Estimation of the hepatitis E assay-dependent seroprevalence among Croatian blood donors.

BACKGROUND: Seroprevalence of hepatitis E virus (HEV) in blood donors presenting to the Croatian Institute of Transfusion Medicine was assessed with 4 available tests (3 ELISA tests and 1 immunoblot (IB) test). MATERIALS AND METHODS: In October and November 2014, a total of 1,036 serum samples of blood donors were collected for the study. Samples were primarily tested for total HEV antibodies by Dia.Pro HEV Ab test (a). All reactive samples were tested by ELISA tests: Dia.Pro HEV IgG (b) and IgM (c), Mikrogen recomWell HEV IgG_old (d) and IgM_old (e), recomWell HEV IgG_new (f) and IgM_new (g), and IB Mikrogen recomLine HEV IgG (h) and IgM (i). HEV IgM reactive samples also positive by the IB were further tested for HEV RNA. RESULTS: There were 21.5% of samples reactive for total HEV antibodies (a). Seroprevalence of HEV IgG according to the b, d, f and h tests was 20.2%, 9.6%, 18.1% and 17.8%, respectively. Seroprevalence of HEV IgM according to the c, e, g and i tests was 4.4%, 1.5%, 2.0% and 1.7%, respectively. Out of 46 HEV IgM (Dia.Pro HEV IgM) positive samples, 18 (39.1%) were also positive by IB. HEV RNA was not detected in any of those samples. There was a significant association between age and HEV seroprevalence (P<0.001). CONCLUSION: Different HEV antibody detection assays showed a high HEV IgG seroprevalence in Croatian blood donors. Among HEV IgG and HEV IgM positive samples HEV RNA was not detected.

Characterisation of antioxidants in photosynthetic and non-photosynthetic leaf tissues of variegated Pelargonium zonale plants.

Hydrogen peroxide is an important signalling molecule, involved in regulation of numerous metabolic processes in plants. The most important sources of H2 O2 in photosynthetically active cells are chloroplasts and peroxisomes. Here we employed variegated Pelargonium zonale to characterise and compare enzymatic and non-enzymatic components of the antioxidative system in autotrophic and heterotrophic leaf tissues at (sub)cellular level under optimal growth conditions. The results revealed that both leaf tissues had specific strategies to regulate H2 O2 levels. In photosynthetic cells, the redox regulatory system was based on ascorbate, and on the activities of thylakoid-bound ascorbate peroxidase (tAPX) and catalase. In this leaf tissue, ascorbate was predominantly localised in the nucleus, peroxisomes, plastids and mitochondria. On the other hand, non-photosynthetic cells contained higher glutathione content, mostly located in mitochondria. The enzymatic antioxidative system in non-photosynthetic cells relied on the ascorbate-glutathione cycle and both Mn and Cu/Zn superoxide dismutase. Interestingly, higher content of ascorbate and glutathione, and higher activities of APX in the cytosol of non-photosynthetic leaf cells compared to the photosynthetic ones, suggest the importance of this compartment in H2 O2 regulation. Together, these results imply different regulation of processes linked with H2 O2 signalling at subcellular level. Thus, we propose green-white variegated leaves as an excellent system for examination of redox signal transduction and redox communication between two cell types, autotrophic and heterotrophic, within the same organ.

Cortisol and testosterone levels on a weekend and a work day in three mountain villages in the Selska Valley of northwest Slovenia.

