RESULTS

1.Double inactivation of Xrcc1 and Atm in the central nervous system

Deletion of theXrcc1 gene increased the number of endogenous DNA Single strand breaks(Khoronenkova and Dianov, 2015). To determine the role of ATM upon DNA damage during brain development, we generatedXrcc1 and Atm double knockout mice.Xrcc1 germline knockout mice showed early embryonic lethality before brain development (Tebbs et al., 1999). To resolve this, we used a Nestin-Cre system to generate Xrcc1conditional knockout mice which induce tissue specific DNA damage, particularlyin the central nervous system during development.This Xrcc1conditional knockout mouse modelhasbeen previously described (Lee et al., 2009). Xrcc1conditionally targeted mice (hereafter referred to as Xrcc1^nes-cre) were crossbred with Atm germline knockout mice (hereafter referred to as Atm^-/-) (Herzog et al., 1998)(Figure 1). Generated Xrcc1 and Atm double knockout mice (hereafter referred to as Xrcc1^nes-cre; Atm^-/-) died beforepostnatal week 3. The genotype of mouse in these experimentswas confirmed at the DNA level (Figure 2A). Xrcc1^nes-cre; Atm -/-mice had smaller brain than those of littermates of WT and Atm^-/- especially the cerebellum (Figure 2B). Gross behavior observation indicated that unlike WT and Atm^-/- mice, Xrcc1

nes-creanimals showed mild ataxia with episodic spasms as previously reported(Lee et al., 2009).

StrikinglyXrcc1^nes-cre; Atm^-/- animals showed severe ataxia and exacerbated neurological phenotypeswhich were not observed either in Xrcc1^nes-creor Atm^-/-animals (Figure. 2C).

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Figure 1. Strategy of generatingXrcc1 and Atm knockout mouse model.

(A) The conditional targeting construct for the Xrcc1 gene was engineered with loxP sites flanking exons 4-10. The Atm germline targeting construct was engineered with Neo gene to interrupt exon 57 and replace a part of exon 58 of the Atm gene. The targeted region is the phosphoinositol 3-kinase domain (PI3K domain) which is catalytic activity domain of Atm.

(B) To inactivate the Xrcc1 and Atmgene in the nervous system, first we gained the Xrcc1^loxP/+;Atm^+/- mice. And then Xrcc1^loxP/loxP;Atm^-/-;Nestin-Cre⁺mice were obtained bybreeding male Xrcc1^loxP/+;Atm^+/-with femaleXrcc1^loxP/+;Atm^+/-;Nestin-Cre⁺mice.We maintained Nestin-Crein female mice to excludeectopic Cre recombinase activity.

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Figure 2. Generation of an Xrcc1 and Atm double knockout mouse.

(A) PCR based genotyping showed that Xrcc1 and Atm double knockout mouse has a LoxP floxed Xrcc1, Atm neo-cassette (Atm mutant) and Cre recombinase alleles. (B) Small brains were found in postnatal day 15 (P15)Xrcc1^Nes-Creanimals and much smaller brains in Xrcc1

Nes-Cre; Atm^-/- mice compared with those of WT and Atm^-/- mice. (C) Comparative view of WT and Xrcc1^Nes-Cre; Atm^-/- mice. Xrcc1^Nes-Cre; Atm^-/- mice (red arrows) showed exacerbated neurological behavior phenotypes such as progressive severe ataxia.

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2. Loss of Xrcc1 and Atm results in DNA repair deficiency in the brain

Xrcc1 and ATM are critical for DNA single strand break repair and double strand break repair respectively. To estimate the role of Xrcc1 and Atmin repairing DNA damage in the nervous system, we analyzed the brains of Xrcc1^nes-cre; Atm^-/-mice for the accumulation of DNA strand breaks. We used gH2AXas a marker for DNA damage that could be detected as foci formation in the nucleus. gH2AXis the phosphorylatedform of histone H2AXthatis accumulated at the sites of DNA strand breaks to act as a sensor for DNA damage and to initiate DNA damage repair responses(Rogakou et al., 1998).Although gH2AX typically involves in DNA double strand breaks, DNA single strand breaks by Xrcc1 deficiency can be converted to DNA double strand break that arise during replication or random damage accumulation leading to adjacent breaks.

