New insights into the immunologic role of oligodendrocyte lineage cells in demyelination diseases

Oligodendrocyte lineage cells(OL-lineage cells)are a cell population that are crucial for mammalian central nervous system(CNS)myelination.OL-lineage cells go through developmental stages,initially differentiating into oligodendrocyte precursor cells(OPCs),before becoming immature oligodendrocytes,then mature oligodendrocytes(OLs).While the main function of cell lineage is in myelin formation,and increasing number of studies have turned to explore the immunological characteristics of these cells.Initially,these studies focused on discovering how OPCs and OLs are affected by the immune system,and then,how these immunological changes influence the myelination process.However,recent studies have uncovered another feature of OL-lineage cells in our immune systems.It would appear that OL-lineage cells also express immunological factors such as cytokines and chemokines in response to immune activation,and the expression of these factors changes under various pathologic conditions.Evidence suggests that OL-lineage cells actually modulate immune functions.Indeed,OL-lineage cells appear to play both"victim"and"agent"in the CNS which raises a number of questions.Here,we summarize immunologic changes in OL-lineage cells and their effects,as well as consider OL-lineage cell changes which influence immune cells under pathological conditions.We also describe some of the underlying mechanisms of these changes and their effects.Finally,we describe several studies which use OL-lineage cells as immunotherapeutic targets for demyelination diseases.

Anodal transcranial direct current stimulation alleviates cognitive impairment in an APP/PS1 model of Alzheimer’s disease in the preclinical stage

Anodal transcranial direct current stimulation(AtDCS)has been shown to alleviate cognitive impairment in an APP/PS1 model of Alzheimer’s disease in the preclinical stage.However,this enhancement was only observed immediately after AtDCS,and the long-term effect of AtDCS remains unknown.In this study,we treated 26-week-old mouse models of Alzheimer’s disease in the preclinical stage with 10 AtDCS sessions or sham stimulation.The Morris water maze,novel object recognition task,and novel object location test were implemented to evaluate spatial learning memory and recognition memory of mice.Western blotting was used to detect the relevant protein content.Morphological changes were observed using immunohistochemistry and immunofluorescence staining.Six weeks after treatment,the mice subjected to AtDCS sessions had a shorter escape latency,a shorter path length,more platform area crossings,and spent more time in the target quadrant than sham-stimulated mice.The mice subjected to AtDCS sessions also performed better in the novel object recognition and novel object location tests than sham-stimulated mice.Furthermore,AtDCS reduced the levels of amyloid-β42 and glial fibrillary acidic protein,a marker of astrocyte activation,and increased the level of neuronal marker NeuN in hippocampal tissue.These findings suggest that AtDCS can improve the spatial learning and memory abilities and pathological state of an APP/PS1 mouse model of Alzheimer’s disease in the preclinical stage,with improvements that last for at least 6 weeks.

Dynamic changes of glial fibrillary acidic protein and nestin in the hippocampus of adult rat brain following ischemic vascular dementia

Vascular dementia produced by permanent ligation of bilateral common carotid arteries involves progressive deterioration of intellectual and cognitive function in rats, which are closely associated with the hippocampus. This study used immunohistochemical analysis to detect the expression of glial fibrillary acidic protein and nestin in the hippocampus in a vascular dementia model. The results revealed that both glial fibrillary acidic protein and nestin expression were increased 1 day after permanent ligation of the bilateral common carotid arteries, compared with a sham-operated group. The expression of glial fibrillary acidic protein peaked at 7 days post-surgery. The expression of nestin was a little weaker than that of glial fibrillary acidic protein, and peaked at 14 days （P 〈 0.01）. The expression of both proteins slightly decreased at 21 and 28 days, accompanied by recovery of cerebral blood flow. In conclusion, this study demonstrated that glial fibrillary acidic protein and nestin exhibited dynamic expression in the rat hippocampus after permanent ligation of bilateral common carotid arteries. This finding suggests that dynamic alterations in protein expression play an important role in the pathogenesis of vascular dementia.