BACKGROUND: In contemporary western populations, a week commonly involves 5 days of paid work (work days) and two non-working days (weekend). Work days are usually perceived as being more stressful than non-work days and this hypothesis has been tested in several studies, most of which selected subjects with jobs that are perceived to have high stress. AIM: The study measured salivary cortisol and testosterone on a work day and a weekend in a community-based sample of people going about their everyday lives and tested the hypothesis that hormone levels will be higher on a work day. SUBJECTS AND METHODS: Slovenian alpine villagers (30 females and 25 males) were sampled without reference to their occupation. Each individual was measured on two occasions, a day on a weekend and a work day as they went about their usual activities in the afternoon. RESULTS: Cortisol (mean = 3.32 ng ml(-1), range 0.4-27.9) and testosterone (mean = 121 pg ml(-1), range 17-424) values were similar to other populations. Neither the age of subjects nor the time in the afternoon of sample collection were associated with hormone concentrations. On each day of collection, cortisol and testosterone values were correlated for each sex, with the estimate of the correlation coefficient ranging from 0.57 to 0.88. For females, testosterone values were higher on the weekend than the work day (102 pg ml(-1) and 60 pg ml(-1), respectively) but not for males (mean across both days 134 pg ml(-1)). Independent of this effect, the presence of a spouse or other adult in the house was significantly associated with lower testosterone levels in both sexes. Husband and wife testosterone values are correlated on the weekend (r = 0.67, p = 0.02) but not on the work day. Mean cortisol values for the weekend and work day were not different and there was no correlation between levels on these two days. CONCLUSIONS: These results, although based upon a small sample size, reveal potential relationships between testosterone, work-rest activities, and the presence-absence of a social partner that warrant further investigation.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala.

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group I metabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4.

Latent structure of dermatoglyphs in the population of Selska Valley.

The historical records of Selska Valley reveal that the eastern part of this area was first settled by Slovene agrarian colonists, the western part by German colonists and the central part by Friulians. These were later followed by Slovene and Slovenized settlers, who penetrated the valley from north to south. Because of its reproductive isolation, the population of Selska Valley is highly suitable for the study of population structures. The quantitative traits of the digital and palmar dermatoglyphs are polygenetically determined characteristics, which, due to their selective inertness to changes, may provide an insight into microevolutionary processes. The purpose of our study was to identify the possible differences between the populations of villages in the valley and the mountain villages attributable to various migration flows through history. Altogether 340 finger and palm prints of 163 males and 177 females were collected in two groups of villages: (1) the lowland villages (Praprotno, Bukovica, Sevlje, Dolenja vas, Selca, Zelezniki and Zali log), and (2) the mountain villages (Podlonk, Prtovc, Spodnje Danje, Zgornja Sorica and Spodnja Sorica). The 18 dermatoglyphic variables were analyzed. A statistical analysis using standard methods was performed and the latent structure evaluated using factor analysis. The discriminant analysis and latent structure of the quantitative properties of dermatoglyphs suggest the presence of certain differences in gene pools of two studied populations (the group of villages in the valley and the group of mountain villages). It is highly probable that these differences can be attributed to low migration in the Selska Valley and to the 'selective inertness' of quantitative dermatoglyphic traits. In a previous study, no significant biological differences between the studied populations were found in qualitative dermatoglyphic traits. This indicates that Selska Valley and its village populations represent a specific isolate, and therefore expanded studies of this type could significantly contribute to a better understanding of the population concerned and isolates in general.

Brain-derived neurotrophic factor is expressed in a gradient in the superior colliculus during development of the retinocollicular projection.

Abstract Theoretical models of topographic map formation have postulated a gradient of attractant in addition to a gradient of repulsion in the target. In species where many axons grow past their correct positions initially, it has also been argued that a parallel gradient of attractant or branching signal is required to ensure collateral formation at the correct position (O'Leary et al., 1999). Brain-derived neurotrophic factor (BDNF) is a known attractant and promotes branching of retinal axons. We have examined its distribution in the superior colliculus and that of its receptor, trkB, in the retina, using immunohistochemistry and in situ hybridization, respectively, during the development of the topographic retinocollicular projection in the wallaby, a marsupial mammal. The number of glial endfeet expressing BDNF at the surface of the colliculus was found to be in a high caudal-to-low rostral gradient during the time when the retinocollicular projection was developing. When the projection was mature the rostrocaudal gradient had disappeared and the number of detectable endfeet expressing BDNF was very low. Messenger RNA for TrkB was expressed in the retinal ganglion cell layer throughout the time when the retinocollicular projection was developing, with no difference in expression across the nasotemporal axis of the retina. The low rostral to high caudal distribution of BDNF in glial endfeet supports the idea that it is providing a parallel gradient of attractant or branching signal in the colliculus.

