The association between physical activity and cognition in men with and without HIV infection.

OBJECTIVES: HIV-associated neurocognitive disorders are highly prevalent, and physical activity (PA) is a modifiable behaviour that may affect neurocognitive function. Our objective was to determine the association between PA and neurocognitive function and the effect of HIV on this association. METHODS: PA was assessed in the Multicenter AIDS Cohort Study with the International Physical Activity Questionnaire. A neuropsychological test battery assessed global impairment and domain-specific impairment (executive function, speed of processing, working memory, learning, memory, and motor function) every 2 years. Semiannually, the Symbol Digit Modalities Test and Trail Making Test Parts A and B were performed. Adjusted logistic regression models were used to assess the PA-neurocognitive function association. Using longitudinal data, we also assessed the PA category-decline of neurocognitive function association with multivariate simple regression. RESULTS: Of 601 men, 44% were HIV-infected. Low, moderate, and high PA was reported in 27%, 25%, and 48% of the HIV-infected men vs. 19%, 32% and 49% of the HIV-uninfected men, respectively. High PA was associated with lower odds of impairment of learning, memory, and motor function [odds ratio (OR) ranging from 0.52 to 0.57; P < 0.05 for all]. The high PA-global impairment association OR was 0.63 [95% confidence interval (CI) 0.39, 1.02]. Among HIV-infected men only, across multiple domains, the high PA-impairment association was even more pronounced (OR from 0.27 to 0.49). Baseline high/moderate PA was not associated with decline of any domain score over time. HIV infection was marginally associated with a higher speed of decline in motor function. CONCLUSIONS: A protective effect of high PA on impairment in neurocognitive domains was observed cross-sectionally. Longitudinal PA measurements are needed to elucidate the PA-neurocognitive function relationship over time.

Improvements in brain and behavior following eradication of hepatitis C.

Despite recent advances in treatment, hepatitis C remains a significant public health problem. The hepatitis C virus (HCV) is known to infiltrate the brain, yet findings from studies on associated neurocognitive and neuropathological changes are mixed. Furthermore, it remains unclear if HCV eradication improves HCV-associated neurological compromise. This study examined the longitudinal relationship between neurocognitive and neurophysiologic markers among healthy HCV- controls and HCV+ adults following successful HCV eradication. We hypothesized that neurocognitive outcomes following treatment would be related to both improved cognition and white matter integrity. Participants included 57 HCV+ participants who successfully cleared the virus at the end of treatment (sustained virologic responders [SVRs]) and 22 HCV- controls. Participants underwent neuropsychological testing and, for a nested subset of participants, neuroimaging (diffusion tensor imaging) at baseline and 12 weeks following completion of HCV therapy. Contrary to expectation, group-level longitudinal analyses did not reveal significant improvement in neurocognitive performance in the SVRs compared to the control group. However, a subgroup of SVRs demonstrated a significant improvement in cognition relative to controls, which was related to improved white matter integrity. Indeed, neuroimaging data revealed beneficial effects associated with clearing the virus, particularly in the posterior corona radiata and the superior longitudinal fasciculus. Findings suggest that a subgroup of HCV+ patients experienced improvements in cognitive functioning following eradication of HCV, which appears related to positive changes in white matter integrity. Future research should examine whether any additional improvements in neurocognition and white matter integrity among SVRs occur with longer follow-up periods.

Environmental Toxins Found Historically in the Polycythemia Vera Cluster Area and their Potential for Inducing DNA Damage.

In 2006, the Agency for Toxic Substances and Disease Registry received a request to determine whether a cluster of polycythemia vera patients existed in a northeast Pennsylvania community. A significant cluster of PV cases was identified at the nexus of three counties near several hazardous waste sites. The current study evaluated the potential for a select number of environmental contaminants previously detected in the cluster area to induce DNA damage using in vitro assays with hematopoietic stem-cell derived progenitor cells. CD34+ cells were isolated from normal cord blood samples and were cultured for 48-72 hours to generate erythroid progenitor cells. Eighteen compounds were chosen for the assay; arsenic trioxide, benzo(a)pyrene, benzene, methylene chloride, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), trichloroethylene, potassium chloride, ethylbenzene, benzo[k]fluoranthene, styrene, cadmium chloride, hydroquinone, 1,1,1-trichloroethane, sodium cyanide, manganese chloride, chromium oxide, lead oxide, and sodium arsenite. Genotoxicity of the compounds was determined using the comet assay, and toxicity determined via the cell viability assay. Using the comet assay, 16 compounds at 10 nM concentration, induced a significant amount of DNA damage compared to the control. When evaluating whether a dose-dependent relationship was present, seventeen of the eighteen compounds led to greater DNA damage with increasing exposure concentrations. 2,3,7,8-TCDD was particularly potent, inducing DNA damage in virtually all cells at 1 µM. In conclusion, most of the toxins evaluated using the comet assay showed potential to induce DNA damage in hematopoietic cells, and the genotoxic effects were dose-dependent.

