Annotated chromosome counts of czechoslovak plants (31–60) (Materials for “Flóra ČSSR”—3)

Chromosome counts of the following 30 taxa (106 populations) are given:Betonica officinalis (2n=16);Bidens frondosus (2n=48);Calamagrostis arundinacea (2n=28+0–2B);Dianthus carthusianorum subsp.latifolius (2n=30);Festuca gigantea (2n=42, 42+2B);Hypericum perforatum (2n=32);Koeleria macrantha (2n=28);Kohlrauschia prolifera (2n=30);Lilium martagon (2n=24+0–2B);Melica ciliata (2n=18);Poa remota (2n=14);Ranunculus polyanthemos (2n=16);R. sardous subsp.sardous (2n=16);Roegneria canina (2n=28+0–1B);Rudbeckia laciniata (2n=76);Scabiosa canescens (2n=16);Serratula tinctoria (2n=22);Seseli elatum subsp.heterophyllum var.beckii (2n=18);S. hippomarathrum (2n=20);Thlaspicaerulescens caerulescens subsp.tatrense (2n=14);Trifolium alpestre (2n=16);T. avense (2n=14);T. medium (2n=79, 80+0–2B, 82);T. rubens (2n=16);Veronica officinalis subsp. alpestris (2n=36);Vincetoxicum hirundinaria (2n=22);Vulpia bromoides (2n=14);Zerna benekenii (2n=28)Z. monoclada (2n=28+0–8B);Z. ramosa (2n=42). Remarks on taxonomy, nomenclature and chorology for some of these taxa are given.     

Functional differentiation of bmp2a and bmp2b genes in zebrafish

Bone morphogenetic protein 2 plays an important role in the regulation of osteoblast proliferation and differentiation. Phylogenetic analysis showed that the bmp2 ortholog evolved from the same ancestral gene family in vertebrates and was duplicated in teleost, which were named bmp2a and bmp2b. The results of whole-mount in situ hybridization showed that the expression locations of bmp2a and bmp2b in zebrafish were different in different periods (24 hpf, 48 hpf, 72 hpf), which revealed potential functional differentiation between bmp2a and bmp2b. Phenotypic analysis showed that bmp2a mutations caused partial rib and vertebral deformities in zebrafish, while bmp2b^−/− embryos died massively after 12 hpf due to abnormal somite formation. We further explored the expression pattern changes of genes (bmp2a, bmp2b, smad1, fgf4, runx2b, alp) related to skeletal development at different developmental stages (20 dpf, 60 dpf, 90 dpf) in wild-type and bmp2a^−/− zebrafish. The results showed that the expression of runx2b in bmp2a^−/− was significantly downregulated at three stages and the expression of other genes were significantly downregulated at 90 dpf compared with wild-type zebrafish. The study revealed functional differentiation of bmp2a and bmp2b in zebrafish embryonic and skeletal development.     

Positioning the expanded akirin gene family of Atlantic salmon within the transcriptional networks of myogenesis

Vertebrate akirin genes usually form a family with one-to-three members that regulate gene expression during the innate immune response, carcinogenesis and myogenesis. We recently established that an expanded family of eight akirin genes is conserved across salmonid fish. Here, we measured mRNA levels of the akirin family of Atlantic salmon (Salmo salar L.) during the differentiation of primary myoblasts cultured from fast-skeletal muscle. Using hierarchical clustering and correlation, the data was positioned into a network of expression profiles including twenty further genes that regulate myogenesis. akirin1(2b) was not significantly regulated during the maturation of the cell culture. akirin2(1a) and 2(1b), along with IGF-II and several igfbps, were most highly expressed in mononuclear cells, then significantly and constitutively downregulated as differentiation proceeded and myotubes formed/matured. Conversely, akirin1(1a), 1(1b), 1(2a), 2(2a) and 2(2b) were expressed at lowest levels when mononuclear cells dominated the culture and highest levels when confluent layers of myotubes were evident. However, akirin1(2a) and 2(2a) were first upregulated earlier than akirin1(1a), 1(1b) and 2(2b), when rates of myoblast proliferation were highest. Interestingly, akirin1(1b), 1(2a), 2(2a) and 2(2b) formed part of a module of co-expressed genes involved in muscle differentiation, including myod1a, myog, mef2a, 14-3-3β and 14-3-3γ. All akirin paralogues were expressed ubiquitously across ten tissues, although mRNA levels were regulated between cell-types and family members. Gene expression patterns were often highly correlated between akirin paralogues, suggesting that natural selection has maintained an intricate network of co-regulation among family members. We concluded that the Atlantic salmon akirin family performs a multifaceted role during myogenesis and has physiological functions spanning many cell-types.     