Depressed female cynomolgus monkeys(Macaca fascicularis) display a higher second-to-fourth(2D:4D)digit ratio

This research aimed to provide evidenee of a relationship between digit ratio and depression status in the cynomolgus monkey(Macaca fascicularis).In stable cyno molgus mon key social groups,we selected 15 depressed monkeys based on depressive-like behavioral criteria and 16 normal control mon keys.All animals were video recorded for two weeks,with the duration and frequency of the core depressive behaviors and 58 other behaviors in 12 behavioral categories then evaluated via behavioral analysis.Fin ger len gths from the right and left forelimb hands of both groups were measured by X-ray imagi ng.Fin ger length and digit ratio comparisons between the two groups were con ducted using Stude nt's Mest.In terms of the durati on of each behavior,signifies nt differences emerged in“Huddling”and five other behavioral categories,including Ingestive,Amicable,Parental,Locomotive,and Resting.In addition to the above five behavioral categories,we found that depressed mon keys spent less time in parental and rubbing back and forth behaviors than the control group.Furthermore,the 4th fin gers were significantly Ion ger in the left and right hands in the control group relative to the depressed mon keys.The sec ond?to?fourth(2D:4D)digit ratio in the left and right forelimb hands was significantly lower in the control group tha n that in the depressed group.Our fin dings revealed significant differences in finger lengths and digit ratios between depressed mon keys and healthy controls,which concords with our view that relatively high fetal testosterone exposure may be a protective factor against developing depressive symptoms(or that low fetal testosterone exposure is a risk factor).

Clinic Pathological Study of an Eccrine Spiradenoma

Eccrine Spiradenoma (ES) is an exceedingly rare sweat-gland tumor, it usually presents as a solitary lesion and painful nodule. ES is a kind of neoplasm with distinct histological characteristics and nonspecific clinical manifestations. Most ES cases have a benign course;however, malignant transformation would occur after a long period of latency. The diagnosis mostly depends on the clinic symptom, histological features and immunohistochemistry. Here, we report a case of ES and literature review. The aim of this study is to understand clinic and histological features for ES.

The Expression of PI3K, AKT, β-Catenin and VEGFR2 in Medulloblastoma

Medulloblastoma (MB) is common tumor of the central nervous system in children. It’s reported that PI3K/AKT and Wnt signal pathway have important roles in MB. This study aims to investigate the expression of PI3K, AKT, Β-catenin and VEGFR-2 in MB to find a new pathway for MB. A total of 33 MB and 17 control brain cases were retrospectively evaluate d for PI3K, AKT, β-catenin and VEGFR-2 expression by immunohistochemical staining, and the relationship with clinical feature were analyzed. The positive rate of PI3K, AKT, β-catenin and VEGFR-2 in 33 MB were significantly greater than those in control group

FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury

Background:Neutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury(TBI).However,the heterogeneity,multifunctionality,and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood.Methods:Using the combined single-cell transcriptomics,metabolomics,and proteomics analysis from TBI patients and the TBI mouse model,we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests.We also characterized the underlying mechanisms both invitro and invivo through molecular simulations,signaling detections,gene expression regulation assessments[including dual-luciferase reporter and chromatin immunoprecipitation(ChIP)assays],primary cultures or co-cultures of neutrophils and oligodendrocytes,intracellular iron,and lipid hydroperoxide concentration measurements,as well as forkhead box protein O1(FOXO1)conditional knockout mice.Results:We identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model.Infiltration of these FOXO1high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI,aggravating acute brain inflammatory damage and promoting late TBI-induced depression.In the acute stage,FOXO1 upregulated cytoplasmic Versican(VCAN)to interact with the apoptosis regulator B-cell lymphoma-2(BCL-2)-associated X protein(BAX),suppressing the mitochondrial translocation of BAX,which mediated the antiapoptotic effect companied with enhancing interleukin-6(IL-6)production of FOXO1high neutrophils.In the chronic stage,the“FOXO1-transferrin receptor(TFRC)”mechanism contributes to FOXO1high neutrophil ferroptosis,disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein,which contributes to the progression of late depression after TBI.Conclusions:FOXO1high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI,which provides insight into the heterogeneity,reprogramming activity,and versatility of neutrophils in TBI.