Diffusion alterations in corpus callosum of patients with HIV.

BACKGROUND AND PURPOSE: Diffusion alterations have been identified in the corpus callosum and frontal white matter of patients infected with human immunodeficiency virus (HIV), though the relevance of these findings to cognitive deterioration has not yet been determined. This study tested the hypothesis that diffusion tensor imaging can detect tissue status alterations in these regions in cognitively impaired patients infected with HIV and the acquired measurements correlate with the severity of cognitive impairment. METHODS: Fractional anisotropy (FA) and mean diffusivity (MD) were determined for corpus callosum (genu and splenium) and frontal white matter (FWM). The DTI measurements were compared in 11 HIV and 11 control participants. Patterns of relationship were examined with cognitive status measures from concurrent neurologic and neuropsychologic evaluations. RESULTS: FA values for the splenium were significantly reduced in the patients infected with HIV and correlated with dementia severity and deficits in motor speed. MD values for the splenium were significantly increased in the patients infected with HIV and correlated with deficits in motor speed. FA measurements were also significantly correlated with performance on visual memory (genu), visuoconstruction (FWM), and verbal memory (FWM) tasks. CONCLUSION: Diffusion abnormalities were identified in the splenium of the corpus callosum in patients infected with HIV, and these alterations were associated with dementia severity and motor speed losses. In vivo assessment of callosal integrity by using quantitative neuroimaging may have potential utility as a marker of brain injury in patients infected with HIV.

Bone marrow diffusion measures correlate with dementia severity in HIV patients.

BACKGROUND AND PURPOSE: Escalation in monocyte trafficking from the bone marrow into the brain may play a critical role in central nervous system injury and cognitive deterioration in patients with HIV infection. This study tested the hypothesis that the mean diffusivity is sensitive to marrow changes in HIV patients and that these quantitative imaging measurements correlate with the severity of dementia. METHODS: The mean diffusivity (MD), determined for clival and calvarial marrow regions, was compared in 11 HIV-infected patients and 9 control subjects. The imaging measurements were also evaluated for relationships with dementia severity and markers of disease progression (CD4 and viral load in plasma). RESULTS: The MD was significantly reduced in both clival and calvarial marrow in HIV-infected patients (P =.006). Diffusion measurements for clival (P =.02) and for calvarial (P =.03) regions were significantly correlated with the severity of dementia. CONCLUSION: The results of this investigation support the utility of diffusion strategies for monitoring the marrow and provide further evidence of a relationship between marrow status changes and neurologic progression in HIV patients.

Randomized trial of antenatal dexamethasone in surfactant-treated infants delivered before 30 weeks' gestation.

