Functional near-infrared spectroscopy in non-invasive neuromodulation

Non-invasive cerebral neuromodulation technologies are essential for the reorganization of cerebral neural networks,which have been widely applied in the field of central neurological diseases,such as stroke,Parkinson’s disease,and mental disorders.Although significant advances have been made in neuromodulation technologies,the identification of optimal neurostimulation paramete rs including the co rtical target,duration,and inhibition or excitation pattern is still limited due to the lack of guidance for neural circuits.Moreove r,the neural mechanism unde rlying neuromodulation for improved behavioral performance remains poorly understood.Recently,advancements in neuroimaging have provided insight into neuromodulation techniques.Functional near-infrared spectroscopy,as a novel non-invasive optical brain imaging method,can detect brain activity by measuring cerebral hemodynamics with the advantages of portability,high motion tole rance,and anti-electromagnetic interference.Coupling functional near-infra red spectroscopy with neuromodulation technologies offe rs an opportunity to monitor the cortical response,provide realtime feedbac k,and establish a closed-loop strategy integrating evaluation,feedbac k,and intervention for neurostimulation,which provides a theoretical basis for development of individualized precise neuro rehabilitation.We aimed to summarize the advantages of functional near-infra red spectroscopy and provide an ove rview of the current research on functional near-infrared spectroscopy in transcranial magnetic stimulation,transcranial electrical stimulation,neurofeedback,and braincomputer interfaces.Furthermore,the future perspectives and directions for the application of functional near-infrared spectroscopy in neuromodulation are summarized.In conclusion,functional near-infrared spectroscopy combined with neuromodulation may promote the optimization of central pellral reorganization to achieve better functional recovery form central nervous system diseases.

Epileptic brain network mechanisms and neuroimaging techniques for the brain network

Epilepsy can be defined as a dysfunction of the brain network,and each type of epilepsy involves different brain-network changes that are implicated diffe rently in the control and propagation of interictal or ictal discharges.Gaining more detailed information on brain network alterations can help us to further understand the mechanisms of epilepsy and pave the way for brain network-based precise therapeutic approaches in clinical practice.An increasing number of advanced neuroimaging techniques and electrophysiological techniques such as diffusion tensor imaging-based fiber tra ctography,diffusion kurtosis imaging-based fiber tractography,fiber ball imagingbased tra ctography,electroencephalography,functional magnetic resonance imaging,magnetoencephalography,positron emission tomography,molecular imaging,and functional ultrasound imaging have been extensively used to delineate epileptic networks.In this review,we summarize the relevant neuroimaging and neuroelectrophysiological techniques for assessing structural and functional brain networks in patients with epilepsy,and extensively analyze the imaging mechanisms,advantages,limitations,and clinical application ranges of each technique.A greater focus on emerging advanced technologies,new data analysis software,a combination of multiple techniques,and the construction of personalized virtual epilepsy models can provide a theoretical basis to better understand the brain network mechanisms of epilepsy and make surgical decisions.

Structural and functional alterations in the brains of patients with anisometropic and strabismic amblyopia:a systematic review of magnetic resonance imaging studies