Graded expression of EphA3 in the retina and ephrin-A2 in the superior colliculus during initial development of coarse topography in the wallaby retinocollicular projection.

We describe the expression of EphA3 and EphA7 receptors and ephrin-A2 ligand in the retina and the superior colliculus during the development of the retinocollicular projection in the marsupial wallaby (Macropus eugenii), using immunoblotting and immunohistochemistry. EphA3 in the retina was in a striking, low central to high peripheral gradient, superimposed on which was a high temporal to low nasal level of expression. This distribution was evident from postnatal day 30, when axons are growing into the colliculus and forming a coarsely organized topographic projection, to postnatal day 65, when axons have arborized in their correct retinotopic positions. EphA7 showed a shallow centroperipheral gradient with no nasotemporal differences in expression. In the superior colliculus no rostrocaudal differences in distribution were detected for either of these receptors. Ephrin-A2 was distributed in a gradient increasing from the rostral to the caudal pole in the superficial layers of the superior colliculus only up to postnatal day 30. Ephrin-A2 was evenly distributed in the retina throughout development of the projection. Expression of EphA3 in the retina increased, while the expression of ephrin-A2 in the colliculus was downregulated over time. The graded expression of EphA3 and ephrin-A2 early in the development of the projection suggests that they play a role in establishment of coarse topography of retinal axons along the rostrocaudal axis of the superior colliculus. However, the gradients were not complementary, meaning that EphA3 alone cannot mediate the repulsive interactions with ephrin-A2 that have been postulated to underlie formation of the topographic map.

[Methemoglobinemia caused by accidental poisoning with metolachlor and metobromuron]. / Methemoglobinemija uzrokovana akcidentalnom intoksikacijom metolaklorom i metobromuronom.

An 81-year-old man was admitted in the emergency department approximately four hours after accidental, ingestion of an unknown quantity of herbicide "Galex 500 EC". This product contains 25% of metolachlor and 25% of metobromuron dissolved in xylene. In spite of the fact that the combination of aniline and urea-substituted derivatives is widely used in agriculture as herbicide, there are very few data available about their harmful effects on humans. These agents appear to be mildly toxic, and rarely has a major systemic effect been reported after the poisoning. On admittance, our patient showed remarkable cyanosis and his methaemoglobin level was 38.4% of the total haemoglobin, rising next day to 46.2%. Only mild transient signs of hypoxic effects on central nervous system were observed and the laboratory findings indicated mild haemolysis. Methylene blue was applied intravenously in a dose of 1.5 mg/kg (10 ml, 1% solution) on the second day of admission. Administration of methylene blue was very effective and the patient was discharged from the hospital fully recovered.

Marsupial retinocollicular system shows differential expression of messenger RNA encoding EphA receptors and their ligands during development.

The protracted development of the wallaby (Macropus eugenii) has allowed study of messenger RNAs encoding Eph receptors EphA3 and EphA7 and ligands ephrin-A2 and -A5 in the retina and superior colliculus at intervals throughout the development of the retinocollicular projection: from birth, before retinal innervation, to postnatal day 95, when the projection is mature. Reverse transcription-polymerase chain reaction showed messenger RNAs for both receptors and ligands were expressed at all ages. EphA7 was expressed more highly in the rostral superior colliculus. Ephrin-A2 and -A5 were expressed more highly in the caudal colliculus. EphA3 was expressed in a complementary manner, more highly in temporal than in nasal retina. There are higher levels of expression of the ligands when the projection is only coarsely topographically organised. This suggests a role for them and their receptor EphA3 in this stage, by repulsive interactions which restrict temporal axons to rostral superior colliculus. This is the first account in a marsupial mammal of the appearance of this molecular family, substantiating its ubiquitous role in topographically organised neuronal connections. Nevertheless, expression is not the same as in the mouse, suggesting differences in the details of topographic coding between species.