Combined treatment with valproic acid and estrogen has neuroprotective effects in ovariectomized mice with Alzheimer’s disease

Postmenopausal women with Alzheimer’s disease exhibit dramatically reduced sensitivity to estrogen replacement therapy,which is though to be related to an estrogen receptor(ER)α/ERβratio imbalance arising from a significantly decreased level of ERs of the brain.The aim of our study was to investigate whether valproic acid(VPA)can enhance the beneficial effects of estrogen on cognitive function through restoration of ERαand ERβexpression in the brain.We removed the ovaries of female APP/PS1 mice to simulate the low estrogen levels present in postmenopausal women and then administered VPA(30 mg/kg,intraperitoneal injection,once daily),17β-estradiol(E2)(2.4μg,intraperitoneal injection,once daily),liquiritigenin(LG)(50μg/kg,intragastric infusion,once daily),VPA+E2,or VPA+LG for 4 successive weeks.Compared with treatment with a single drug,treatment with VPA+E2 or VPA+LG significantly increased the level of glycogen synthase kinase 3β,increased the expression of estrogen receptorα,reduced the expression of small ubiquitin-like modifiers,and increased the level of estrogen receptorβ.This resulted in enhanced sensitivity to estrogen therapy,reduced amyloidβaggregation,reduced abnormal phosphorylation of the tau protein,reduced neuronal loss,increased dendritic spine and postsynaptic density,and significantly alleviated memory loss and learning impairment in Alzheimer’s disease.This study was approved by the Chongqing Medical University Animal Protection and Ethics Committee,China on March 6,2013.

Angioplasty and stenting for severe vertebral artery orifice stenosis: effects on cerebellar function remodeling verified by blood oxygen level-dependent functional magnetic resonance imaging

Vertebral artery orifice stenting may improve blood supply of the posterior circulation of the brain to regions such as the cerebellum and brainstem. However, previous studies have mainly focused on recovery of cerebral blood flow and perfusion in the posterior circulation after interventional therapy. This study examined the effects of functional recovery of local brain tissue on cerebellar function remodeling using blood oxygen level-dependent functional magnetic reso- nance imaging before and after interventional therapy. A total of 40 Chinese patients with severe unilateral vertebral artery orifice stenosis were enrolled in this study. Patients were equally and randomly assigned to intervention and control groups. The control group received drug treat- ment only. The intervention group received vertebral artery orifice angioplasty and stenting ＋ identical drug treatment to the control group. At 13 days after treatment, the Dizziness Handicap Inventory score was compared between the intervention and control groups. Cerebellar function remodeling was observed between the two groups using blood oxygen level-dependent functional magnetic resonance imaging. The improvement in dizziness handicap and cerebellar function was more obvious in the intervention group than in the control group. Interventional therapy for severe vertebral artery orifice stenosis may effectively promote cerebellar function remodeling and exert neuroprotective effects.

Alterations in expression and localization of POMGNT1 in the APP/PS1 mouse model of Alzheimer’s disease

Alzheimer’s disease(AD)is a complex and progressive neurodegenerative disorder that causes dementia in the aging population.One of the characteristic pathologic hallmarks of AD is the senile plaques,composed of the accumulation of b-amyloid.Neurotoxic b-amyloid results from the cleavage of transmembrane b-amyloid precursor protein(APP)by b-secretases and g-secretases,sequentially.O-mannosylation was found essential for the normal function of APP in Saccharomyces cerevisiae.1 Protein Olinked mannose b1,2-N-acetyl-glucosaminyltransferase 1(POMGNT1)is a glycosyltransferase crucial for the elongation of O-mannosyl glycans and catalyzes the transfer of Nacetylglucosamine from uridine 50-diphosphate-N-acetylglucosamine to O-mannose of glycoproteins.