Amblyopia is the most common cause of vision loss in children and can persist into adulthood in the absence of effective intervention.Previous clinical and neuroimaging studies have suggested that the neural mechanisms underlying strabismic amblyopia and anisometropic amblyopia may be different.Therefore,we performed a systematic review of magnetic resonance imaging studies investigating brain alterations in patients with these two subtypes of amblyopia;this study is registered with PROSPERO(registration ID:CRD42022349191).We searched three online databases(PubMed,EMBASE,and Web of Science) from inception to April 1,2022;39 studies with 633 patients(324patients with anisometropic amblyo pia and 309 patients with strabismic amblyopia) and 580 healthy controls met the inclusion criteria(e.g.,case-control designed,pee r-reviewed articles) and were included in this review.These studies highlighted that both strabismic amblyopia and anisometropic amblyopia patients showed reduced activation and distorted topological cortical activated maps in the striate and extrastriate co rtices during tas k-based functional magnetic resonance imaging with spatial-frequency stimulus and retinotopic representations,respectively;these may have arisen from abnormal visual experiences.Compensations for amblyopia that are reflected in enhanced spontaneous brain function have been reported in the early visual cortices in the resting state,as well as reduced functional connectivity in the dorsal pathway and structural connections in the ventral pathway in both anisometro pic amblyopia and strabismic amblyopia patients.The shared dysfunction of anisometro pic amblyopia and strabismic amblyopia patients,relative to controls,is also chara cterized by reduced spontaneous brain activity in the oculomotor co rtex,mainly involving the frontal and parietal eye fields and the cerebellu m;this may underlie the neural mechanisms of fixation instability and anomalous saccades in amblyopia.With regards to specific alterations of the two forms of amblyo pia,anisometropic amblyo pia patients suffer more microstructural impairments in the precortical pathway than strabismic amblyopia patients,as reflected by diffusion tensor imaging,and more significant dysfunction and structural loss in the ventral pathway.Strabismic amblyopia patients experience more attenuation of activation in the extrastriate co rtex than in the striate cortex when compared to anisometropic amblyopia patients.Finally,brain structural magnetic resonance imaging alterations tend to be lateralized in the adult anisometropic amblyopia patients,and the patterns of brain alterations are more limited in amblyopic adults than in childre n.In conclusion,magnetic resonance imaging studies provide important insights into the brain alterations underlying the pathophysiology of amblyopia and demonstrate common and specific alte rations in anisometropic amblyo pia and strabismic amblyopia patients;these alterations may improve our understanding of the neural mechanisms underlying amblyopia.

Brain Functional Network Changes in Patients with Poststroke Cognitive Impairment Following Acupuncture Therapy

Background: The mechanisms by which acupuncture affects poststroke cognitive impairment (PSCI) remain unclear. Objective: To investigate brain functional network (BFN) changes in patients with PSCI after acupuncture therapy. Methods: Twenty-two PSCI patients who underwent acupuncture therapy in our hospital were enrolled as research subjects. Another 14 people matched for age, sex, and education level were included in the normal control (HC) group. All the subjects underwent resting-state functional magnetic resonance imaging (rs-fMRI) scans;the PSCI patients underwent one scan before acupuncture therapy and another after. The network metric difference between PSCI patients and HCs was analyzed via the independent-sample t test, whereas the paired-sample t test was employed to analyze the network metric changes in PSCI patients before vs. after treatment. Results: Small-world network attributes were observed in both groups for sparsities between 0.1 and 0.28. Compared with the HC group, the PSCI group presented significantly lower values for the global topological properties (γ, Cp, and Eloc) of the brain;significantly greater values for the nodal attributes of betweenness centrality in the CUN. L and the HES. R, degree centrality in the SFGdor. L, PCG. L, IPL. L, and HES. R, and nodal local efficiency in the ORBsup. R, ORBsupmed. R, DCG. L, SMG. R, and TPOsup. L;and decreased degree centrality in the MFG. R, IFGoperc. R, and SOG. R. After treatment, PSCI patients presented increased degree centrality in the LING.L, LING.R, and IOG. L and nodal local efficiency in PHG. L, IOG. R, FFG. L, and the HES. L, and decreased betweenness centrality in the PCG. L and CUN. L, degree centrality in the ORBsupmed. R, and nodal local efficiency in ANG. R. Conclusion: Cognitive decline in PSCI patients may be related to BFN disorders;acupuncture therapy may modulate the topological properties of the BFNs of PSCI patients.

Recent progress in the applications of presynaptic dopaminergic positron emission tomography imaging in parkinsonism

Nowadays,presynaptic dopaminergic positron emission tomography,which assesses deficiencies in dopamine synthesis,storage,and transport,is widely utilized for early diagnosis and differential diagnosis of parkinsonism.This review provides a comprehensive summary of the latest developments in the application of presynaptic dopaminergic positron emission tomography imaging in disorders that manifest parkinsonism.We conducted a thorough literature search using reputable databases such as PubMed and Web of Science.Selection criteria involved identifying peer-reviewed articles published within the last 5 years,with emphasis on their relevance to clinical applications.The findings from these studies highlight that presynaptic dopaminergic positron emission tomography has demonstrated potential not only in diagnosing and differentiating various Parkinsonian conditions but also in assessing disease severity and predicting prognosis.Moreover,when employed in conjunction with other imaging modalities and advanced analytical methods,presynaptic dopaminergic positron emission tomography has been validated as a reliable in vivo biomarker.This validation extends to screening and exploring potential neuropathological mechanisms associated with dopaminergic depletion.In summary,the insights gained from interpreting these studies are crucial for enhancing the effectiveness of preclinical investigations and clinical trials,ultimately advancing toward the goals of neuroregeneration in parkinsonian disorders.