OBJECTIVE: To determine if an additive effect exists between antenatal corticosteroid administration and postnatal surfactant therapy in the prevention of respiratory distress syndrome (RDS) in preterm infants. METHODS: A randomized, double-blind trial was conducted from April 1990 to June 1994, in which dexamethasone (5 mg every 12 hours for a total of four doses) or saline was given to women at risk for delivery at 24-29 weeks' gestation. At birth, prophylactic surfactant was administered to all study infants. Main outcome measures were RDS occurrence and severity. Secondary clinical end points included bronchopulmonary dysplasia, pneumothorax, patent ductus arteriosus, necrotizing enterocolitis, retinopathy, intraventricular hemorrhage, and death. RESULTS: Seventy-five of the 124 randomized subjects delivered 96 infants within the studied gestational age range (dexamethasone, n = 54; placebo, n = 42). Similar maternal demographics and obstetric complications were noted between study groups. A greater population of infants were delivered from multi-fetal gestations in the dexamethasone cohort (26 of 54 versus 12 of 42 newborns; P = .05). There were no significant differences in the occurrence or severity of RDS between the dexamethasone and placebo infants (none or mild, 67 versus 67%; moderate, 24 versus 26%; severe, 9 versus 7%, respectively), or differences in any of the secondary clinical outcomes. The study size was sufficient to exclude a 50% reduction in RDS incidence as a consequence of dexamethasone exposure. An analysis restricted to singletons (dexamethasone, n = 28; placebo, n = 30) revealed similar overall occurrence of intraventricular hemorrhage (12 of 28 versus ten of 30; P = .63), but significantly fewer grade 3 and 4 intraventricular hemorrhages in dexamethasone-exposed neonates (two of 12 versus six of ten; P = .048). CONCLUSION: Antenatal dexamethasone does not appear to decrease the incidence or severity of RDS in surfactant-treated infants delivered at 24-29 weeks' gestation, but may be associated with reduced severity of intraventricular hemorrhages in surfactant-treated singletons in this gestational age range.

A comparison of pregnancy loss between transcervical and transabdominal chorionic villus sampling.

OBJECTIVE: To evaluate the comparative safety of transcervical and transabdominal chorionic villus sampling (CVS). METHODS: From May 1988 to January 1992, CVS was performed by two operators at 9-12 weeks' gestation in 1048 singleton pregnancies. The sampling method for each patient, transabdominal or transcervical, was chosen primarily based upon placental location; the transabdominal route was used for anterior or fundal location and the transcervical route for posterior placentation. Perinatal outcome was assessed by post-procedure patient telephone contact, mid-gestation ultrasound evaluation, postpartum questionnaire completed by the referring obstetrician, and telephone interview with each patient after delivery. RESULTS: Complete follow-up was available in 1012 cases (97%). Excluding 39 elective abortions, 35 of 973 euploid pregnancies aborted spontaneously. The difference in fetal loss rate between transcervical and transabdominal CVS approached statistical significance (5.2 versus 2.9%; P = .058). Bleeding before CVS (P = .006) and multiple placental aspirations (P = .022) were associated with fetal loss for the entire study group. An interaction between uterine position and sampling method was also indicated; an increased loss rate was associated with transcervical CVS in the presence of uterine retroversion (P = .0017). CONCLUSION: Despite choosing the preferred CVS method for each patient, an increased loss rate may be associated with transcervical sampling in the presence of uterine retroversion.

Multifetal reduction increases the risk of preterm delivery and fetal growth restriction in twins: a case-control study.

OBJECTIVE: To compare pregnancy outcome in twin gestations resulting from multifetal reduction to "primary" twin pregnancies derived from either spontaneous conception or infertility therapy. DESIGN: Case-control study. SETTING: University-affiliated tertiary center. PATIENT(S): Multifetal pregnancies (quadruplets or more) reduced to twins (group A) compared with twin gestations conceived either spontaneously (group B) or through infertility therapy (group C). INTERVENTION(S): Multifetal reduction for group A; perinatal care for groups A, B, and C. MAIN OUTCOME MEASURE(S): Comparison of perinatal complications between groups including antepartum bleeding, premature membrane rupture, and preterm labor. Neonatal outcomes compared including gestational age at delivery, birth weight, incidence of fetal growth restriction, and twin discordancy. RESULT(S): A higher incidence of idiopathic preterm labor was noted in group A cases (14/18) compared with either of the control groups (B: 26/54, or C: 24/54). As a consequence, group A had the lowest gestational age at delivery (32.6 +/- 3.9 weeks) compared with groups B (33.6 +/- 4.4 weeks) and C (36.0 +/- 3.4 weeks). Corresponding birth weights of both first- and second-born twins were significantly lower in group A compared with group C, whereas the birth weight comparison between groups A and B showed a nonsignificant difference. The proportion of pregnancies in which one or both twins weighted less than the 10th percentile was greatest in group A pregnancies (A: 5/18 versus C: 5/54). Discordant birth weight among twin pairs was proportionately greater for group A cases at both the 20% and 30% discordance levels. CONCLUSION(S): Twin gestations resulting from multifetal reduction are at increased risk for preterm birth, fetal growth restriction, and discordancy when compared with fertility therapy-derived, nonreduced twins.

