective flattening, alogia, apathy, anhedonia and inattention were found to be inversely associated with CHRFAM7A expression. Furthermore, analysis of repeated measures revealed a significant increase in CHRFAM7A expression in patients after short-term administration of antipsychotic medication. Our study observations support the role for CHRFAM7A gene in schizophrenia pathogenesis and suggest a potential novel link between deficient CHRFAM7A expression and negative psychopathology. Furthermore, up-regulation of CHRFAM7A gene expression by antipsychotics suggests that it could be a potential state marker for clinical severity.

Gene polymorphisms and response to transcranial direct current stimulation for auditory verbal hallucinations in schizophrenia.

OBJECTIVE: Recent observations demonstrate a significant ameliorative effect of add-on transcranial direct current stimulation (tDCS) on auditory verbal hallucinations (AVHs) in schizophrenia. Of the many SNPs, NRG1 rs35753505 and catechol-o-methyl transferase (COMT) rs4680 polymorphisms have shown to have a strong association with neuroplasticity effect in schizophrenia. METHODS: Schizophrenia patients (n=32) with treatment resistant auditory hallucinations were administered with an add-on tDCS. The COMT (rs4680) and NRG1 (rs35753505) genotypes were determined. The COMT genotypes were categorised into Val group (GG; n=15) and Met group (GG/AG; n=17) and NRG1 genotypes were categorised into AA group (n=12) and AG/GG group (n=20). RESULTS: The reduction in auditory hallucination sub-scale score was significantly affected by COMT-GG genotype [Time×COMT interaction: F(1,28)=10.55, p=0.003, ɳ2=0.27]. Further, COMT-GG effect was epistatically influenced by the co-occurrence of NRG1-AA genotype [Time×COMT×NRG1 interaction: F(1,28)=8.09, p=0.008, ɳ2=0.22]. Irrespective of genotype, females showed better tDCS response than males [Time×Sex interaction: F(1,21)=4.67, p=0.04, ɳ2=0.18]. CONCLUSION: COMT-GG and NRG1-AA genotypes aid the tDCS-induced improvement in AVHs in schizophrenia patients. Our preliminary observations need replication and further systematic research to understand the neuroplastic gene determinants that modulate the effect of tDCS.

Transcranial direct current stimulation and neuroplasticity genes: implications for psychiatric disorders.

BACKGROUND AND AIM: Transcranial direct current stimulation (tDCS) is a non-invasive and well-tolerated brain stimulation technique with promising efficacy as an add-on treatment for schizophrenia and for several other psychiatric disorders. tDCS modulates neuroplasticity; psychiatric disorders are established to be associated with neuroplasticity abnormalities. This review presents the summary of research on potential genetic basis of neuroplasticity-modulation mechanism underlying tDCS and its implications for treating various psychiatric disorders. METHOD: A systematic review highlighting the genes involved in neuroplasticity and their role in psychiatric disorders was carried out. The focus was on the established genetic findings of tDCS response relationship with BDNF and COMT gene polymorphisms. RESULT: Synthesis of these preliminary observations suggests the potential influence of neuroplastic genes on tDCS treatment response. These include several animal models, pharmacological studies, mentally ill and healthy human subject trials. CONCLUSION: Taking into account the rapidly unfolding understanding of tDCS and the role of synaptic plasticity disturbances in neuropsychiatric disorders, in-depth evaluation of the mechanism of action pertinent to neuroplasticity modulation with tDCS needs further systematic research. Genes such as NRG1, DISC1, as well as those linked with the glutamatergic receptor in the context of their direct role in the modulation of neuronal signalling related to neuroplasticity aberrations, are leading candidates for future research in this area. Such research studies might potentially unravel observations that might have potential translational implications in psychiatry.

Clinical correlates of superior temporal gyrus volume abnormalities in antipsychotic-naïve schizophrenia.

In this study, the authors report superior temporal gyrus (STG) and Heschl's gyrus (HG) volume deficits in a large sample of medication-naïve patients with schizophrenia (N=55) in comparison with healthy control subjects (N=45) with structural MRI using voxel-based morphometry. Patients had significantly smaller volumes of left HG [X=-41, Y=-22, Z=11; Brodmann's area (BA)-41), right HG (X=47, Y=-18, Z=11; BA-41), and left STG (X=-50, Y=-34, Z=11; BA-42] compared with healthy control subjects. In addition, Positive and Negative Syndrome Scale positive score had a significant negative correlation with left HG. Findings observed in a large sample of antipsychotic-naive patients with schizophrenia emphasize the role of HG and STG in the pathophysiology of schizophrenia.