Synchronous measurements of prefrontal activity and pulse rate variability during online video game playing with functional near-infrared spectroscopy

Interactions between the central nervous system(CNS)and autonomic nervous system(ANS)play a crucial role in modulating perception,cognition,and emotion production.Previous studies on CNS–ANS interactions,or heart–brain coupling,have often used heart rate variability(HRV)metrics derived from electrocardiography(ECG)recordings as empirical measurements of sympathetic and parasympathetic activities.Functional near-infrared spectroscopy(fNIRS)is a functional brain imaging modality that is increasingly used in brain and cognition studies.The fNIRS signals contain frequency bands representing both neural activity oscillations and heartbeat rhythms.Therefore,fNIRS data acquired in neuroimaging studies can potentially provide a single-modality approach to measure task-induced responses in the brain and ANS synchronously,allowing analysis of CNS–ANS interactions.In this proof-of-concept study,fNIRS was used to record hemodynamic changes from the foreheads of 20 university students as they each played a round of multiplayer online battle arena(MOBA)game.From the fNIRS recordings,neural and heartbeat frequency bands were extracted to assess prefrontal activities and shortterm pulse rate variability(PRV),an approximation for short-term HRV,respectively.Under the experimental conditions used,fNIRS-derived PRV metrics showed good correlations with ECG-derived HRV golden standards,in terms of absolute measurements and video game playing(VGP)-related changes.It was also observed that,similar to previous studies on physical activity and exercise,the PRV metrics closely related to parasympathetic activities recovered slower than the PRV indicators of sympathetic activities after VGP.It is concluded that it is feasible to use fNIRS to monitor concurrent brain and ANS activations during online VGP,facilitating the understanding of VGP-related heart–brain coupling.

Functional brain imaging of gastrointestinal sensation in health and disease

It has since long been known, from everyday experience as well as from animal and human studies, that psychological processes-both affective and cognitive- exert an influence on gastrointestinal sensorimotor function. More specifically, a link between psychological factors and visceral hypersensitivity has been suggested, mainly based on research in functional gastrointestinal disorder patients. However, until recently, the exact nature of this putative relationship remained unclear, mainly due to a lack of non-invasive methods to study the （neurobiological） mechanisms underlying this relationship in non-sleeping humans. As functional brain imaging, introduced in visceral sensory neuroscience some 10 years ago, does provide a method for in vivo study of brain-gut interactions, insight into the neurobiological mechanisms underlying visceral sensation in general and the influence of psychological factors more particularly, has rapidly grown. In this article, an overview of brain imaging evidence on gastrointestinal sensation will be given, with special emphasis on the brain mechanisms underlying the interaction between affective ＆ cognitive processes and visceral sensation. First, the reciprocal neural pathways between the brain and the gut （brain- gut axis） will be briefly outlined, including brain imaging evidence in healthy volunteers. Second, functional brain imaging studies assessing the influence of psychological factors on brain processing of visceral sensation in healthy humans will be discussed in more detail. Finally, brain imaging work investigating differences in brain responses to visceral distension between healthy volunteers and functional gastrointestinal disorder patients will be highlighted.

Abnormal brain activity in adolescents with Internet addiction who attempt suicide: an assessment using functional magnetic resonance imaging

Internet addiction is associated with an increased risk of suicidal behavior and can lead to brain dysfunction among adolescents.However,whether brain dysfunction occurs in adolescents with Internet addiction who attempt suicide remains unknown.This observational cross-sectional study enrolled 41 young Internet addicts,aged from 15 to 20 years,from the Department of Psychiatry,the First Affiliated Hospital of Chongqing Medical University,China from January to May 2018.The participants included 21 individuals who attempted suicide and 20 individuals with Internet addiction without a suicidal attempt history.Brain images in the resting state were obtained by a 3.0 T magnetic resonance imaging scanner.The results showed that activity in the gyrus frontalis inferior of the right pars triangularis and the right pars opercularis was significantly increased in the suicidal attempt group compared with the non-suicidal attempt group.In the resting state,the prefrontal lobe of adolescents who had attempted suicide because of Internet addiction exhibited functional abnormalities,which may provide a new basis for studying suicide pathogenesis in Internet addicts.The study was authorized by the Ethics Committee of Chongqing Medical University,China(approval No.2017 Scientific Research Ethics(2017-157))on December 11,2017.