Complications of transsphenoidal surgery: results of a national survey, review of the literature, and personal experience.

OBJECTIVE: The primary objectives of this report were, first, to determine the number and incidence of complications of transsphenoidal surgery performed by a cross-section of neurosurgeons in the United States and, second, to ascertain the influence of the surgeon's experience with the procedure on the occurrence of these complications. The secondary objective was to review complications of transsphenoidal surgery from the standpoint of their causation, treatment, and prevention. METHODS: Questionnaires regarding 14 specific complications of transsphenoidal surgery were mailed to 3172 neurosurgeons. The data reported were analyzed from the 958 respondents (82%) who reported performing the operation. The neurosurgeons surveyed were asked to estimate the number of transsphenoidal operations performed, to identify any complications observed, and to estimate the percentage of operations that had resulted in any of the 14 specific complications. The 958 respondents were placed into three experience groups, based on the number of transsphenoidal operations performed. The data were analyzed by using chi 2 tests and Spearman correlation coefficients. The secondary objectives were met through a detailed review of the literature, in light of our experience. RESULTS: Of the respondents, 87.3% reported having performed < 200 operations and 9.7% reported 200 to 500 previous operations. The remaining 3% reported more than 500 previous operations. More extensive previous experience with transsphenoidal surgery was associated with a greater likelihood of having witnessed each specific complication. The mean operative mortality rate for all three groups was 0.9%. Anterior pituitary insufficiency (19.4%) and diabetes insipidus (17.8%) were complications with the highest incidence of occurrence. The overall incidence of cerebrospinal fluid fistulas was 3.9%. Other significant complications, such as carotid artery injuries, hypothalamic injuries, loss of vision, and meningitis, occurred with incidence rates between 1 and 2%. An inverse relationship was found between the experience group and the likelihood of complications, as indicated by significant negative Spearman correlation coefficients for all but 2 of the 14 complications listed in the survey (P < 0.05). Thus, increased experience with transsphenoidal surgery seems to be associated with a decreased percentage of operations resulting in complications. Some caution should be exercised in interpreting these data, because they are based on the respondents' estimates. CONCLUSION: Transsphenoidal surgery seems to be a reasonably safe procedure, with a mortality rate of less than 1%. However, a significant number of complications do occur. The incidence of these complications seems to be higher, with statistical significance, in the hands of less experienced surgeons. The learning curve seems to be relatively shallow, because a statistically significantly decreased incidence of morbidity and death could be documented after 200 and even 500 transsphenoidal operations. Better understanding of the indications for transsphenoidal surgery and improved familiarity with the regional anatomy should further lower the incidence of death and morbidity resulting from this procedure in the hands of all neurosurgeons.

Association of white matter hyperintensities with low serum 25-hydroxyvitamin D levels.

BACKGROUND AND PURPOSE: Vitamin D deficiency is associated with cognitive impairment in the elderly and with increased white matter T2 hyperintensities in elderly debilitated patients. We investigated the relationship between serum vitamin D and brain MR findings in adult outpatients. MATERIALS AND METHODS: Brain MR studies of 56 patients ages 30-69 years were selected when vitamin D level had been obtained within 90 days of the MRI. White matter T2 hyperintensities were characterized by size and location by two neuroradiologists. Manual volumetric analysis was assessed in patients more than 50 years of age. RESULTS: The entire cohort showed a significant negative relationship between serum 25-hydroxyvitamin D and the number of confluent juxtacortical white matter T2 hyperintensities (P = .047). The cohort ages 50 years and older showed stronger correlation between confluent white matter T2 hyperintensities and serum 25-hydroxyvitamin D in the juxtacortical region; number (P = .015) and size of white matter T2 hyperintensities (P = .048). Atrophy was not significantly related to serum 25-hydroxyvitamin D by radiologist visual analysis or by the bicaudate ratio. CONCLUSIONS: We found a significant relationship between vitamin D and white matter T2 hyperintensities in independent adult outpatients, especially over the age of 50 years.

Vascular risk factors, HIV serostatus, and cognitive dysfunction in gay and bisexual men.