Pituitary volume in medication-naïve adults with obsessive compulsive disorder.

Pituitary volume is considered to reflect hypothalamic-pituitary-adrenal axis dysregulation, and this has been studied in various psychiatric disorders. This study demonstrates that pituitary volume as assessed through the region of interest manual tracing method in 50 medication-naïve adult patients with obsessive-compulsive disorder was not significantly different compared with 40 healthy control subjects (687.80 ± 126.60 versus 694.73 ± 131.59, F=0.55, p=0.46). The authors also compared the patients with obsessive-compulsive disorder without any comorbid axis I conditions (N=35) with healthy control subjects and found no difference in the pituitary volumes (681.62 ± 130.85 versus 694.72 ± 131.59, F=0.90, p=0.35). This emphasizes the need to examine hypothalamo-pituitary axis structures after taking into consideration various potential confounders such as medications and depression.

Neural basis of tDCS effects on auditory verbal hallucinations in schizophrenia: a case report evidence for cortical neuroplasticity modulation.

Transcranial direct current stimulation (tDCS) has been reported to ameliorate auditory hallucinations that are nonresponsive/minimally responsive to antipsychotic treatment in schizophrenia. The neurobiological basis of the tDCS effects in ameliorating auditory hallucinations is yet to be explored. In this case report, for the first time, using the novel method for noninvasive assessment of cortical plasticity, we demonstrate potential neuroplasticity effect of tDCS in improving treatment-resistant auditory hallucinations in a schizophrenic patient.

Rapid improvement of auditory verbal hallucinations in schizophrenia after add-on treatment with transcranial direct-current stimulation.

Treatment of nonresponsive auditory hallucinations in schizophrenia have been reported to improve with transcranial direct-current stimulation. This case description illustrates the use of add-on transcranial direct-current stimulation for rapid amelioration of auditory hallucinations in schizophrenia during the acute phase. Because transcranial direct-current stimulation is safe, largely well tolerated, and relatively inexpensive, this add-on treatment option is worth exploring through further rigorous studies.

Resting-state functional connectivity correlates of antipsychotic treatment in unmedicated schizophrenia.

BACKGROUND: Antipsychotics may modulate the resting state functional connectivity(rsFC) to improve clinical symptoms in schizophrenia(Sz). Existing literature has potential confounders like past medication effects and evaluating preselected regions/networks. We aimed to evaluate connectivity pattern changes with antipsychotics in unmedicated Sz using Multivariate pattern analysis(MVPA), a data-driven technique for whole-brain connectome analysis. METHODS: Forty-seven unmedicated patients with Sz(DSM-IV-TR) underwent clinical evaluation and neuroimaging at baseline and after 3-months of antipsychotic treatment. Resting-state functional MRI was analysed using group-MVPA to derive 5-components. The brain region with significant connectivity pattern changes with antipsychotics was identified, and post-hoc seed-to-voxel analysis was performed to identify connectivity changes and their association with symptom changes. RESULTS: Connectome-MVPA analysis revealed the connectivity pattern of a cluster localised to left anterior cingulate and paracingulate gyri (ACC/PCG) (peak coordinates:x = -04,y = +30,z = +26;k = 12;cluster-pFWE=0.002) to differ significantly after antipsychotics. Specifically, its connections with clusters of precuneus/posterior cingulate cortex(PCC) and left inferior temporal gyrus(ITG) correlated with improvement in positive and negative symptoms scores, respectively. CONCLUSION: ACC/PCG, a hub of the default mode network, seems to mediate the antipsychotic effects in unmedicated Sz. Evaluating causality models with data from randomised controlled design using the MVPA approach would further enhance our understanding of therapeutic connectomics in Sz.

Analyzing biomarker discovery: Estimating the reproducibility of biomarker sets.

Many researchers try to understand a biological condition by identifying biomarkers. This is typically done using univariate hypothesis testing over a labeled dataset, declaring a feature to be a biomarker if there is a significant statistical difference between its values for the subjects with different outcomes. However, such sets of proposed biomarkers are often not reproducible - subsequent studies often fail to identify the same sets. Indeed, there is often only a very small overlap between the biomarkers proposed in pairs of related studies that explore the same phenotypes over the same distribution of subjects. This paper first defines the Reproducibility Score for a labeled dataset as a measure (taking values between 0 and 1) of the reproducibility of the results produced by a specified fixed biomarker discovery process for a given distribution of subjects. We then provide ways to reliably estimate this score by defining algorithms that produce an over-bound and an under-bound for this score for a given dataset and biomarker discovery process, for the case of univariate hypothesis testing on dichotomous groups. We confirm that these approximations are meaningful by providing empirical results on a large number of datasets and show that these predictions match known reproducibility results. To encourage others to apply this technique to analyze their biomarker sets, we have also created a publicly available website, https://biomarker.shinyapps.io/BiomarkerReprod/, that produces these Reproducibility Score approximations for any given dataset (with continuous or discrete features and binary class labels).