During early neural development,there was widespread accumulation of DNA damage throughout theXrcc1^nes-cre; Atm^-/-mouse brain more than that of the Xrcc1^nes-cre brain.gH2AXfoci formation occurred in proliferating cells in the various areas of both Xrcc1^nes-creand Xrcc1^nes-cre; Atm^-/- mice brain.WhenXrcc1^nes-creand Xrcc1^nes-cre; Atm^-/- animals were compared,the Xrcc1^nes-cre; Atm^-/-brain showedmore accumulation of gH2AX signal than the Xrcc1^nes-crebrain, suggesting that Xrcc1^nes-cre; Atm^-/- animals faced increased challenge to DNA damage and by inactivating Atm, the load of DNA damage was affected (Figure 3A,B).

Moreover, these DNA strand breakswas not repaired until proliferating cells becoming post-mitotic differentiated cells.WT andAtm^-/-mice brain did not show any noticeable gH2AX positive signal besides small gH2AX foci that occur during normal proliferation (data not

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shown).This sign of DNA damage accumulation was also confirmed with 53BP1 foci;

another DNA damage marker (Figure 3A). 53BP1 is one of many early responding proteins to DNA damage which is phosphorylated upon DNA damage, and could be detected as foci formation in the nucleus (Dimitrova et al., 2008).

3. Cerebellar interneurons are not rescued inthe Xrcc1^nes-cre; Atm^-/-brain

To analyze the function of ATMformaintenance of genomic stability during brain development,we had surveyed the Xrcc1^nes-cre; Atm^-/- mouse brain using various markers of differentiated neural cells. In the previous study, Xrcc1^nes-crebrains showed profound and widespread loss of cerebellar interneurons in the white matter (Lee et al., 2009). Interneuron progenitors of the Xrcc1^nes-crecerebellumunderwent DNA damage induced p53 dependent cell cycle arrest rather than apoptosis resulting in absence of interneurons. To confirm this, theXrcc1^nes-cre; p53^-/-mouse had been generated and rescue of interneuron loss in the cerebellum was observed. p53 wasidentified as the first substrate of ATM in vitro and in vivo(Banin et al., 1998; Canman et al., 1998; Khanna et al., 1998).DNA damage triggers several signal transduction pathways that lead either to damage repair coupled with attenuation of cell cycle arrest, or to programmed cell death (apoptosis) (Choi et al., 2012;

Sullivan et al., 2012). Upon DNA damage, p53 is stabilized and activated through various posttranslational modifications including phosphorylation by ATM(Banin et al., 1998).

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Figure 3. The Xrcc1^Nes-Cre;Atm^-/-deficiency accumulates DNA damage during brain development. (A-B) The embryonic day 15.5 (E15.5) cerebellum (A) and cerebral cortex (CTX) (B) from Xrcc1^Nes-Creand Xrcc1^Nes-Cre;Atm^-/- mice. Endogenous DNA damage was accumulated more in proliferating cells of the Xrcc1^Nes-Cre;Atm^-/-developing brain, as shown by γH2AX and 53BP1 foci formation in the nucleus. Nuclei were stained with DAPI. VZ, Ventricular Zone; EGL. External granule cell layer; RL, rhombic lip; CP, cortical plate.

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Thus, we had speculatedthatXrcc1 and Atm deficiency also rescued p53-dependent cell-cycle arrest in the cerebellar interneurons similar toXrcc1^nes-cre; p53^-/-cerebellar phenotype.