Cytogenetic characterization of ten araneomorph spiders (Araneae): Karyotypes and meiotic features

Karyotypes, sex chromosome systems and meiotic characteristics are reported for ten spider species belonging to the families Gnaphosidae, Philodromidae, Salticidae, Oxyopidae and Sicariidae by using standard Giemsa staining. The male diploid numbers (2n) and sex chromosome systems are as follows: Berinda hakani 2n = 22 (X₁X₂), Berinda ensigera 2n = 22 (X₁X₂), Trachyzelotes lyonneti 2n = 22 (X₁X₂), Trachyzelotes malkini 2n = 22 (X₁X₂), Zelotes caucasius 2n = 22 (X₁X₂) (Gnaphosidae); Thanatus pictus 2n = 28 (X₁ X₂), Tibellus macellus 2n = 24 (X₁ X₂) (Philodromidae); Neon reticulatus 2n = 21 (X₀) (Salticidae); Peucetia virescens 2n = 28 (X₁X₂) (Oxyopidae) and Loxosceles rufescens 2n = 21 (X₁ X₂Y) (Sicariidae). All species have monoarmed chromosomes with the exception of L. rufescens that has biarmed (metacentric and submetacentric) chromosomes. The obtained data are the first results for the genera Berinda, Trachyzelotes and Neon. Additionally, with the exception of L. rufescens, all species are being chromosomally analyzed for the first time.     

Altered clearance of human α2-macroglobulin complexes following reaction with cis-dichlorodiamineplatinum(II)

Clearance studies were performed in mice using α₂-macroglobulin (α₂M), α₂M-trypsin comlex and α₂M-CH₃NH₂ complex. All three species were incubated with cis-dichlorodiamine platinum(II) (cis-DDPt) at concentrations between 9.0 μM and 1.67 mM for 4 h and then dialyzed. The clearance rate of native α₂M was unchanged following incubation with cis-DDPt. α₂M-trypsin and α₂M-CH₃NH₂ cleared rapidly from the ciruculation; however, reaction with cis-DDPt significantly decreased the plasma elimination rate of both complexes. Non-denaturing gel electrophoresis and α₂M activity assays demonstrated relative stability following incubations with cis-DDPt which markedly altered clearance. Evidence for cis-DDPt crosslinking of α₂M subunits was obtained: however, whether this crosslinking is involved in altered clearance remains undetermined. Iodoacetamide treatment of α₂M did not duplicate the effect of cis-DDPton α₂M clearance, nor did it inhibit the effect of cis-DDPt on α₂M clearance. Plasma elimination of α₂M complex was also unaltered by pretreatment of mice with intravenous free cis-DDPt.     

Production of anti-self-H-2 antibodies by C3D2F1 mice hyperimmune to L cell/L1210 hybrids and L1210 leukemia cells

During the course of immunization of (C3H × DBA/2)F₁ mice (genotype H-2^k/b) with L cell (H-2^k/k)/L1210 leukemia cell (H-2^d/d) hybrids and L1210 leukemia cells, some of them produced a good titer of anti-self-H-2 (H-2^d) antibodies. Antigens recognized by this anti-self-H-2 antiserum were shown to be controlled by the H-2K-IA-IB-IJ-IE subregions of the H-2^d but not H-2^k nor H-2^b haplotypes of parental as well as F₁ origins and to have a tissue distribution identical to that of class 1 H-2 (H-2K/D) antigens.     

Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2)

The Tm-2 gene of tomato and its allelic gene, Tm-2², confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2², Tm-2² confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2². Although resistance induced by Tm-2 and Tm-2² is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2² induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2² but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2² is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2² are involved in HR cell death.     

Discovery of a new fragrance allele and development of functional markers for identifying diverse fragrant genotypes in rice

Discovery of new fragrance alleles provides important genetic resources for breeding fragrant rice. In this study, a hybrid complementation test demonstrated the association of a new fragrance allele without mutation in the coding region with flavor formation in a fragrant rice variety Nankai 138. The new allele (badh2-p-5′UTR) has a 3-bp deletion in the 5′ untranslated region and an 8-bp insertion in the promoter (?1,314 site upstream from the initiation codon). Surprisingly, we found that there is also an 8-bp insertion in the promoter of the badh2-E7 allele. We developed a new sequence tagged site functional marker to identify the badh2-p-5′UTR and badh2-E7 alleles according to the 8-bp insertion in their promoters. A cleaved amplified polymorphic sequence (AluI) functional marker targeting a common base substitution in the intron 2 of three badh2 alleles, viz. badh2-p-5′UTR, badh2-E7 and badh2-E2, was developed to identify diverse genotypes for fragrance in rice. Based on the results of sequence alignments among the three badh2 alleles, we suggest that the badh2-E7 and badh2-p-5′UTR alleles may have the same genetic origin. In addition, the genetic distance between the badh2-E7 and badh2-p-5′UTR alleles may be closer than that between the badh2-E2 and the badh2-p-5′UTR alleles, or between the badh2-E2 and the badh2-E7 alleles.     

Multinuclear NMR study and crystal structures of complexes of the types cis- and trans-Pt(amine)2I2

Complexes of the types cis- and trans-Pt(amine)₂I₂ were studied by spectroscopic methods, especially by multinuclear NMR spectroscopy. In ¹⁹⁵Pt NMR, the cis diiodo compounds with primary amines were observed between −3342 and −3357 ppm in acetone, while the trans compounds were found between −3336 and −3372 ppm. For the secondary amines, the chemical shifts were observed at lower fields. In ¹H NMR, the trans complexes were observed at higher fields than the cis compounds, while in ¹³C NMR, the reverse was observed. The ²J(¹⁹⁵Pt-¹H) and ³J(¹⁹⁵Pt-¹H) coupling constants are larger for the cis compounds (ave. 67 and 45 Hz, respectively) than for the trans isomers (ave. 59 and 38 Hz). In ¹³C NMR, the values of ²J(¹⁹⁵Pt-¹³C) and ³J(¹⁹⁵Pt-¹³C) were also found to be larger for the cis complexes (ave. 17 and 39 Hz versus 11 and 28 Hz). There seems to be a slight dependence of the pK_a values of the protonated amines or the proton affinity in the gas phase with the δ(Pt) chemical shifts. The crystal structures of eight diiodo complexes were determined. These compounds are cis-Pt(CH₃NH₂)₂I₂, cis-Pt(n-C₄H₉NH₂)₂I₂, cis-Pt(Et₂NH)₂I₂, trans-Pt(n-C₃H₇NH₂)₂I₂, trans-Pt(iso-C₃H₇NH₂)₂I₂, trans-Pt(n-C₄H₉NH₂)₂I₂, trans-Pt(t-C₄H₉NH₂)₂I₂ and trans-Pt(Me₂NH)₂I₂. The Pt-N bond distances located in trans position to the iodo ligands were compared to those located in trans position to the amines. The Pt-N bond in cis-Pt(Et₂NH)₂I₂ are much longer than the others, probably caused by the steric hindrance of the two very bulky ligands located in cis positions.     