Factors associated with SARS-CoV-2 test positivity in long-term care homes: A population-based cohort analysis using machine learning.

BACKGROUND: SARS-Cov-2 infection rates are high among residents of long-term care (LTC) homes. We used machine learning to identify resident and community characteristics predictive of SARS-Cov-2 infection. METHODS: We linked 26 population-based health and administrative databases to identify the population of all LTC residents tested for SARS-Cov-2 infection in Ontario, Canada. Using ensemble-based algorithms, we examined 484 factors, including individual-level demographics, healthcare use, comorbidities, functional status, and laboratory results; and community-level characteristics to identify factors predictive of infection. Analyses were performed separately for January to April (early wave 1) and May to August (late wave 1). FINDINGS: Among 80,784 LTC residents, 64,757 (80.2%) were tested for SARS-Cov-2 (median age 86 (78-91) years, 30.6% male), of whom 10.2% of 33,519 and 5.2% of 31,238 tested positive in early and late wave 1, respectively. In the late phase (when restriction of visitors, closure of communal spaces, and universal masking in LTC were routine), regional-level characteristics comprised 33 of the top 50 factors associated with testing positive, while laboratory values and comorbidities were also predictive. The c-index of the final model was 0.934, and sensitivity was 0.887. In the highest versus lowest risk quartiles, the odds ratio for infection was 114.3 (95% CI 38.6-557.3). LTC-related geographic variations existed in the distribution of observed infection rates and the proportion of residents at highest risk. INTERPRETATION: Machine learning informed evaluation of predicted and observed risks of SARS-CoV-2 infection at the resident and LTC levels, and may inform initiatives to improve care quality in this setting. FUNDING: Funded by a Canadian Institutes of Health Research, COVID-19 Rapid Research Funding Opportunity grant (# VR4 172736) and a Peter Munk Cardiac Centre Innovation Grant. Dr. D. Lee is the Ted Rogers Chair in Heart Function Outcomes, University Health Network, University of Toronto. Dr. Austin is supported by a Mid-Career investigator award from the Heart and Stroke Foundation. Dr. McAlister is supported by an Alberta Health Services Chair in Cardiovascular Outcomes Research. Dr. Kaul is the CIHR Sex and Gender Science Chair and the Heart & Stroke Chair in Cardiovascular Research. Dr. Rochon holds the RTO/ERO Chair in Geriatric Medicine from the University of Toronto. Dr. B. Wang holds a CIFAR AI chair at the Vector Institute.

Antisaccade task performance in obsessive-compulsive disorder and its clinical correlates.

OBJECTIVE: Obsessive-Compulsive Disorder (OCD) is characterized by abnormalities in the cortico-striato-thalamo-cortical (CSTC) circuitry of the brain. Antisaccade eye movement tasks measure aspects of the voluntary control of behaviour that are sensitive to CSTC circuitry dysfunction. METHOD: In this study, we examined antisaccade eye movement parameters of OCD patients in comparison with healthy controls (HC). In addition, we also examined the relationship between the antisaccade eye movement parameters and the severity of OCD. Antisaccade performance among right handed OCD patients (N = 65) was compared to matched right handed HC (N = 57). Eye tracking data during the task performance were collected using an Eye-Link eye-tracker at 1000-Hz sampling rate. OCD symptom severity was evaluated using Yale-Brown obsessive compulsive scale. RESULTS: The antisaccade error percentage was significantly greater in OCD patients than HC (p < 0.001). In addition, OCD patients had less accurate final eye position compared to HC (p < 0.001). There were no significant correlation between antisaccade parameters and OCD severity measures. CONCLUSION: Deficient performance in antisaccade task supports CSTC abnormality in OCD and this appears to be independent of the illness severity. Examining this in remitted participants with OCD and in unaffected first degree relatives could help ascertaining their endophenotype validity.

Predictors of mortality among long-term care residents with SARS-CoV-2 infection.