Histopathological analysis of both the Xrcc1^nes-cre and Xrcc1^nes-cre; Atm^-/-cerebellum showed the dramatic reduction in size and disruption of the cellular architecture of the cerebellum in the Xrcc1^nes-cre; Atm^-/-animals (Figure 4A). Furthermore, there was no sign of proper interneuron genesis in the Xrcc1^nes-cre; Atm^-/-cerebellum.So in contrast tothe Xrcc1^nes-cre; p53 -/-cerebellum, cerebellar interneurons were not rescued in the Xrcc1^nes-cre; Atm^-/-cerebellum.

These interneurons are critical for attenuating the output signal of granule and Purkinje cell thereby maintaining the balance of electrical activity between cerebellar neurons (Barmack and Yakhnitsa, 2008). The cerebellumcontains five different types of interneurons: the Stellate cell, Basket cell, Golgi cell, Lugaro cell and Unipolar brush cell(Harriman; John C.

Eccles, 1967; Voogd and Glickstein, 1998; Ramnani, 2006).The Stellate and Basket cells are inhibitory GABAergic interneurons, found in the molecular layer of the cerebellum.

Accordingly, we found thatthe expression level of Glutamic acid decarboxylase (GAD), the enzyme that catalyzes synthesis of g-aminobutyric acid (GABA) was almost lack in the Xrcc1^nes-cre and Xrcc1^nes-cre; Atm^-/-cerebellum (Figure4B). Cerebellar interneurons are originated from progenitors in the white matter of the cerebellum and begin to differentiateafter birth(Yamanaka et al., 2004; Weisheit et al., 2006). To define the differentiation stage of interneurons, we measured cerebellar interneurons in differentiation using a interneuron early differentiation maker; Pax2 immunostaining.

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Figure 4. Atm inactivation does not rescue cerebellar interneuron loss in the Xrcc1

nes-crecerebellum

(A) The P15 cerebellum from WT, Xrcc1^Nes-Cre and Xrcc1^nes-cre; Atm^-/-mice. Nissl staining identified defects in the cerebellar structure. Interneurons of theXrcc1^nes-creand Xrcc1^nes-cre; Atm^-/- cerebellum were markedly reduced in the molecular layer (MO). (B) GAD (Glutamate decarboxylase) immunostaining identifies GABAergic neurons, which were absent in the molecular layer of the Xrcc1^nes-creand Xrcc1^nes-cre; Atm^-/-cerebellum (white arrow). (C) Immunostaining for Pax2 identifies differentiating interneurons in the white matter (WM) of the developing postnatal cerebellum. Cerebellar interneuron populations were reduced in both Xrcc1^nes-creand Xrcc1^nes-cre; Atm^-/-suggesting that ATM signaling is not involved in interneuron genesis.

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Compared with wild-type and Atm^-/-(data notshown) tissue, there was a marked reduction of Pax2-positive interneuron progenitors in both theXrcc1^nes-cre and Xrcc1^nes-cre; Atm -/-cerebellum(Figure 4C).These datasuggest two important outcomes that in the cerebellum Xrcc1 is necessary to maintain genomic stability for interneuron progenitors and to suppress DNA damage induced Atm independent p53-mediated cell cycle checkpoint activation.At least, ATM is not a sole p53 upstream kinase in cerebellar interneuron progenitors. This is distinct from the well-known for DNA damage signaling pathway.

4. Purkinje cells are susceptible to Xrcc1 and Atm loss

The most prominentclinical symptom of A-T patients is progressive neurodegeneration due to Purkinje cell loss (Boder, 1985; Gatti et al., 1991; Crawford, 1998).Purkinje cells are functionally important since they provide solo neuronal output signalsto the deep cerebellar nucleus. Their neuronal activity is controlled by the neuronal input from granule cells as well as interneurons. They are arranged as a monolayer and make up to 10⁴dendritic connections.

Most Purkinje cell axons make inhibitory synaptic connections utilizing GABA as neurotransmitter to inhibit neurons in the deep cerebellar nuclei, which are located deep within the cerebellar white matter. As a result, the cerebellum regulate movement via Purkinje cell activity(Cerminara et al., 2015).