Structural Asymmetry of Dorsolateral Prefrontal Cortex Correlates with Depressive Symptoms： Evidence from Healthy Individuals and Patients with Major Depressive Disorder

In this study,we investigated the role of structural asymmetry of the dorsolateral prefrontal cortex（DLPFC） in the continuum of depression from healthy individuals to patients.Structural magnetic resonance imaging was performed in 70 patients with major depressive disorder（MDD）,49 matched controls,and 349 healthy university students to calculate structural asymmetry indexes of the DLPFC.First-episode,treatment-naive MDD patients showed a relatively lower asymmetry index than healthy controls,and their asymmetry index was negatively correlated with the depressive symptoms.This abnormality was normalized by antidepressants in medicated MDD patients.Furthermore,the asymmetry index was negatively correlated with the depressive symptoms in university students;this was replicated at two time points in a subgroup of students,suggesting good test-retest reliability.Our findings are consistent with previous studiesthat support the imbalance hypothesis of MDD and suggest a potential structural basis underlying the functional asymmetry of the DLPFC in depression.In future,the structural index of the DLPFC may become a potential biomarker to evaluate individuals＇ risk for the onset of MDD.

A multiple-reaction-monitoring mass spectrometric method for simultaneous quantitative analysis of five plasma apolipoproteins

A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysregulated in neuropsychiatric disorders and cognitive dysfunction:apolipoprotein H(APOH),apolipoprotein J(APOJ),apolipoprotein A4(APOA4),apolipoprotein E(APOE),and apolipoprotein D(APOD).The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOFTM 5600,followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500.Our results showed that the collision energies of mTRAQ-labeled peptides were approximately 15%–20%higher than corresponding non-labeled peptides.Through optimized transitions and parameters,we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins.The results indicated that the MRM signals of four target APOs were significantly increased after depletion,while the MRM signal of one APO,APOD,was decreased.Furthermore,the relative abundances of the five target APOs in healthy human plasma were stable,and the ranking of these proteins according to their MS responses changed slightly.Therefore,we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma.

Clemastine rescues behavioral changes and enhances remyelination in the cuprizone mouse model of demyelination

Increasing evidence suggests that white matter disorders based on myelin sheath impairment may underlie the neuropathological changes in schizophrenia.But it is unknown whether enhancing remyelination is a beneficial approach to schizophrenia.To investigate this hypothesis,we used clemastine,an FDA-approved drug with high potency in promoting oligodendroglial differentiation and myelination,on a cuprizone-induced mouse model of demyelination.The mice exposed to cuprizone（0.2%in chow） for 6 weeks displayed schizophrenia-like behavioral changes,including decreased exploration of the center in the open field test and increased entries into the arms of the Y-maze,as well as evident demyelination in the cortex and corpus callosum.Clemastine treatment was initiated upon cuprizone withdrawal at 10 mg/kg per day for3 weeks.As expected,myelin repair was greatly enhanced in the demyelinated regions with increased mature oligodendrocytes（APC-positive） and myelin basic protein.More importantly,the clemastine treatment rescued the schizophrenia-like behavioral changes in the open field test and the Y-maze compared to vehicle,suggesting a beneficial effect via promoting myelin repair.Our findings indicate that enhancing remyelination may be a potential therapy for schizophrenia.

Versatile flexible micelles integrating mucosal penetration and intestinal targeting for effectively oral delivery of paclitaxel

The extremely low bioavailability of oral paclitaxel(PTX)mainly due to the complicated gastrointestinal environment,the obstruction of intestinal mucus layer and epithelium barrier.Thus,it is of great significance to construct a coordinative delivery system which can overcome multiple intestinal physicochemical obstacles simultaneously.In this work,a high-density PEGylation-based glycocholic acid-decorated micelles(PTX@GNPs)was constructed by a novel polymer,9-Fluorenylmethoxy carbonyl-poly ethylene glycocholic acid(Fmoc-PEG-GCA).The Fmoc motif in this polymer could encapsulate PTX viaπ-πstacking to form the core of micelles,and the low molecular weight and non-long hydrophobic chain of Fmoc ensures the high-density of PEG.Based on this versatile and flexible carriers,PTX@GNPs possess mucus trapping escape ability due to the flexible PEG,and excellent intestine epithelium targeting attributed to the high affinity of GCA with apical sodium-dependent bile acid transporter.The in vitro and in vivo results showed that this oral micelle could enhance oral bioavailability of PTX,and exhibited similar antitumor efficacy to Taxol injection via intravenous route.In addition,oral PTX@GNPs administered with lower dosage within shorter interval could increase in vivo retention time of PTX,which supposed to remodel immune microenvironment and enhance oral chemotherapy efficacy by synergistic effect.