Allelic variation and distribution independence of Puroindoline b-B2 variants and their association with grain texture in wheat

In this study, we identify the allelic variation of the Pinb-B2v3 variant, which could be divided into three different alleles, Pinb-B2v3a, Pinb-B2v3b and Pinb-B2v3c. The result of χ² tests showed that the distribution of Puroindoline b-2 variants has different frequencies in common and durum wheats. Analysis of the association of Pinb-B2v with grain hardness indicated that wheat cultivars with Pinb-B2v3b possessed relatively higher single kernel characterization system (SKCS) hardness indices in soft wheat in the 2006–2007 cropping season. Further analysis of SKCS hardness among different Puroindoline B-b2 variants by an F₈ recombinant inbred line (RIL) population containing 350 RILs indicated that lines with Pinb-2v3b were on average 5.4 SKCS hardness index units harder than those carrying the Pinb-2v2 haplotype. Derived cleaved amplified polymorphic sequence markers were developed for identification of Pinb-B2v3b and Pinb-B2v3c alleles and will be useful for screening early generation materials by marker-assisted selection during wheat breeding. The results of quantitative real-time PCR indicated that the relative expression level of Pinb-B2v3b was significantly higher than those of Pinb-B2v2, Pinb-B2v3a and Pinb-B2v3c, that four Pinb-B2 alleles showed the highest relative expression level on the 14th day after anthesis during grain development, and that relative expression levels of Pinb-B2v3b and Pinb-B2v2 in leaf were significantly higher than those in root, suggesting that PINB-2 are possibly not seed-specific proteins and that the expression level of Pinb-B2v3 was possibly positively correlated with grain hardness.     

NMDA receptors interact with flotillin-1 and -2, lipid raft-associated proteins

N-methyl-d-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the brain. Here we demonstrate interactions between the NR2A and NR2B subunits of NMDARs with flotillin-1 (flot-1), a lipid raft-associated protein. When mapped, analogous regions in the far distal C-termini of NR2A and NR2B mediate binding to flot-1, and the prohibitin homology domain of flot-1 contains binding sites for NR2A and NR2B. Although NR2B can also directly bind to flot-2 via a separate region of its distal C-terminus, NMDARs were significantly more colocalized with flot-1 than flot-2 in cultured hippocampal neurons. Overall, this study defines a novel interaction between NMDARs and flotillins.

Structured summary

MINT-7013094: NR2A (uniprotkb:Q00959), NR2B (uniprotkb:Q00960) and Flot2 (uniprotkb:Q9Z2S9) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7013147: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with NR2A (uniprotkb:Q00959) by anti bait coimmunoprecipitation (MI:0006)MINT-7013189: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013033: NR2A (uniprotkb:Q00959) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by two hybrid (MI:0018)MINT-7013178: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013197, MINT-7013210: NR2B (uniprotkb:Q00960) and NR2A (uniprotkb:Q00959) physically interact (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013002: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot1 (uniprotkb:O08917) by two hybrid (MI:0018)MINT-7013117: Flot1 (uniprotkb:Q9Z1E1), NR2B (uniprotkb:Q00960) and NR2A (uniprotkb:Q00959) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7013171: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by anti bait coimmunoprecipitation (MI:0006)MINT-7013017: NR2A (uniprotkb:Q00959) physically interacts (MI:0218) with Flot1 (uniprotkb:O08917) by two hybrid (MI:0018)MINT-7013054: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by two hybrid (MI:0018)MINT-7013129: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with NR2B (uniprotkb:Q00960) by anti bait coimmunoprecipitation (MI:0006)MINT-7013155: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with NR2B (uniprotkb:Q00960) by anti bait coimmunoprecipitation (MI:0006)MINT-7013074: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by two hybrid (MI:0018)MINT-7013162: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with NR2A (uniprotkb:Q00959) by anti bait coimmunoprecipitation (MI:0006)     