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Figure 5. Cerebellar atrophy and astrogliosis in the Xrcc1^Nes-Cre; Atm^-/-mice brain

(A) The P16 cerebellum (the vermis part) from WT, Xrcc1^Nes-Cre and Xrcc1^Nes-Cre; Atm^-/- mice.

Staining for calbindin identifies the Purkinje cell layer. Bottom, the cerebellum hemisphere – no proper layer of granule cells (green color) was found in Atm^-/-; Xrcc1^Nes-Cre, leading to several ataxia. (B) The P21 corpus callosum (Cc) and cerebellum (Cb) from WT, Atm^-/- , Xrcc1^Nes-Cre and Xrcc1^Nes-Cre; Atm^-/- mice. Staining for GFAP identifies astrocytes. The Xrcc1^Nes-Crebrain displayed increased GFAP (Glial fibrillary acidic protein) immunoreactivity, and further increment in the Xrcc1^Nes-Cre; Atm^-/- brain, indicating the sign of astrogliosis.

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The Purkinje cell layer in the experimental animals was visualized with Calbindin immuno-staining. In WT, Atm^-/-andXrcc1^nes-cre mouse, Purkinje cells are densely packed as a single cell layer forming the proper Purkinje cell layer and exhibit dense dendritic arborizationwhich fill the molecular layer. Whereasin the Xrcc1^nes-cre; Atm^-/- mouse, Purkinje cell density was markedly reduced and they appear heterotopically located in the molecular and granule cell layers. Also Purkinje cell bodies (nucleus) formed the irregular arrangement and their dendritic branches are lost in the Xrcc1^nes-cre; Atm^-/-cerebellum (Figure 5A). For these reasonsXrcc1^nes-cre;Atm^-/-cerebellum showed unclear boundaries between the molecular layer and granular layer. Overall the Xrcc1^nes-cre;Atm^-/-animalsdid not have proper cerebellum structure which is most likely non-functional, resulting in severe ataxia. This Xrcc1

nes-cre;Atm^-/-animal model is actually the first animal model displaying ataxia by deletion of the Atm gene, mimicking ataxic phenotype of A-T patients.

Next, we analyzedastrocytes – one of glial cell populations in the nervous system – using glial fibrillary acid protein (GFAP)immunostaining. The Xrcc1^nes-cre cortex(the Corpus callosum is the white matter structure rich with astrocytes.) displayed increased immunoreactivity of GFAP suggestive of astrogliosis (Figure 5B). And the Xrcc1^nes-cre;Atm -/-brainshowed further increased immunoreactivity suggesting that the condition of the chronic DNA damage status was worsened by Atm inactivation. This astrogliosis has been implicated in both perturbation of brain homeostasis and neurodegenerative disease (Cirillo et al., 2011), Furthermore, astrogliosis occurs in many type of neurological disorders including spasms(Weidenheim et al., 2009).In case of the cerebellum (Cb), specialized glia

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population called Bergmann glia is critical for migration of granule cells once granule cell precursor cells exit cell cycle to form granule cell layers (Sild and Ruthazer, 2011).The WT, Atm^-/- and Xrcc1^nes-cre cerebellum showed a dense parallel array of GFAP immunoreactivity in the molecular layer, typical Bergmann glia formation (Figure 5B). However, the Xrcc1

nes-cre;Atm^-/-cerebellum displayed total disruption of Bergmann glia that indicates granule cell migration is abnormal leading to malformation of the Purkinje cell layer.

5. Xrcc1 and Atmare required for oligodendrocyte differentiation

We approachedto another kind of differentiated glial cells in the central nervous system (CNS), oligodendrocyte. In the CNS, oligodendrocytes synthesize large quantities of myelin, a lipid-rich, multi-layered spiral-wrapped membrane structure which insulates nerve axons, so that neuronal electric impulse can be conducted with minimal loss. Myelination segments by oligodendrocytes refer to as the internode, which enables rapid and highly efficient neural conduction in neuronal networks.(Gieselmann et al., 1994; Miller and Mi, 2007;

Haroutunian et al., 2014).