Structure,function,and pathology of Neurexin-3

Neurexin-3 is primarily localized in the presynaptic membrane and forms complexes with various ligands located in the postsynaptic membrane.Neurexin-3 has important roles in synapse development and synapse functions.Neurexin-3 mediates excitatory presynaptic differentiation by interacting with leucine-rich-repeat transmembrane neuronal proteins.Meanwhile,neurexin-3 modulates the expression of presynapticα-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors andγ-aminobutyric acid A receptors by interacting with neuroligins at excitatory and inhibitory synapses.Numerous studies have documented the potential contribution of neurexin-3 to neurodegenerative and neuropsychiatric disorders,such as Alzheimer's disease,addiction behaviors,and other diseases,which raises hopes that understanding the mechanisms of neurexin-3 may hold the key to developing new strategies for related illnesses.This review comprehensively covers the literature to provide current knowledge of the structure,function,and clinical role of neurexin-3.

Comparative proteomic analysis of plasma from bipolar depression and depressive disorder:identification of proteins associated with immune regulatory

Bipolar disorder(BD)is a debilitating psychiatric mood dis-order affecting approximately 1%-3%of the population worldwide(Merikangas,2007).Bipolar disorder is characterized by recurrent episodes of depression,hypomania,mania,or mixed states,and it has a poor outcome,with high rates of relapse,lingering residual symptoms,cognitive impairment,and functional impairment(Moreno et al.,2007)Although various etiopathological hypotheses concerning the disease have been reported,the pathophysiology underlying BD remains poody understood(Gawryluk and Young,2011).

The DIRECT consortium and the REST-meta-MDD project:towards neuroimaging biomarkers of major depressive disorder

Despite a growing neuroimaging literature on the pathophysiology of major depressive disorder(MDD),repro-ducible findings are lacking,probably reflecting mostly small sample sizes and heterogeneity in analytic approaches.To address these issues,the Depression Imaging REsearch ConsorTium(DIRECT)was launched.The REST-meta-MDD project,pooling 2428 functional brain images processed with a standardized pipeline across all participating sites,has been the first effort from DIRECT.In this review,we present an overview of the moti-vations,rationale,and principal findings of the studies so far from the REST-meta-MDD project.Findings from the first round of analyses of the pooled repository have included alterations in functional connectivity within the default mode network,in whole-brain topological properties,in dynamic features,and in functional lat-eralization.These well-powered exploratory observations have also provided the basis for future longitudinal hypothesis-driven research.Following these fruitful explorations,DIRECT has proceeded to its second stage of data sharing that seeks to examine ethnicity in brain alterations in MDD by extending the exclusive Chinese original sample to other ethnic groups through international collaborations.A state-of-the-art,surface-based preprocessing pipeline has also been introduced to improve sensitivity.Functional images from patients with bipolar disorder and schizophrenia will be included to identify shared and unique abnormalities across diag-nosis boundaries.In addition,large-scale longitudinal studies targeting brain network alterations following antidepressant treatment,aggregation of diffusion tensor images,and the development of functional magnetic resonance imaging-guided neuromodulation approaches are underway.Through these endeavours,we hope to accelerate the translation of functional neuroimaging findings to clinical use,such as evaluating longitudinal effects of antidepressant medications and developing individualized neuromodulation targets,while building an open repository for the scientific community.