Functional magnetic resonance imaging and the brain: A brief review

Functional magnetic resonance imaging(fMRI) is em-ployed in many behavior analysis studies, with blood oxygen level dependent-(BOLD-) contrast imaging being the main method used to generate images. The use of BOLD-contrast imaging in f MRI has been refined over the years, for example, the inclusion of a spin echo pulse and increased magnetic strength were shown to produce better recorded images. Taking careful precautions to control variables during measurement, comparisons between different specimen groups can be illustrated by f MRI imaging using both quantitative and qualitative methods. Differences have been observed in comparisons of active and resting, developing and aging, and defective and damaged brains in various studies. However, cognitive studies using f MRI still face a number of challenges in interpretation that can only be overcome by imaging large numbers of samples. Furthermore, f MRI studies of brain cancer, lesions and other brain pathologies of both humans and animals are still to be explored.

Activation of brain areas following ankle dorsiflexion versus plantar flexion Functional magnetic resonance imaging verification

Changes in activated areas of the brain during ankle active dorsiflexion and ankle active plantar flexion were observed in six healthy subjects using functional magnetic resonance imaging. Excited areas of ankle active dorsiflexion involved the bilateral primary motor area and the primary somatosensory area, as well as the bilateral supplementary sensory area, the primary visual area, the right second visual area, and the vermis of cerebellum. Excited areas of ankle active plantar flexion included the ipsilateral supplementary motor area, the limbic system, and the contralateral corpus striatum. Fine movements of the cerebral cortex control the function of the ankle dorsiflexion to a larger extent than ankle plate flexion, and the function of ankle plate flexion is more controlled by the subcortical area.

Altered spontaneous brain activity in patients with diabetic optic neuropathy:A resting-state functional magnetic resonance imaging study using regional homogeneity

BACKGROUND Diabetes is a common chronic disease.Given the increasing incidence of diabetes,more individuals are affected by diabetic optic neuropathy(DON),which results in decreased vision.Whether DON leads to abnormalities of other visual systems,including the eye,the visual cortex,and other brain regions,remains unknown.AIM To investigate the local characteristics of spontaneous brain activity using regional homogeneity(ReHo)in patients with DON.METHODS We matched 22 patients with DON with 22 healthy controls(HCs).All subjects underwent resting-state functional magnetic resonance imaging.The ReHo technique was used to record spontaneous changes in brain activity.Receiver operating characteristic(ROC)curves were applied to differentiate between ReHo values for patients with DON and HCs.We also assessed the correlation between Hospital Anxiety and Depression Scale scores and ReHo values in DON patients using Pearson correlation analysis.RESULTS ReHo values of the right middle frontal gyrus(RMFG),left anterior cingulate(LAC),and superior frontal gyrus(SFG)/left frontal superior orbital gyrus(LFSO)were significantly lower in DON patients compared to HCs.Among these,the greatest difference was observed in the RMFG.The result of the ROC curves suggest that ReHo values in altered brain regions may help diagnose DON,and the RMFG and LAC ReHo values are more clinically relevant than SFG/LFSO.We also found that anxiety and depression scores of the DON group were extremely negatively correlated with the LAC ReHo values(r=-0.9336,P<0.0001 and r=-0.8453,P<0.0001,respectively).CONCLUSION Three different brain regions show ReHo changes in DON patients,and these changes could serve as diagnostic and/or prognostic biomarkers to further guide the prevention and treatment of DON patients.