Transient expression of alpha-1B adrenoceptor messenger ribonucleic acids in the rat superior cervical ganglion during postnatal development.

We have examined the developmental profile of the alpha-1 and alpha-2 adrenergic receptor messenger ribonucleic acids expression in the rat superior cervical ganglion. The expression of the six messenger ribonucleic acids was studied using reverse transcription-polymerase chain reaction. At four weeks, the dominant messenger ribonucleic acids transcripts in this sympathetic ganglion were alpha-1C, alpha-2A, alpha-2B and alpha-2C. The expression of alpha-1 genes in the superior cervical ganglion appears to be regulated during postnatal development in that two alpha-1 (alpha-1B, alpha-1C) genes were expressed at birth, three, seven and 14 days postnatal but no amplified product for alpha-1B was detected at 28 days and in the aged animals, while the alpha-1C transcript continued to be expressed. No amplified product for alpha-1D was detected in superior cervical ganglion at any of the ages studied. While all three alpha-2 genes were expressed in the superior cervical ganglion at four weeks the dominant alpha-2 messenger ribonucleic acids transcript expressed in the superior cervical ganglion was alpha-2A. This pattern of alpha-2 adrenoceptor gene expression was maintained from birth, throughout development and into old age. These results suggest that the expression of alpha-1 adrenergic receptors in the superior cervical ganglion is regulated developmentally while the expression of alpha-2 genes remains unchanged.

Variation in mRNA expression of alpha-adrenergic, neurokinin and muscarinic receptors amongst four arteries of the rat.

Different mechanisms mediate constriction and dilation in different vascular beds. We have used reverse transcription-polymerase chain reaction to investigate whether specific patterns of receptor gene expression may underlie these variable responses. Total RNA, from the basilar, pulmonary, mesenteric and tail arteries of anaesthetised adult Wistar rats, was reverse transcribed and amplified using primers specific for the molecular subtypes of the alpha 1(A, B, D)- and alpha 2(A, B, C)-adrenergic, neurokinin (NK1-NK3) and muscarinic (m1-m5), receptors. Results showed that the pattern of gene expression was variable with no two arteries having the same receptor profile. Messenger RNA for the alpha 1A, alpha 1B, alpha 2B, NK1, NK3, m3 and m5 receptor subtypes were detected in all vessels studied while the remaining subtypes showed a variable expression amongst the arteries. This is the first description of mRNA for the m5 muscarinic receptor in peripheral tissue. The NK3 receptor was the major neurokinin receptor expressed in all vessels except the pulmonary artery, in which the NK1 receptor was also strongly expressed. We conclude that each artery expressed a specific receptor array which may permit some unique neural and hormonal controls.

Alpha-adrenergic, neurokinin and muscarinic receptors in rat mesenteric artery; an mRNA study during postnatal development.

Developmental studies show that the innervation of autonomic targets is accompanied by increases in the density of receptors, maturation of receptor-signalling pathways and changes in receptor subtype. The innervation of the rat mesenteric artery occurs over the first 3 postnatal weeks. In this study, we investigated whether alterations in receptor gene expression may underlie physiological changes recorded during development and maturity in this vessel. Total RNA, from mesenteric arteries of rats at birth and postnatal days 7, 14, 28, 240 and 360, was reverse transcribed and amplified using primers specific for the alpha 1 (A, B, D)- and alpha 2 (A, B, C)-adrenergic, neurokinin (NK1-NK3) and muscarinic (m1-m5) receptors. Results showed that all receptor genes expressed at 28 days, except the alpha 1D-adrenergic receptor, were already expressed at birth. Some receptor subtypes showed no change in their relative expression, always being either strongly (alpha 1A, alpha 2B, NK3) or weakly (alpha 2A, alpha 2C, NK1) expressed. Relative to the expression of these receptors, others showed a developmental increase in expression up to 14 days postnatal (alpha 1B, alpha 1D, m2, m3, m5) but no further change with maturity. These latter changes coincide with the development of sympathetic and sensory nerve plexuses in the mesenteric artery, but do not correlate with the physiological changes seen during development and ageing.