A New Acquaintance of Oligodendrocyte Precursor Cells in the Central Nervous System

Oligodendrocyte precursor cells(OPCs)are a heterogeneous multipotent population in the central nervous system(CNS)that appear during embryogenesis and persist as resident cells in the adult brain parenchyma.OPCs could generate oligodendrocytes to participate in myelination.Recent advances have renewed our knowledge of OPC biology by discovering novel markers of oligodendroglial cells,the myelin-independent roles of OPCs,and the regulatory mechanism of OPC development.In this review,we will explore the updated knowledge on OPC identity,their multifaceted roles in the CNS in health and diseases,as well as the regulatory mechanisms that are involved in their developmental stages,which hopefully would contribute to a further understanding of OPCs and attract attention in the field of OPC biology.

Resveratrol provides neuroprotection by regulating the JAK2/STAT3/PI3K/AKT/mTOR pathway after stroke in rats

Ischemic stroke is a common disease with high mortality and morbidity worldwide.One of the important pathophysiological effects of ischemic stroke is apoptosis.A neuroprotective effect is defined as the inhibition of neuronal apoptosis to rescue or delay the infarction in the surviving ischemic penumbra.Resveratrol is a natural polyphenol that reportedly prevents cerebral ischemia injury by regulating the expression of PI3K/AKT/mTOR.Therefore,this study aimed to elucidate the neuroprotective effect of resveratrol on cerebral ischemia/reperfusion injury and to investigate the signaling pathways and mechanisms through which resveratrol regulates apoptosis in the ischemic penumbra.Rats were subjected to middle cerebral artery occlusion for 2 h followed by 24 h reperfusion.Cerebral infarct volume was measured using 2%TTC staining.TUNEL staining was conducted to evaluate neuronal apoptosis.Western blotting and immunohistochemistry were used to detect the proteins involved in the JAK2/STAT3/PI3K/AKT/mTOR pathway.The results suggested that resveratrol significantly improved neurological function,reduced cerebral infarct volume,decreased neuronal damage,and markedly attenuated neuronal apoptosis;these effects were attenuated by the inhibition of PI3K/AKT with LY294002 and JAK2/STAT3 with AG490.We also found that resveratrol significantly upregulated the expression of p-JAK2,p-STAT3,p-AKT,p-mTOR,and BCL-2 and downregulated expression of cleaved caspase-3 and BAX,which was partially reversed by LY294002 and AG490.These results suggested that resveratrol provides a neuroprotective effect against cerebral ischemia/reperfusion injury,which is partially mediated by the activation of JAK2/STAT3 and PI3K/AKT/mTOR.Resveratrol may indirectly upregulate the PI3K/AKT/mTOR pathway by activating JAK2/STAT3.

Glomerular Filtration Rate is Associated with Hemorrhagic Transformation in Acute Ischemic Stroke Patients without Thrombolytic Therapy

Background： Whether there is a relationship between glomerular filtration rate IGFR） and hcnlorrhagic transformation （HT） after acute ischemic stroke （A1S） is still under debate. The aim of our study was to determine whether the GFR level is a predictor of HT in AIS patients without thrombolytic therapy （TT）. Methods： Consecutive A IS patients without TT were included in this prospective study fiom January 2014 to December 2016 in the First Affiliated Hospital of Chongqiug Medical University. We divided them into two groups （HT and non-HT group） and meticulously collected baseline characteristics and laboratory and imaging data of interested individuals. Multivariate regression analysis was performed to assess the correlation between GFR and HT in stroke patients without TT. Results： Among 426 consecutive patients, 74 （17.3%） presented HT （mean age： 65 ± 12 years, number of male patients： 47） on the follow-up scans. In multivariate regression analysis, HT was significantly associated with low GFR （odds ratio [OR] = 3.708, confidence interval [CI] = 1.326-10.693, P = 0.013）, atrial fibrillation （AF; OR = 2.444, CI= 1.087-5.356, P = 0.027）, large cerebral inthrction （OR = 2.583, CI= 1.236-5.262, P= 0.010）, and hypoalbuminemia （HA OR = 4.814, CI= 1.054 22.153, P = 0.037） for AIS patients without TT. Conclusions： The present study strongly showed that lower GFR is an independently predictor of HT： in addition, large inlhrct volume, AF, and HA are also important risks of HT for AIS patients without TT, which offered a practical information that risk factors should be paid attention or eliminated to prevent HT for stroke patients though the level of evidence seems to be unstable.