BACKGROUND: While individuals living in long-term care (LTC) homes have experienced adverse outcomes of SARS-CoV-2 infection, few studies have examined a broad range of predictors of 30-day mortality in this population. METHODS: We studied residents living in LTC homes in Ontario, Canada, who underwent PCR testing for SARS-CoV-2 infection from January 1 to August 31, 2020, and examined predictors of all-cause death within 30 days after a positive test for SARS-CoV-2. We examined a broad range of risk factor categories including demographics, comorbidities, functional status, laboratory tests, and characteristics of the LTC facility and surrounding community were examined. In total, 304 potential predictors were evaluated for their association with mortality using machine learning (Random Forest). RESULTS: A total of 64,733 residents of LTC, median age 86 (78, 91) years (31.8% men), underwent SARS-CoV-2 testing, of whom 5029 (7.8%) tested positive. Thirty-day mortality rates were 28.7% (1442 deaths) after a positive test. Of 59,702 residents who tested negative, 2652 (4.4%) died within 30 days of testing. Predictors of mortality after SARS-CoV-2 infection included age, functional status (e.g., activity of daily living score and pressure ulcer risk), male sex, undernutrition, dehydration risk, prior hospital contacts for respiratory illness, and duration of comorbidities (e.g., heart failure, COPD). Lower GFR, hemoglobin concentration, lymphocyte count, and serum albumin were associated with higher mortality. After combining all covariates to generate a risk index, mortality rate in the highest risk quartile was 48.3% compared with 7% in the first quartile (odds ratio 12.42, 95%CI: 6.67, 22.80, p < 0.001). Deaths continued to increase rapidly for 15 days after the positive test. CONCLUSIONS: LTC residents, particularly those with reduced functional status, comorbidities, and abnormalities on routine laboratory tests, are at high risk for mortality after SARS-CoV-2 infection. Recognizing high-risk residents in LTC may enhance institution of appropriate preventative measures.

Differential impact of interleukin-6 promoter gene polymorphism on hippocampal volume in antipsychotic-naïve schizophrenia patients.

BACKGROUND: Differential susceptibility model hypothesizes that a genotype need not be unfavorable all the time as postulated in stress-diathesis model but can be beneficial in a supportive context. Single-nucleotide polymorphism (SNP) (rs18000795) within the promoter region of interleukin-6 (IL-6) gene was earlier noted to have a differential susceptibility on hippocampal volume in schizophrenia (SCZ). MATERIALS AND METHODS: We examined antipsychotic-naïve/free SCZ patients (n = 35) in comparison with healthy controls (n = 68). Hippocampus volumes were assessed in 3 Tesla magnetic resonance imaging using voxel-based morphometry. Region of interest analysis was done using hippocampus mask. IL-6 SNP (rs1800795) was genotyped using TaqMan allelic discrimination assay. RESULTS: A significantly deficient right (T = 3.03; K E= 392; P SVC-FWE= 0.04) and left (T = 3.03; K E= 47; P uncorr= 0.03) hippocampal gray matter volumes were noted in SCZ patients after controlling for the potential confounding effects of age, sex, and total brain volume. There was a significant diagnosis x rs1800795 genotype interaction involving both left (T = 2.17, K E= 95, P uncorr= 0.02) and right (T = 1.82, K E= 29, P uncorr= 0.04) hippocampal volumes. Patients with GG (left: F =5.78; P = 0.02; right: F =6.21; P = 0.01) but not GC/CC genotype (left: F =0.89; P = 0.34; right: F <0.01; P = 0.95) had volume depletion. CONCLUSION: A paradoxical smaller hippocampal volume with GG genotype was noted in SCZ. Further elucidation of its mechanistic basis might have translational implications.

Impact of NRG1 HapICE gene variants on digit ratio and dermatoglyphic measures in schizophrenia.

Multiple lines of evidence have suggested a potential role of Neuregulin-1 (NRG1) in the neurodevelopmental pathogenesis of schizophrenia. Interaction between genetic risk variants present within NRG1 locus and non-specific gestational putative insults can significantly impair crucial processes of brain development. Such genetic effects can be analyzed through the assessment of digit ratio and dermatoglyphic patterns. We examined the role of two well-replicated polymorphisms of NRG1 (SNP8NRG221533 and SNP8NRG243177) on schizophrenia risk and its probable impact on the digit ratio and dermatoglyphic measures in patients (N = 221) and healthy controls (N = 200). In schizophrenia patients, but not in healthy controls, a significant association between NRG1 SNP8NRG221533 C/C genotype with lower left 2D:4D ratio, as well as with higher FA_TbcRC and DA_TbcRC. The substantial effect of SNP8NRG221533 on both digit ratio and dermatoglyphic measures suggest a potential role for NRG1 gene variants on neurodevelopmental pathogenesis of schizophrenia.