Acupuncture at Waiguan(SJ5) and sham points influences activation of functional brain areas of ischemic stroke patients: a functional magnetic resonance imaging study

Most studies addressing the specificity of meridians and acupuncture points have focused mainly on the different neural effects of acupuncture at different points in healthy individuals. This study examined the effects of acupuncture on brain function in a pathological context. Sixteen patients with ischemic stroke were randomly assigned to true point group （true acupuncture at right Waiguan （SJ5）） and sham point group （sham acupuncture）. Results of functional magnetic resonance imaging revealed activation in right parietal lobe （Brodmann areas 7 and 19）, the right temporal lobe （Brodmann area 39）, the right limbic lobe （Brodmann area 23） and bilateral oc-cipital lobes （Brodmann area 18）. Furthermore, inhibition of bilateral frontal lobes （Brodmann area 4, 6, and 45）, right parietal lobe （Brodmann areas 1 and 5） and left temporal lobe （Brodmann area 21 ） were observed in the true point group. Activation in the precuneus of right parietal lobe （Brodmann area 7） and inhibition of the left superior frontal gyrus （Brodmann area 10） was observed in the sham group. Compared with sham acupuncture, acupuncture at Waiguan in stroke patients inhibited Brodmann area 5 on the healthy side. Results indicated that the altered specificity of sensation-associated cortex （Brodmann area 5） is possibly associated with a central mechanism of acupuncture at Waiguan for stroke patients.

Functional brain imaging studies on specificity of meridian and acupoints

At present, the specificity of meridians and acupoints has been studied using functional brain imaging techniques from many standpoints, including meridians, acupoints, and sham acupoints, as well as different meridians and acupoints, coordination of acupoints, and factors influencing meridian and acupoint specificity Preliminary experimental data have demonstrated that acupuncture at meridians and acupoints is specific with regard to brain neural information. However, research findings are contradictory, which may be related to brain functional complexity, resolution of functional brain imaging techniques, and experimental design. Future studies should further improve study method, and should strictly control experimental conditions to better analyze experimental data and acquire more beneficial data. Because of its many advantages, the functional brain imaging technique is a promising method for studying meridian and acupoint specificity.

Functional brain imaging with use of a new and powerful neuroimaging technique

Most of the information available on the human brain came from subjects who had sustained major head wounds, or who suffered from various mental disorders. By determining the extent of brain damage, and the nature of the loss of function, it was possible to infer which regions of the brain were responsible for which function. With the development of the imaging techniques of computerised tomography (CT) and magnetic resonance imaging it was possi-ble to be more specific as to the location of damage in brain injured patients. The meas-urement of the electrical signals on the scalp, arising from the synchronous firing of the neu-rons in response to a stimulus, known as elec-troencephalography (EEG), opened up new possibilities in studying brain function in nor-mal subjects. However it was the advent of the functional imaging modalities of positron emis-sion tomography (PET), single photon emission computed tomography (SPECT), functional magnetic resonance imaging (fMRI), and mag-netoencephalography (MEG) that led to a new era in the study of brain function. In this paper the mechanisms of the techniques mentioned above are outlined, together with an assess-ment of their strengths and weaknesses. Then an introduction to the Metabolism and Blood Flow in the Brain is given. This is followed by a more detailed explanation of functional MRI and how such experiments are performed.

Makeup Activates Brain Activity in Visually Impaired Persons: Evaluation by Functional Magnetic Resonance Imaging

Cosmetics are used to improve physical appearance, but the benefits may be limited to people without visual impairment. The importance of attractiveness among blind persons has not been assessed. We investigated the influence of makeup on brain activity of blind persons using functional magnetic resonance imaging (fMRI). Participants were 7 blind females (BFs) who learned to fully apply makeup and 9 mostly age-matched normally sighted females (NSFs). Brain activity was measured using fMRI before and after application of makeup and during a makeup image task in each state. In the default mode network at rest, there was no difference between the BFs and NSFs. However, a lateral visual network to the opposite side was observed in the NSFs, whereas no such network was noted in the BFs. A weak network was noted in the BFs in the occipital fusiform gyrus and temporal occipital fusiform cortex, and an extensive visual area network defect was noted. Also, activity after makeup application was significantly higher in the nucleus accumbens, pallidum, and hippocampus. Activity in the right middle cingulate gyrus, right cerebral white matter, and right anterior cingulate gyrus was higher before makeup in both BFs and NSFs, and the activity was significantly higher and more extensive in the BFs. In conclusion, applying makeup is a personally rewarding activity, even for BFs, as it strongly activates the reward system and the reward/memory system network, even in the absence of a visual area network.