When we examined the brain tissues using Myelin basic protein (MBP) antibody, a marker for fully mature oligodendrocyte,surprisingly we found that the fully mature oligodendrocytes were absent in the cerebral cortex including the Corpus Callosum and to the minor extent in the cerebellum of the Xrcc1^nes-cre; Atm^-/-brain (Figure 6).Compared with the matched brain areas in the WT, Atm^-/-and Xrcc1^nes-creanimals, there was a marked

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reduction of MBP-positive oligodendrocyte in the Xrcc1^nes-cre; Atm^-/-brain at postnatal day7(P7) (Figure 6A). Even at postnatal day 14 (P14), MBP was not observed in the Xrcc1^nes-cre; Atm^-/-brain (Figure 6B).Deficit of fully mature oligodendrocytes was further confirmed by the expression of another oligodendrocyte marker CNPase at the protein level determined by western blotting.2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase) is a critical enzyme for generation of myelin. The expression level of CNPase was completely diminished in the Xrcc1^nes-cre; Atm^-/-cerebral cortexat P14.This failure of oligodendrocyte differentiation persisted into postnatal 21 days that is the time point when the Xrcc1^nes-cre; Atm^-/-animals rarely survived.The Xrcc1^nes-cre; Atm^-/-mouse cerebellum showed minor reduction of CNPase protein level. In contrast, the expression of neuronal marker, NeuN showed compatible expression pattern in theXrcc1^nes-cre; Atm^-/-cortex to that of the WT, Atm -/-and Xrcc1^nes-creanimals(Figure 6C). These data imply the strong possibility of involvement of Atm and its signaling during oligodendrocyte genesis in the context of DNA damage particularly induced by Xrcc1 inactivation during brain development. This Atm involvement is quite exclusive to oligodendrocytes since other neural populations such as neurons and astrocytes were not affected in the Xrcc1^nes-cre; Atm^-/-brain.

Similar to the in vivofinding, we observed a complete absence of CNPase expression at the protein level in differentiated cells from neural stem cells (NSC) in vitro. NSC from WT, Atm^-/- andXrcc1^nes-creembryonic forebrainexpressed CNPase 14 days (DIV14) after plating in differentiation medium. However, we could not detect CNPase expression in cells differentiated from Xrcc1^nes-cre; Atm^-/-NSC (Figure 6D). Next, we checked the transcription level of MBP and PLP (Proteolipid protein), mature oligodendrocyte markers. We

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appliedquantitative real-time PCR to quantify the MBP and PLP mRNA levels in the P17 cortex and cerebellum. The expression level of target proteins was measured after synthesizing cDNA from total RNA. The MBP and PLP expression at the transcriptional level was low at P17 in the Xrcc1^nes-cre; Atm^-/- samples compared to the control groups (Figure 6E,F). These data suggest that the very low protein level of MBP and PLP is not due to the matter of target protein stability, rather the diminished transcriptional level.

This phenomenon which is lack of mature oligodendrocytes in the brain, called leukodystrophy,was also found in several A-T patients(Chung et al., 1994; Sardanelli et al., 1995; Opeskin et al., 1998; Ciemins and Horowitz, 2000; Habek et al., 2008; Lin et al., 2014). So far, the underlying molecular mechanisms for leukodystrophy resulting from Atm mutation are not known. This is the first animal model with Atm deficiency to show defects in oligodendrocyte genesis. In future, it is warranty that the molecular and biochemical mechanisms for leukodystrophy will be revealed by analyzing further this animal model.

Furthermore, a possible pharmaceutical application to relieve neurological symptoms will be explored using this animal model.

6. Oligodendrocyte progenitor cell formation in the Xrcc1^nes-cre; Atm^-/-brain.

Oligodendrogenesis begins with multipotent embryonic neural stem cells (NSC), which can be differentiated into neurons, astrocytes, and oligodendrocytes. The successive stages of oligodendrocyte differentiation include oligodendrocyte progenitor cells (OPC), pre-oligodendrocyte, and fully mature oligodendrocytes.

