Sarin-Induced Neuroinflammation in Mouse Brain Is Attenuated by the Caspase Inhibitor Q-VD-OPh.

Organophosphates cause hyperstimulation of the central nervous system, leading to extended seizures, convulsions, and brain damage. Sarin is a highly toxic organophosphate nerve agent that has been employed in several terrorist attacks. The prolonged toxicity of sarin may be enhanced by the neuroinflammatory response initiated by the inflammasome, caspase involvement, and generation/release of proinflammatory cytokines. Since neurodegeneration and neuroinflammation are prevalent in sarin-exposed animals, we were interested in evaluating the capacity of quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh), a pan caspase inhibitor to attenuate neuroinflammation following sarin exposure. To test this hypothesis, sarin-exposed C57BL/6 mice were treated with Q-VD-OPh or negative control quinolyl-valyl-O-methylglutamyl-[-2,6-difluorophenoxy]-methyl ketone, sacrificed at 2- and 14-day time points, followed by removal of the amygdala and hippocampus. A Bio-Rad 23-Plex cytokine analysis was completed on each tissue. The results suggest that exposure to sarin induced a dramatic increase in interleukin-1ß and 6 other cytokines and a decrease in 2 of the 23 cytokines at 2 days in the amygdala compared with controls. Q-VD-OPh attenuated these changes at the 2-day time point. At 14 days, six of these cytokines were still significantly different from controls. Hippocampus was less affected at both time points. Diazepam, a neuroprotective drug against nerve agents, caused an increase in several cytokines but did not have a synergistic effect with Q-VD-OPh. Treatment of sarin exposure with apoptosis inhibitors appears to be a worthwhile approach for further testing as a comprehensive counteragent against organophosphate exposure. SIGNIFICANCE STATEMENT: A pan inhibitor of caspases (Q-VD-OPh) was proposed as a potential antidote for sarin-induced neuroinflammation by reducing the level of inflammation via inflammasome caspase inhibition. Q-VD-OPh added at 30 minutes post-sarin exposure attenuated the inflammatory response of a number of cytokines and chemokines in the amygdala and hippocampus, two brain regions sensitive to organophosphate exposure. Apoptotic marker reduction at 2 and 14 days further supports further testing of inhibitors of apoptosis as a means to lessen extended organophosphate toxicity in the brain.

An individual data-driven virtual resection model based on epileptic network dynamics in children with intractable epilepsy: a magnetoencephalography interictal activity application.

Epilepsy surgery continues to be a recommended treatment for intractable (medication-resistant) epilepsy; however, 30-70% of epilepsy surgery patients can continue to have seizures. Surgical failures are often associated with incomplete resection or inaccurate localization of the epileptogenic zone. This retrospective study aims to improve surgical outcome through in silico testing of surgical hypotheses through a personalized computational neurosurgery model created from individualized patient's magnetoencephalography recording and MRI. The framework assesses the extent of the epileptic network and evaluates underlying spike dynamics, resulting in identification of one single brain volume as a candidate for resection. Dynamic-locked networks were utilized for virtual cortical resection. This in silico protocol was tested in a cohort of 24 paediatric patients with focal drug-resistant epilepsy who underwent epilepsy surgery. Of 24 patients who were included in the analysis, 79% (19 of 24) of the models agreed with the patient's clinical surgery outcome and 21% (5 of 24) were considered as model failures (accuracy 0.79, sensitivity 0.77, specificity 0.82). Patients with unsuccessful surgery outcome typically showed a model cluster outside of the resected cavity, while those with successful surgery showed the cluster model within the cavity. Two of the model failures showed the cluster in the vicinity of the resected tissue and either a functional disconnection or lack of precision of the magnetoencephalography-MRI overlapping could explain the results. Two other cases were seizure free for 1 year but developed late recurrence. This is the first study that provides in silico personalized protocol for epilepsy surgery planning using magnetoencephalography spike network analysis. This model could provide complementary information to the traditional pre-surgical assessment methods and increase the proportion of patients achieving seizure-free outcome from surgery.

Multi-View Kernel Learning for Identification of Disease Genes.