Differential effects of histamine on the N-methyl-D-aspartate channel in hippocampal slices and cultures.

The effect of histamine on N-methyl-D-aspartate currents was investigated in pyramidal neurons in the CA1 region of acute hippocampal slices from juvenile rats. The objective was to compare histamine effects in the slice with those previously reported in acutely dissociated and cultured hippocampal neurons. Micromolar concentrations of histamine had no effect on N-methyl-D-aspartate mediated excitatory postsynaptic currents in the slice, in contrast to the large enhancement seen in culture under identical conditions. However, millimolar concentrations of histamine blocked these currents both in the slice and in culture. Possible reasons for the lack of enhancement in the slice were explored as follows. (1) Histamine could not penetrate the slice or was already present at high concentrations inside the slice. This was tested by recording N-methyl-D-aspartate currents elicited in outside-out patches pulled from the somas of CA1 slice neurons. Histamine still had no effect in patches, whereas the corresponding experiment for cultured neurons showed robust enhancement. (2) Slices release an endogenous ligand that binds with high affinity to the histamine site on the N-methyl-D-aspartate receptor, blocking its activation. This was tested by superfusing cultures with supernatant from homogenized slice tissue. Histamine enhancement was maintained in these cultures. (3) CA1 slices and cultures express different N-methyl-D-aspartate receptor subtypes. The reverse transcription-polymerase chain reaction technique was used to examine the expression of messenger RNA encoding N-methyl-D-aspartate receptor subunits in the two systems. No difference was found in the whole-tissue expression of messenger RNA for the NR2A, 2B or 2C subunits or for the eight known splice variants of the NR1 subunit. It is hypothesized that the differential enhancing effect of histamine in slices and culture involves posttranslational modifications or other factors that modulate the N-methyl-D-aspartate receptor/ion channel according to its environment.

Sensory nerves play an efferent role in the function of the arterioles, but not the dilator muscle, of the rat iris.

We have studied the expression, distribution and function of receptors for the sensory neurotransmitters, substance P (SP) and calcitonin gene-related peptide (CGRP) in the dilator muscle and arterioles of the rat iris. Immunohistochemical studies showed that the sensory fibres containing these peptides are distributed throughout the connective tissue stroma of the iris and in association with the larger arterioles, but do not come into close association with the dilator muscle cells. Using reverse-transcription polymerase chain reaction, we have shown that both NK1 and NK3 receptor message is expressed by iris tissue, comprising both dilator muscle and stromal tissue. Binding sites for the NK1 agonist, [Sar9, Met(O2)11]-substance P (SarSP), and for CGRP are confined to the stromal layer and to the larger arterioles within that layer and do not appear to be associated with the dilator muscle itself. Application of either SarSP or CGRP produced both a vasodilatation and an inhibition of sympathetic nerve-induced vasoconstriction of the larger arterioles. Neither SarSP nor CGRP altered the resting tone of the dilator nor were they capable of modulating the contractions due to sympathetic nervous activity. These results suggest that the sensory fibres perform an efferent role in the larger irideal arterioles while their presence in the irideal stroma appears not to modulate the activity of the dilator muscle.

Alpha adrenoceptor gene expression in the rat iris during development and maturity.

We have examined the developmental profile of the alpha-1 and alpha-2 adrenergic receptor mRNA expression in the rat iris. The expression of the six mRNAs was studied using reverse transcription-polymerase chain reaction. At 4 weeks, the dominant mRNA transcripts in the rat iris were alpha-1B, alpha-1C and alpha-2A. This pattern of alpha adrenoceptor expression was seen from birth, throughout development, and maintained into old age.

Cross talk between receptors mediating contraction and relaxation in the arterioles but not the dilator muscle of the rat iris.