BACKGROUND: The purpose of this study was to evaluate the relationship between cognitive performance, risk factors for cardiovascular and cerebrovascular disease (CVD), and HIV infection in the era of highly active antiretroviral therapy. METHODS: We evaluated the cognitive functions of men enrolled in the cardiovascular disease substudy of the Multicenter AIDS Cohort Study who were aged > or =40 years, with no self-reported history of heart disease or cerebrovascular disease. Results from comprehensive neuropsychological evaluations were used to construct composite scores of psychomotor speed and memory performance. Subclinical CVD was assessed by measuring coronary artery calcium and carotid artery intima-media thickness (IMT), as well as laboratory measures, including total cholesterol, fasting glucose, glycosylated hemoglobin, glomerular filtration rate (estimated), and standardized blood pressure and heart rate measures. RESULTS: After accounting for education, depression, and race, carotid IMT and glomerular filtration rate were significantly associated with psychomotor speed, whereas IMT was associated with memory test performance. HIV serostatus was not significantly associated with poorer cognitive test performance. However, among the HIV-infected individuals, the presence of detectable HIV RNA in plasma was linked to lower memory performance. CONCLUSIONS: These findings suggest that HIV infection may not be the most important predictor of cognitive performance among older gay and bisexual men in the post-highly active antiretroviral therapy era, at least among those with access to medical care and to appropriate medications. Medical factors associated with normal aging are significantly associated with performance on neuropsychological tests, and good clinical management of these factors both in HIV-infected individuals and those at risk for infection may have beneficial effects in the short term and could reduce the risk of subsequent cognitive decline.

Whole brain and localized magnetization transfer measurements are associated with cognitive impairment in patients infected with human immunodeficiency virus.

BACKGROUND AND PURPOSE: Patients infected with human immunodeficiency virus (HIV) are susceptible to cognitive deterioration. This study investigated the utility of magnetization transfer (MT) imaging for quantification of brain tissue alterations associated with cognitive deficits in patients with HIV. MATERIALS AND METHODS: MT ratios (MTR) were derived for whole brain and for regions of interest (ROIs) in the basal ganglia and white matter in 11 HIV and 12 control subjects. Relationships with severity of cognitive impairment and specific neuropsychological deficits were also evaluated. RESULTS: MTR values for normalized whole brain histogram peak height, whole brain histogram mean, and all examined ROIs were reduced in the HIV subjects. Normalized histogram peak height and mean for whole brain, as well as means for the corpus callosum, basal ganglia, and frontal white matter (FWM), were significantly correlated with severity of cognitive impairment. MTR values for white matter regions (corpus callosum, FWM, and centrum semiovale) were correlated with specific cognitive deficits. CONCLUSION: Quantitative MTR measurements, determined for the whole brain and for vulnerable ROIs, are sensitive to neuropathologic changes associated with cognitive impairment in HIV-infected patients.

Monocyte chemoattractant protein-1 correlates with subcortical brain injury in HIV infection.

Various biomarkers have been suggested as associative or predictive of HIV-associated neurocognitive impairment. Plasma levels of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-alpha), and hematocrit were evaluated for relationships with diffusion tensor imaging measurements of centrum semiovale, caudate, and putamen. MCP-1 levels correlated with tissue status (mean diffusivity) in all examined regions. Plasma markers were also significantly correlated with anisotropy measurements in centrum semiovale (TNF-alpha) and putamen (hematocrit).

Probing essential residues for cellular uptake with a cationic nuclear localization signal sequence.

The nuclear localization signal sequence (NLS) of the transcription factor NF-kappaB is a cationic peptide with the ability to cross the cytoplasmic membrane and facilitate the delivery of attached cargo, such as DNA and proteins, to cells. Previous research had pointed to the essential role of cationic residues, therefore, the importance of residues within the NLS of NF-kappaB was evaluated for cellular uptake using an alanine replacement strategy. Although it was expected that removal of the cationic groups would have the greatest effect on membrane translocation, the most significant decreases in cellular uptake occurred with the replacement of the hydrophilic Q6 (80%) and the hydrophobic L8 (70%) residues. Replacement of the positively charged residues resulted in 30-40% decrease in cellular uptake, indicating that electrostatic interactions are not the primary driving force for membrane translocation.

Communicability and thought disorder in schizophrenics and other diagnostic groups. A follow-up study.