Gene expression data sets and protein-protein interaction (PPI) networks are two heterogeneous data sources that have been extensively studied, due to their ability to capture the co-expression patterns among genes and their topological connections. Although they depict different traits of the data, both of them tend to group co-functional genes together. This phenomenon agrees with the basic assumption of multi-view kernel learning, according to which different views of the data contain a similar inherent cluster structure. Based on this inference, a new multi-view kernel learning based disease gene identification algorithm, termed as DiGId, is put forward. A novel multi-view kernel learning approach is proposed that aims to learn a consensus kernel, which efficiently captures the heterogeneous information of individual views as well as depicts the underlying inherent cluster structure. Some low-rank constraints are imposed on the learned multi-view kernel, so that it can effectively be partitioned into k or fewer clusters. The learned joint cluster structure is used to curate a set of potential disease genes. Moreover, a novel approach is put forward to quantify the importance of each view. In order to demonstrate the effectiveness of the proposed approach in capturing the relevant information depicted by individual views, an extensive analysis is performed on four different cancer-related gene expression data sets and PPI network, considering different similarity measures.

Corrigendum to "A new pathogenic POLG variant" [Molecular Genetics and Metabolism Reports 32 (2022) 100890].

[This corrects the article DOI: 10.1016/j.ymgmr.2022.100890.].

A new pathogenic POLG variant.

POLG gene mutations are the most common causes of inherited mitochondrial disorders. The enzyme produced by this gene is responsible for the replication and repair of mitochondrial DNA. To date, around 300 pathogenic variants have been described in this gene. The resulting clinical outcomes of POLG mutations are widely variable in both phenotype and severity. There is considerable overlap in the phenotype of the so-called POLG syndromes with no clear genotype-phenotype correlation. Here we describe a newly discovered pathogenic variant in the POLG gene in a 7-year-old male that died of uncontrollable refractory status epilepticus. Genetic epilepsy panel sequencing identified two variants in the POLG gene, the common p.A467T pathological mutation and a novel p.S809R POLG variant found in trans with the p.A467T POLG that accompanied a severely reduced mitochondrial DNA level in the patient's tissues.

Scalable Non-Linear Graph Fusion for Prioritizing Cancer-Causing Genes.

In the past few decades, both gene expression data and protein-protein interaction (PPI)networks have been extensively studied, due to their ability to depict important characteristics of disease-associated genes. In this regard, the paper presents a new gene prioritization algorithm to identify and prioritize cancer-causing genes, integrating judiciously the complementary information obtained from two data sources. The proposed algorithm selects disease-causing genes by maximizing the importance of selected genes and functional similarity among them. A new quantitative index is introduced to evaluate the importance of a gene. It considers whether a gene exhibits a differential expression pattern across sick and healthy individuals, and has a strong connectivity in the PPI network, which are the important characteristics of a potential biomarker. As disease-associated genes are expected to have similar expression profiles and topological structures, a scalable non-linear graph fusion technique, termed as ScaNGraF, is proposed to learn a disease-dependent functional similarity network from the co-expression and common neighbor based similarity networks. The proposed ScaNGraF, which is based on message passing algorithm, efficiently combines the shared and complementary information provided by different data sources with significantly lower computational cost. A new measure, termed as DiCoIN, is introduced to evaluate the quality of a learned affinity network. The performance of the proposed graph fusion technique and gene selection algorithm is extensively compared with that of some existing methods, using several cancer data sets.

Association of preterm low-birth-weight infants and maternal periodontitis during pregnancy: An interventional study.

CONTEXT: The impact of periodontal disease during pregnancy and its effect on adverse pregnancy outcomes is seen in the literature. When it comes to the link of disease related to periodontium to that of adverse pregnancy outcomes, a need can arise if a significant cause-effect relationship does exist or not between them. AIM: The study was aimed to determine the association of periodontal health status in pregnant women with the occurrence of preterm low birth weight (LBW) infants in Vadodara, Gujarat. SETTINGS AND DESIGN: An interventional study with 100 patients was conducted, of which 67 participants were included in the control group and 33 participants were included in the intervention group. A total of 12 participants dropped out from the study and 88 were analyzed for the outcome. SUBJECTS AND METHODS: The Community periodontal Index of Treatment needs index was taken for all enrolled participants and then were divided into interventional group and control group. Participants in the interventional group underwent scaling and root planning. Data related to the time of delivery and weight of the baby was taken from the hospital records. STATISTICAL ANALYSIS: A comparison of baseline characteristics was made using unpaired t-test. Chi-square test was used for the analysis of intergroup comparison. The odds ratio and the relative risk calculation were also done. P ≤ 0.05 was considered for statistical significance. RESULTS: The odds ratio for both preterm and LBW were 3.86 times and 2.96, respectively. The Chi-square statistical test analysis was statistically significant for both preterm and LBW infants on the intergroup comparison. CONCLUSION: Periodontal disease can be considered as one of the risk factors for preterm LBW babies as not only the presence of disease condition causes an increase in inflammatory mediator but also the elimination of the disease condition reduces the adverse pregnancy outcomes.