Obstacles Facing the Implementation of Functional Magnetic Resonance Imaging （fMRI） of Brain in Jeddah City

fMRI （Functional Magnetic Resonance Imaging） is a relatively new technique that uses MRI （Magnetic Resonance Imaging） to measure the hemodynamic response （change in blood flow） related to neural activity in the brain. This paper aims to explore and identify the obstacles facing the implementation and applications of IMRI in radiology departments within Jeddah city by analyzing related data received by direct questionnaires and interviews with all the people working in MRI units in Jeddah city and finds that the major obstacle is lacking of awareness of fMRI among medical professionals and their training.

Functional magnetic resonance imaging evidence for activated functional brain areas following acupoint needling in the extremities

Totally three articles focusing on functional magnetic resonance imaging features of brain function in the activated brain regions of stroke patients undergoing acupuncture on the healthy limbs and healthy controls undergoing acupuncture on the lower extremities are published in three issues. We hope that our readers find these papers useful to their research.

Emotion processing in Parkinson's disease: a blood oxygenation level-dependent functional magnetic resonance imaging study

Parkinson's disease is a neurodegenerative disorder caused by loss of dopamine neurons in the substantia nigra pars compacta. Tremor, rigidity, and bradykinesia are the major symptoms of the disease. These motor impairments are often accompanied by affective and emotional dysfunctions which have been largely studied over the last decade. The aim of this study was to investigate emotional processing organization in the brain of patients with Parkinson's disease and to explore whether there are differences between recognition of different types of emotions in Parkinson's disease. We examined 18 patients with Parkinson's disease(8 men, 10 women) with no history of neurological or psychiatric comorbidities. All these patients underwent identical brain blood oxygenation level-dependent functional magnetic resonance imaging for emotion evaluation. Blood oxygenation level-dependent functional magnetic resonance imaging results revealed that the occipito-temporal cortices, insula, orbitofrontal cortex, basal ganglia, and parietal cortex which are involved in emotion processing, were activated during the functional control. Additionally, positive emotions activate larger volumes of the same anatomical entities than neutral and negative emotions. Results also revealed that Parkinson's disease associated with emotional disorders are increasingly recognized as disabling as classic motor symptoms. These findings help clinical physicians to recognize the emotional dysfunction of patients with Parkinson's disease.

Functional Imaging of Breast Tissue and Clinical Application

A novel approach to image hemoglobin concentration（△Dhb） and oxygen saturation （△Doxy） of breast tissue is presented. The scenograph of dual-wavelength （760 and 850 nm） near infrared lights through breast tissue is acquired by high sensitive charge coupled device （CCD） camera. The evaluation criterion of the difference of △Dhh and △Doxy between detected and referenced breast tissue can be obtained by a calculation formula without complicate caculation. This approach is applied to clinic detection in breast tissue. The ongoing clinical experiments indicate that malignant tumor usually exhibits characterize of ＂higher △Dhb and lower △Doxy＂, while benign lesion often shows ＂lower △Dhb and higher △Doxy＂ or other characters. So it is useful to assist the diagnosis of breast disease.

Role of functional imaging in the development and refinement of invasive neuromodulation for psychiatric disorders

Deep brain stimulation(DBS) is emerging as a pow-erful tool for the alleviation of targeted symptoms in treatment-resistant neuropsychiatric disorders. Despite the expanding use of neuropsychiatric DBS, the mecha-nisms responsible for its effects are only starting to be elucidated. Several modalities such as quantitative elec-troencephalography as well a intraoperative recordings have been utilized to attempt to understand the under-pinnings of this new treatment modality, but functional imaging appears to offer several unique advantages. Functional imaging techniques like positron emission tomography, single photon emission computed tomog-raphy and functional magnetic resonance imaging have been used to examine the effects of focal DBS on activ-ity in a distributed neural network. These investigations are critical for advancing the field of invasive neuro-modulation in a safe and effective manner, particularly in terms of defining the neuroanatomical targets and refining the stimulation protocols. The purpose of this review is to summarize the current functional neuroim-aging findings from neuropsychiatric DBS implantation for three disorders: treatment-resistant depression, obsessive-compulsive disorder, and Tourette syndrome. All of the major targets will be discussed(Nucleus ac-cumbens, anterior limb of internal capsule, subcallosal cingulate, Subthalamic nucleus, Centromedial nucleus of the thalamus-Parafasicular complex, frontal pole, and dorsolateral prefrontal cortex). We will also address some apparent inconsistencies within this literature, and suggest potential future directions for this promis-ing area.