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Figure 6. Fully mature Oligodendrocytes are missing in the Xrcc1^nes-cre; Atm^-/-brain.

(A-B) The Corpus callosum (Cc) and cerebellum (Cb) from WT, Atm^-/-, Xrcc1^Nes-Cre and Xrcc1^nes-cre; Atm^-/-mice at ages P7 and P14. Staining for MBP (myelin basic protein) identified fully differentiated oligodendrocytes in the brain. Double inactivation of Atm and Xrcc1in the brain leads to complete absence of fully mature oligodendrocytes.

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Figure 6. Fully mature Oligodendrocytes are missing in the Xrcc1^nes-cre; Atm^-/-brain (Cont’d).

(C) Protein extracts from the P14 and P21 mouse cerebral cortex and cerebellum were subjected to western blot analysis for NeuN (neuron marker) and CNPase (3’-cyclic nucleotide 3’-phosphodiesterase, fully mature oligodendrocyte marker). β-actin was used as a loading control. (D) Neural stem cells from E14.5 WT, Atm^-/-, Xrcc1^Nes-Creand Xrcc1^Nes-Cre; Atm^-/-forebrain were cultured in NSC differentiation media. Proteins were extracted from in vitro differentiated cells after DIV (days in vitro) 14 and DIV21. β-actin was used as a loading control.Arrow indicates NeuN positive western band (the upper band is none specific.)dKO, Xrcc1^Nes-Cre; Atm^-/-doubleknockout

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Figure 6. Fully mature Oligodendrocytes are missing in the Xrcc1^nes-cre; Atm^-/-brain (Cont’d).

(E-F)Realtime PCR quantification of MBP and another fully mature oligodendrocyte marker, PLP mRNA expression in the cortex and cerebellum at postnatal day 17 (the mRNA level was normalized to the level of sample WT). These are representative graphs from two repeats that showed the same pattern of the expression difference.dKO, Xrcc1^Nes-Cre; Atm -/-doubleknockout

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Accompanying this morphological change is the stage-specific expression of molecular markers; Platelet-derived growth factor receptor α (PDGFRα) in oligodendrocyte progenitors, Myelin basic protein (MBP), 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNPase) and Proteolipid protein (PLP) in mature myelinating OL. Also Oligodendrocyte transcription factor 1 and 2 (Olig1 and Olig2) are specific transcription factors to confine oligodendrocyte lineage(Eng et al., 1968; Hart et al., 1989; Karthigasan et al., 1996; Qi et al., 2001; Ligon et al., 2006).

To define the differentiation stage of oligodendrocyte susceptible to double inactivation of Xrcc1 and Atm, we immunostained OPC using PDGFRα antibody.In contrast with the lack of fully mature oligodendrocyte marker expression such as MBP and PLP(Figure 6A,B), we observeda normal level of PDGFRα expression in the Xrcc1^nes-cre; Atm^-/-Corpus Callosum where oligodendrocytes are mainly generated at postnatal day 14 (Figure 7A). Consistently with this in vivo data, weconfirmed thatthrough NSC differentiationin vitro. TheXrcc1^nes-cre; Atm^-/-NSC differentiated into PDGFRα+OPC measured byimmunocytochemistry (Figure7 B,C).Also the transcription level of PDGFRα was not reduced in the Xrcc1^nes-cre; Atm -/-oligodendrocytesfrom the P7brain tissue measured by realtime PCR method (Figure7 D).

Despite the normal OPC population, oligodendrocyteswere absent in the Xrcc1^nes-cre; Atm -/-brain.This lackof mature oligodendrocytes in the brain of Xrcc1^nes-cre; Atm^-/- mouse could result from either DNA damage induced cell cycle arrest or apoptosis in PDGFRα positive

Despite the normal OPC population, oligodendrocyteswere absent in the Xrcc1^nes-cre; Atm -/-brain.This lackof mature oligodendrocytes in the brain of Xrcc1^nes-cre; Atm^-/- mouse could result from either DNA damage induced cell cycle arrest or apoptosis in PDGFRα positive