Sex-Differences in Traumatic Brain Injury in the Absence of Tau in Drosophila.

Traumatic brain injuries, a leading cause of death and disability worldwide, are caused by a severe impact to the head that impairs physiological and psychological function. In addition to severity, type and brain area affected, brain injury outcome is also influenced by the biological sex of the patient. Traumatic brain injury triggers accumulation of Tau protein and the subsequent development of Tauopathies, including Alzheimer's disease and Chronic traumatic encephalopathy. Recent studies report differences in Tau network connections between healthy males and females, but the possible role of Tau in sex-dependent outcome to brain injury is unclear. Thus, we aimed to determine if Tau ablation would alleviate sex dependent outcomes in injured flies. We first assessed motor function and survival in tau knock-out flies and observed sex-differences in climbing ability, but no change in locomotor activity in either sex post-injury. Sex differences in survival time were also observed in injured tau deficient flies with a dramatically higher percent of female death within 24 h than males. Additionally, 3'mRNA-Seq studies in isolated fly brains found that tau deficient males show more gene transcript changes than females post-injury. Our results suggest that sex differences in TBI outcome and recovery are not dependent on the presence of Tau in Drosophila.

Indications for Inpatient Magnetoencephalography in Children - An Institution's Experience.

Magnetoencephalography (MEG) is recognized as a valuable non-invasive clinical method for localization of the epileptogenic zone and critical functional areas, as part of a pre-surgical evaluation for patients with pharmaco-resistant epilepsy. MEG is also useful in localizing functional areas as part of pre-surgical planning for tumor resection. MEG is usually performed in an outpatient setting, as one part of an evaluation that can include a variety of other testing modalities including 3-Tesla MRI and inpatient video-electroencephalography monitoring. In some clinical circumstances, however, completion of the MEG as an inpatient can provide crucial ictal or interictal localization data during an ongoing inpatient evaluation, in order to expedite medical or surgical planning. Despite well-established clinical indications for performing MEG in general, there are no current reports that discuss indications or considerations for completion of MEG on an inpatient basis. We conducted a retrospective institutional review of all pediatric MEGs performed between January 2012 and December 2020, and identified 34 cases where MEG was completed as an inpatient. We then reviewed all relevant medical records to determine clinical history, all associated diagnostic procedures, and subsequent treatment plans including epilepsy surgery and post-surgical outcomes. In doing so, we were able to identify five indications for completing the MEG on an inpatient basis: (1) super-refractory status epilepticus (SRSE), (2) intractable epilepsy with frequent electroclinical seizures, and/or frequent or repeated episodes of status epilepticus, (3) intractable epilepsy with infrequent epileptiform discharges on EEG or outpatient MEG, or other special circumstances necessitating inpatient monitoring for successful and safe MEG data acquisition, (4) MEG mapping of eloquent cortex or interictal spike localization in the setting of tumor resection or other urgent neurosurgical intervention, and (5) international or long-distance patients, where outpatient MEG is not possible or practical. MEG contributed to surgical decision-making in the majority of our cases (32 of 34). Our clinical experience suggests that MEG should be considered on an inpatient basis in certain clinical circumstances, where MEG data can provide essential information regarding the localization of epileptogenic activity or eloquent cortex, and be used to develop a treatment plan for surgical management of children with complicated or intractable epilepsy.

Inhibiting Mitochondrial Cytochrome c Oxidase Downregulates Gene Transcription After Traumatic Brain Injury in Drosophila.