Tolerance of transcranial direct current stimulation in psychiatric disorders: An analysis of 2000+ sessions.

Transcranial direct current stimulation (tDCS), a non-invasive, neuromodulatory technique, is being increasingly applied to several psychiatric disorders. In this study, we describe the side-effect profile of repeated tDCS sessions (N = 2005) that were administered to 171 patients (156 adults and 15 adolescents) with different psychiatric disorders [schizophrenia [N = 109], obsessive-compulsive disorder [N = 28], alcohol dependence syndrome [N = 13], mild cognitive impairment [N = 10], depression [N = 6], dementia [N = 2] and other disorders [N = 3]]. tDCS was administered at a constant current strength of 2 mA with additional ramp-up and ramp-down phase of 20 s each at the beginning and end of the session, respectively. Other tDCS protocol parameters were: schizophrenia and obsessive-compulsive disorder: 5-days of twice-daily 20-min sessions with an inter-session interval of 3-h; Mild cognitive impairment/dementia and alcohol dependence syndrome: at least 5-days of once-daily 20-min session; Depression: 10-days of once-daily 30 min session. At the end of each tDCS session, any adverse event observed by the administrator and/or reported by the patient was systematically assessed using a comprehensive questionnaire. The commonly reported adverse events during tDCS included burning sensations (16.2%), skin redness (12.3%), scalp pain (10.1%), itching (6.7%), and tingling (6.3%). Most of the adverse events were noted to be mild, transient and well-tolerated. In summary, our observations suggest that tDCS is a safe mode for therapeutic non-invasive neuromodulation in psychiatric disorders in adults as well as the adolescent population.

Neuro-hemodynamic endophenotypes of emotional interference in OCD: fMRI study using emotion counting stroop task.

BACKGROUND: We sought to examine the endophenotype pattern of neuro-hemodynamic substrates of emotion counting Stroop (ecStroop) paradigm in patients with OCD, their unaffected siblings [first degree relatives-FDR] and healthy controls (HC). METHODS: OCD patients (medication naïve)[N = 16], their unaffected siblings(FDR)[N = 16] and HC [N = 24] were compared using an established ecStroop paradigm in a 3-Tesla fMRI. The relative BOLD signals corresponding to the three types of conditions (neural words-N, words with negative emotional salience-E and words with salience for OCD-O) were examined in the apriori hypothesized brain regions. RESULTS: Both in O minus N contrast and O minus E contrast, the groups demonstrated significant differential activation of right insula (BA 13). The post-hoc analyses showed in patients and FDRs relative to HC the following: significant hyperactivation of insula in O minus E contrast; significant hyperactivation of right insula and right DLPFC (BA 9) in O minus N contrast. CONCLUSIONS: The neuro-hemodynamic responses corresponding to the obsessive words in insula and DLPFC could be potential endophenotypes. "Threat relatedness" might thus have a vulnerability meaning in the pathogenesis and neurobiological basis of OCD.

Efficacy of fronto-temporal transcranial direct current stimulation for refractory auditory verbal hallucinations in schizophrenia: A randomized, double-blind, sham-controlled study.

Persistent auditory verbal hallucinations (AVH) that are refractory to antipsychotic medications are reported in about 20-30% of schizophrenia patients. Transcranial Direct Current Stimulation (tDCS), a non-invasive and safe neuromodulatory technique, has attracted significant interest as an add-on treatment for refractory AVH in schizophrenia. Studies examining the efficacy of tDCS for refractory AVH in schizophrenia have reported inconsistent findings. In this study, using a randomized, double-blind, sham-controlled design (RCT), we sought to examine the effect of add-on tDCS [anode corresponding to left dorsolateral prefrontal cortex and cathode to left temporo-parietal junction; 2-mA, twice-daily sessions for 5-days] to treat refractory AVH in schizophrenia patients (N=25); following this RCT phase, patients that had <30% reduction in AVH severity were offered an open-label extension (OLE) active stimulation to evaluate the effect of cross-over to verum tDCS. In the RCT phase, repeated measures ANOVA with tDCS type [verum (N=12) vs. sham (N=13)] as between subjects factor demonstrated a significant tDCS-type X time-point interaction [F=21.5, p<0.001, partial-η2=0.48] with significantly greater reduction of AVH score in verum tDCS group as compared to sham group. In the OLE phase, sham-to-verum crossed over patients (N=13) showed significantly greater reduction in AVH severity than their corresponding change during RCT phase (t=2.9; p=0.01). Together, these observations add further support to the beneficial effects of add-on tDCS to treat refractory AVH schizophrenia.