To evaluate qualitative differences in the nature of thought disorder, the 'cloze' procedure and the Scale for the Assessment of Thought, Language and Communication were used to compare speech samples from schizophrenic, depressive, manic, schizo-affective and normal subjects at two different times. At the acute phase, thought-disordered subjects (schizophrenics, manics and schizo-affectives) were less communicable than non-thought-disordered subjects (depressives and normals). Communicability increased with remission of the more flagrant features of disturbance. Comparison of the thought-disordered diagnostic groups in the rate and pattern of remission of specific features of thought disorder indicated that factors reflecting goal-disrupted cognition distinguished the groups.

Predictability as an index of impaired verbal communication in schizophrenic and affective disorders.

The cloze procedure was used to examine predictability in speech samples from schizophrenic, depressive, manic, schizoaffective and normal subjects. Each speech sample was also rated for particular indices of thought disorder using the Andreasen Scale for the Assessment of Thought, Language and Communication. Schizophrenics were found to be less predictable than other patient groups or normals. Depressives were found to be the most predictable. Correlational analyses of cloze scores and specific thought disorder ratings suggest that reduced predictability is associated with traditional indicators of thought disorder.

Poverty of speech in schizophrenia and depression during in-patient and post-hospital periods.

Poverty of speech, a prominent feature of the negative symptom construct in schizophrenia, was assessed longitudinally in 12 schizophrenic and 13 depressed subjects at hospital admission and about seven months after discharge in order to evaluate hypotheses concerning course and diagnostic specificity. Multiple measures of the poverty of speech construct were employed, including both clinical and quantitative indices. During the in-patient period, poverty of speech was more pronounced among depressed than schizophrenic subjects. Examination of this specific negative symptom across in-patient and follow-up evaluations indicated that poverty of speech increased among schizophrenic subjects, but remained relatively stable or declined among depressed subjects. These results suggest that the processes underlying poverty of speech may differ in schizophrenia and depression.

Disease burden in HIV-associated cognitive impairment: a study of whole-brain imaging measures.

OBJECTIVE: To study whole-brain MR measures derived from diffusion tensor imaging and magnetization transfer imaging (MTI) for the in vivo assessment of cumulative neuropathologic changes in HIV and to evaluate the quantitative imaging strategies with respect to cognitive status measures including the severity of dementia and the degree of impairment in specific cognitive domains including attention, memory, constructional abilities, and motor speed. METHODS: Quantitative whole-brain measurements, including fractional anisotropy (FA), apparent diffusion coefficient (ADC), and magnetization transfer ratio (MTR), were derived from histograms and compared in HIV and control participants. Relationships between the MR and cognitive status measures were examined. RESULTS: Whole-brain FA and MTR were reduced in patients with HIV and correlated with dementia severity. Whole-brain MTR and ADC were correlated with psychomotor deficits. Evaluation of relationships between the studied MR measures indicated a correlation between ADC and MTR; FA was not correlated with either ADC or MTR. CONCLUSIONS: Findings from this investigation support the use of quantitative whole-brain MR measures for evaluation of disease burden in HIV. Reductions in whole-brain fractional anisotropy and magnetization transfer ratio (MTR) distinguished HIV and control subjects, and these measures were associated with dementia severity. Relationships were identified between whole-brain MTR and apparent diffusion coefficient and psychomotor deficits. Combining these quantitative strategies in neuroimaging examinations may provide more comprehensive information concerning ongoing changes in the brains of HIV patients.

Changes in auditory-vocal reaction times within and across experimental sessions: preliminary observations.

Changes in auditory-vocal reaction times (AVRTs) within and across experimental sessions were studied in 13 healthy university students, all females. Subjects were required to listen to a series of synthesized vowels and utter each of the vowels as soon as they heard it. The vowels were /i/, /u/, /a/, /o/, and /ae/, each presented 14 times and all presented in random order and at irregular intervals (2.5-4.5 sec). The stimuli and the instructions were prerecorded and presented to the subjects binaurally at a comfortable intensity level via headphones in an IAC booth. Each subject performed the experimental task twice, a week apart. The stimuli and the vocal responses were tape recorded and later digitized and computer analyzed. Serial analysis of successive AVRTs revealed significant intra- and intersession decreases in AVRTs in the majority of the subjects. Increases in AVRTs were also seen, but much less frequently. The implications of these findings are discussed.