Traumatic brain injuries (TBIs) caused by a sudden impact to the head alter behavior and impair physical and cognitive function. Besides the severity, type and area of the brain affected, the outcome of TBI is also influenced by the patient's biological sex. Previous studies reporting mitochondrial dysfunction mainly focused on exponential reactive oxygen species (ROS) generation, increased mitochondrial membrane potential, and altered mitochondrial dynamics as a key player in the outcome to brain injury. In this study, we evaluated the effect of a near-infrared (NIR) light exposure on gene expression in a Drosophila TBI model. NIR interacts with cytochrome c oxidase (COX) of the electron transport chain to reduce mitochondrial membrane potential hyperpolarization, attenuate ROS generation, and apoptosis. We subjected w 1118 male and female flies to TBI using a high-impact trauma (HIT) device and subsequently exposed the isolated fly brains to a COX-inhibitory wavelength of 750 nm for 2 hours (hr). Genome-wide 3'-mRNA-sequencing of fly brains revealed that injured w 1118 females exhibit greater changes in transcription compared to males at 1, 2, and 4 hours (hr) after TBI. Inhibiting COX by exposure to NIR downregulates gene expression in injured females but has minimal effect in injured males. Our results suggest that mitochondrial COX modulation with NIR alters gene expression in Drosophila following TBI and the response to injury and NIR exposure varies by biological sex.

The Preoperative Evaluation of Drug-Resistant Epilepsy.

Antiepileptic drugs afford good seizure control for approximately 70% of individuals with epilepsy. Epilepsy surgery is extremely helpful for appropriate individuals with drug resistance. Since antiquity, trephination was a crude and invasive technique to manage epilepsy. The late 1800s saw the advent of a more evidence-based approach with attempts to define seizure foci and determine areas of function. Seizure localization initially required direct brain stimulation during surgery before resection. Fortunately, improved knowledge of seizure semiology and advancements in preoperative investigations have enabled epilepsy specialists to better analyze the benefit of seizure reduction versus risk of functional harm. This preoperative phase and the investigative techniques used to analyze surgical candidacy will be discussed in this article.

Drosophila Exhibit Divergent Sex-Based Responses in Transcription and Motor Function After Traumatic Brain Injury.

Every year, millions of people in the US suffer brain damage from mild to severe traumatic brain injuries (TBI) that result from a sudden impact to the head. Despite TBI being a leading cause of death and disability worldwide, sex differences that contribute to varied outcomes post-injury are not extensively studied and therefore, poorly understood. In this study, we aimed to explore biological sex as a variable influencing response to TBI using Drosophila melanogaster as a model, since flies have been shown to exhibit symptoms commonly seen in other mammalian models of TBI. After inflicting TBI using the high-impact trauma device, we isolated w 1118 fly brains and assessed gene transcription changes in male and female flies at control and 1, 2, and 4 hr after TBI. Our results suggest that overall, Drosophila females show more gene transcript changes than males. Females also exhibit upregulated expression changes in immune response and mitochondrial genes across all time-points. In addition, we looked at the impact of injury on mitochondrial health and motor function in both sexes before and after injury. Although both sexes report similar changes in mitochondrial oxidation and negative geotaxis, locomotor activity appears to be more impaired in males than females. These data suggest that sex-differences not only influence the response to TBI but also contribute to varied outcomes post-injury.

A direct visuosensory cortical connectivity of the human limbic system. Dissecting the trajectory of the parieto-occipito-hypothalamic tract in the human brain using diffusion weighted tractography.

The human hypothalamus is at the center of the human limbic system anatomically and physiologically. The hypothalamus plays pivotal roles in controlling autonomic responses and instinctive behaviors such as regulating fear, aggression, learning, feeding behavior, circadian rhythm, and reproductive activities. The detailed anatomy of the pathways responsible for mediating these responses, however, is yet to be determined. The inhibitory effect of the cerebral cortex on the hypothalamus in many autonomic responses, suggests the presence of direct connection between the cortex and hypothalamic nuclei. While, there is ample information to support the cortico-hypothalamic association between the prefrontal cortex and hypothalamic nuclei, the information regarding a direct posterior cortico-hypothalamic alliance is scant. The visuosensory information may be crucial for the limbic system to regulate some of the important limbic functions. Multiple dissection animal studies revealed direct posterior cortical connectivity with the hypothalamic nuclei. However, a direct cortico-hypothalamic connectivity from the parieto-occipital cortices has not been revealed in the human brain yet. Diffusion weighted imaging (DWI) may be helpful in better visualizing the anatomy of this direct posterior cortico-limbic connectivity noninvasively in the human brain. We studied 30 healthy human subjects. Using a high-spatial and high angular resolution diffusion weighted tractography technique, for the first time, we were able to delineate and reconstruct the trajectory of the parieto-occipito-hypothalamic tract.