1. Sympathetic nerve stimulation causes contraction of the dilator muscle and the large arterioles of the iris via the activation of alpha 1B-adrenoceptors. We have investigated whether increases in adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and the activation of receptors in these tissues can modulate these nerve-mediated contractions. 2. Increasing intracellular cyclic AMP with dibutyryl cyclic AMP (1 mM), forskolin (50 microM) or isobutylmethylxanthine (100 microM) produced relaxation of both the dilator and the arterioles, abolished the nerve-mediated constriction of the arterioles, but potentiated the nerve-mediated contraction of the iris dilator. 3. Pretreatment of the preparations with cholera toxin, to activate Gs permanently, caused a dilatation of the arterioles and abolished the nerve-mediated constriction but had no effect on the dilator muscle. 4. The beta-adrenoceptor agonist, isoprenaline (1 microM), the adenosine-A1,-A2 agonist, N-ethylcarboxamidoadenosine NECA (100 nM), in the presence of the A1-selective antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 10 nM), and calcitonin gene-related peptide (CGRP, 10 nM) all separately caused a dilatation of the arterioles and abolished the nerve-mediated constriction, while only isoprenaline (1 microM) produced an effect on the dilator, i.e. a relaxation but a potentiation of the nerve-mediated contraction. These results suggest the presence of at least 3 types of receptor linked to Gs and an increase in cyclic AMP in the arterioles, i.e. beta-adrenoceptor, adenosine-A2 and CGRP, but only 1 Gs-linked receptor, i.e. beta-adrenoceptors, on the dilator muscle cells.2+ '

Identification of alpha 2 adrenergic receptor gene expression in sympathetic neurones using polymerase chain reaction and in situ hybridization.

alpha 2 Adrenergic receptors are involved in mediating pre- and postsynaptic responses in the sympathetic nervous system. In this study, the expression of alpha 2 genes was examined by the amplification of mRNA, extracted from adult rat superior cervical ganglion through reverse transcription and subsequent amplification of appropriate target sequence using polymerase chain reaction and sequence specific oligonucleotide primers for the three alpha 2 receptor genes. Results from these studies have shown that the major alpha 2 adrenergic mRNA transcript was the one that encodes the alpha 2A receptor. Nucleotide sequence of the 312 base-pair (bp) alpha 2A cDNA was homologous to the RG20 adrenergic receptor, the rat homologue of the human alpha 2A receptor. The 312 bp alpha 2A cDNA was used as a probe in Northern blot analysis of the mRNA from superior cervical ganglion and brain. A 3.9 kb mRNA transcript was present in these extracts. To confirm that the alpha 2A gene expression was in the sympathetic neurones we have used the 312 bp alpha 2A cDNA, biotinylated, as a probe for in situ hybridization studies and have demonstrated that the alpha 2A mRNA was found only in the cell bodies of sympathetic neurones.

Trimestral changes of seizure frequency in pregnant epileptic women.

The aim of this study was to assess the influence of pregnancy on trimestral frequency of epileptic seizures. The seizure frequency was studied retrospectively in 50 pregnant epileptic women compared to one year before the onset of pregnancy, for each trimester of pregnancy separately, and for the whole duration of pregnancy. In the first trimester 18% of the pregnant epileptic women had an increased seizure frequency. There was no change in seizure frequency in 56% of pregnancies. In 26% of the pregnant women the number of seizures was decreased. In the second trimester, pregnancy did not influence seizure frequency in 60% of the pregnancies; 16% of the women had increased frequency of seizures and 24% had decreased frequency. In the third trimester, 20% of the pregnant epileptic women had higher seizure frequency. In 56% of the women no change in seizure frequency was recorded during pregnancy. For the entire period of pregnancy, 22% of the pregnant epileptic women had an increased seizure frequency, 54% had no change, and 24% had lower frequency as compared to one year before conception. No difference of seizure frequency was noted between trimesters and the whole course of pregnancy.

[Unusual localization of an echinococcal cyst]. / Neobicna lokalizacija ehinokokne ciste.

Hardly ever one can find a hydatid cyst in other parts of the organism except in the liver and lungs. The localization in the retroperitoneum is very rare. We have shown an unusually located hydatid cyst in the retroperitoneum. The cyst was firmly integrated with the structure of m. psoas and the muscle resection was necessary.