Mercury-induced autoimmunity: Report of two adolescent siblings with Morvan syndrome "plus" and review of the literature.

Heavy metal toxicity is a global health concern. Mercury intoxication has been implicated in the etiology and pathogenesis of autoimmune disease, including Morvan syndrome. We describe two siblings with overlapping features of distinct autoimmune syndromes following accidental exposure to elemental mercury. Morvan syndrome was the predominant clinical phenotype. In addition to the characteristic anti-leucine-rich glioma-inactivated protein 1 (LGI1) and anti-contactin-associated protein-like 2 (Caspr2) autoantibodies, glutamic acid decarboxylase 65-kilodalton isoform (GAD65), and N-type and P/Q-type voltage-gated calcium channel (VGCC) antibodies were detected. Treatment with chelation therapy, glucocorticoids, and intravenous immunoglobulin was unsuccessful, but complete resolution of symptoms was achieved following treatment with rituximab. Herein, we perform an extensive review of the literature with a focus on the emerging concepts of mercury-induced autoimmunity and the role of mercury in the etiopathogenesis of autoimmune diseases of the nervous system.

Adrenocortical carcinoma: a literature review.

Adrenocortical carcinoma (ACC) is reported to be present in 3-10% of the population with most tumors presenting as benign tumors. Most cases of ACC are a sporadic accumulation of mutations over time. However, studies show a predisposition to various genetic mutations may contribute. Research over the last couple of decades has elucidated causes of ACC to be driven by several molecular changes that include inactivation of tumor suppressor genes and activation of a myriad of different oncogenes, DNA mutations, and epigenetic changes. The widely adopted staging of ACC is by European Network of Study of Adrenal Tumors (ENSAT) due to its correlations with clinical outcomes. At the time of presentation, a detailed history taking with attention to the history of symptoms of hormonal excess and family history of possible hereditary influence is the first step of evaluation. It is followed by a thorough physical examination for evaluation of ACC. Management of ACC poses a unique challenge as it involves oncologic and endocrine issues. Except for one trial, treatment guidelines are based on retrospective studies and non-randomized trials, and therefore the level of evidence is grade II to grade IV. Personalized therapy including identifying the actionable target in each patient is the future of ACC management. The knowledge base of ACC is evolving based on the basic science and clinical trials conducted by worldwide groups such as COMITE of France, ENSAT of Europe, TCGA project and American Australian Asian Adrenal Alliance (A5). Future studies should aim at clear molecular and clinical standardization. Recommended therapeutic strategies should be prospectively recorded.

Mammalian Models of Traumatic Brain Injury and a Place for Drosophila in TBI Research.

Traumatic brain injury (TBI), caused by a sudden blow or jolt to the brain that disrupts normal function, is an emerging health epidemic with ∼2.5 million cases occurring annually in the United States that are severe enough to cause hospitalization or death. Most common causes of TBI include contact sports, vehicle crashes and domestic violence or war injuries. Injury to the central nervous system is one of the most consistent candidates for initiating the molecular and cellular cascades that result in Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Not every TBI event is alike with effects varying from person to person. The majority of people recover from mild TBI within a short period of time, but repeated incidents can have deleterious long-lasting effects which depend on factors such as the number of TBIs sustained, time till medical attention, age, gender and genetics of the individual. Despite extensive research, many questions still remain regarding diagnosis, treatment, and prevention of long-term effects from TBI as well as recovery of brain function. In this review, we present an overview of TBI pathology, discuss mammalian models for TBI and focus on current methods using Drosophila melanogaster as a model for TBI study. The relatively small brain size (∼100,000 neurons and glia), conserved neurotransmitter signaling mechanisms and sophisticated genetics of Drosophila allows for cell biological, molecular and genetic analyses that are impractical in mammalian models of TBI.

Comprehensive evaluation of microalgal based dairy effluent treatment process for clean water generation and other value added products.

The current study demonstrates a comprehensive investigation on clean water generation from raw dairy wastewater (RDW) using a robust microalgal strain, Ascochloris sp. ADW007 and its growth, biomass, and lipid productivities in outdoor conditions. Microalgal treatment studies were conducted in column photobioreactor (CPB) and flat-pate photobioreactor (FPB), where the volumetric algal biomass productivity in RDW was significantly increased in both CPB (0.284 ± 0.0017 g/L/d) and FPB (0.292 ± 0.0121 g/L/d) as compared to synthetic mediums viz., BG11 and TAP, respectively, with enhanced lipid content. Maximum lipid accumulation of 33.40% was obtained within 7 d growth. The volumetric and areal lipid productivities in CPB and FPB were 94 mg/L/d and 5.597 g/m2/d, and 98 mg/L/d and 9.754 g/m2/d, respectively. Chemiflocculation, filtration, and centrifugation techniques were employed for harvesting microalgal biomass. Among the flocculants, 0.08% (w/v) FeCl3 harvested >99% of algal cells within 5 min, while 0.03% (w/v) cetyl trimethyl ammonium bromide and 0.125% (w/v) sodium hydroxide harvested >96% of the cells in 30 and 60 min. After microalgal treatment, >80% of clean and odorless water was obtained with reduction in 94-96% of COD, 72-80% of nitrate and 80-97% of total phosphate, respectively. Highlights Utilization of 100% raw dairy wastewater without any treatment. Production of clean and odorless water for recycle and reuse. COD, nitrate and total phosphate reduction by 94-96%, 72-80%, and 80-97% after treatment. Microalgal treatment studies in simple column and flat-plate photobioreactors. Biomass and lipid production as other value added by-products.

Patient Centered Outcomes in Periodontal Treatment-An Evidenced Based Approach.

Transformation of research into clinical practice is the most challenging step in evidence based dental practice. Designing the most reliable research with applicable endpoint evaluation is very important as it can lead to successful research outcomes that can be accepted in clinical practice. In the periodontal research few accepted endpoints are used frequently as they are believed to be the gold standard in measuring the periodontal disease and the treatment outcomes. However, a wide range of endpoints used are surrogate endpoints and these endpoints have no direct correlation with the patient centered outcomes. Hence, a direct relationship of surrogate endpoints with true endpoints needs to be established. This review highlights the importance of true endpoints and challenges in implementing these in clinical research. Importance of patient's centered outcomes are also reviewed and duly discussed here. Need for conducting research which includes the true endpoints or the surrogate endpoints with clinical applicability and tangible outcomes, was also suggested in this review.

Significance and Functional Similarity for Identification of Disease Genes.

One of the most significant research issues in functional genomics is insilico identification of disease related genes. In this regard, the paper presents a new gene selection algorithm, termed as SiFS, for identification of disease genes. It integrates the information obtained from interaction network of proteins and gene expression profiles. The proposed SiFS algorithm culls out a subset of genes from microarray data as disease genes by maximizing both significance and functional similarity of the selected gene subset. Based on the gene expression profiles, the significance of a gene with respect to another gene is computed using mutual information. On the other hand, a new measure of similarity is introduced to compute the functional similarity between two genes. Information derived from the protein-protein interaction network forms the basis of the proposed SiFS algorithm. The performance of the proposed gene selection algorithm and new similarity measure, is compared with that of other related methods and similarity measures, using several cancer microarray data sets.

Lip reposition surgery: A new call in periodontics.

"Gummy smile" is a major concern for a large number of patients visiting the dentist. Esthetics has now become an integral part of periodontal treatment plan. This article presents a case of a gummy smile in which esthetic correction was achieved through periodontal plastic surgical procedure wherein a 10-12 mm of partial-thickness flap was dissected apical to mucogingival junction followed by approximation of the flaps. This novel technique gave excellent post-operative results with enormous patient satisfaction. This surgical chair-side procedure being one of its kinds with outstanding results is very rarely performed by Periodontists. Thus, a lot of clinical work and literature review with this surgical technique is required. To make it a routine surgical procedure this technique can be incorporated as a part of periodontal plastic surgery in the text. Hence, we have put forward experience of a case with critical analysis of the surgical technique including the limitations of the technique.