Neolignans from an Aniba species

A benzene extract of the trunk of an Aniba species (Lauraceae) contained benzyl benzoate, benzyl salicylate, sitosterol and the neolignans (2S,3S,3aR)-3a-allyl-5-methoxy-3-methyl-2-piperonyl-2,3,3a,6-tetrahydro-6-oxobenzofuran (burchellin); (2S,3S,3aR)-3a-allyl-5-methoxy-3-methyl-2-veratryl-2,3,3a,6-tetrahydro-6-oxobenzofuran; (2S,3S,3aR)-3a-allyl-5,7-dimethoxy-3-methyl-2-veratryl-2,3,3a,6-tetrahydro-6-oxobenzofuran; (2S,3S,5S)-5-allyl-5-methoxy-3-methyl-2-veratryl-2,3,5,6-tetrahydro-6-oxo-benzofuran; (2R,3R)-7-methoxy-3-methyl-5-propenyl-2-veratryl-2,3-dihydrobenzofuran; rel-(1R,5R,6R,7R,8S)-1-allyl-8-hydroxy-3-methoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene (guianin); rel-(1S,5S,6S,7R,8R)-1-allyl-8-hydroxy-3,5-dimethoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene; rel-(1S,5S,6S,7R,8R)-8-acetoxy-1-allyl-3-hydroxy-5-methoxy-7-methyl-4-oxo-6-piperonyl-bicyclo[3,2,1]oct-2-ene; rel-1S,5S,6S,7R,8R)-8-acetoxy-3,5-dimethoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene; rel-(1R,5S,6R,7R)-1-allyl-3-methoxy-7-methyl-4,8-dioxo-6-piperonylbicyclo[3,2,1]oct-2-ene.     

2(S),3(S)-3-hydroxy-4-methyleneglutamic Acid from Gleditsia caspica

A new natural product, 2(S),3(S)-3-hydroxy-4-methyleneglutamic acid (G3) has been isolated from seeds of Gleditsia caspica. The structure has been established by chemical and spectroscopic methods. Catalytic reduction of G3 yields 2(S),4(S)-4-methylglutamic acid and a new amino acid, 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid. Ozonolysis of G3 followed by oxidation gives 2(S),3(R)-3-hydroxyaspartic acid. The S- (or l-) configurations at C₂ in G3 and in 2(S),3(S),4(S)-3-hydroxy-4-methyglutamic acid and the S-configurations at C₃ for G3 and 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid and at C₄ for 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid are inferred from the configurations at C₂ in 2(_S),4(_S)-4-methylglutamic acid and at C₂ and C₃ in 2(S),3(R)-3-hydroxyaspartic acid. The seeds also contain appreciable quantities of 2(S),3(S),4(R)-3-hydroxy-4-methylglutami c acid (G1) and 2(S),4(R)-4-methylglutamic acid.     

Homogeneous CO hydrogenation and transfer hydrogenation catalyzed by ruthenium(II) complexes containing optically active Ph2PCH(Ph)CH(Me)NH2 and 1,2-C5H8(PPh2)2 ligands

Combination of (1S,2S)-cyclopentanediylbis(diphenylphosphine) with [Ru(η⁴-C₈H₁₂){η³-(CH₂)₂CMe}₂] afforded the chelate complex [Ru{η³-(CH₂)₂CMe}₂{(1S,2S)-C₅H₈(PPh₂)₂}] (1), which gave (OC-6-13)-[RuCl₂{(1S,2S)-C₅H₈(PPh₂)₂}{(1S,2S)-Ph₂PCH(Ph)CH(Me)NH₂}] (2) upon reaction with methanolic HCl in acetone, followed by the addition of the β-aminophosphine in DMF. The (P ∩ N)₂-chelated complexes (OC-6-13)-[RuCl₂{(1S,2S)-Ph₂PCH(Ph)CH(Me)NH₂}₂] (3) and (OC-6-13)-[RuCl₂{(1R,2S)-Ph₂PCH(Ph)CH(Me)NH₂}₂] (4) resulted from RuCl₃ · 3H₂O and the P,N ligands under reducing conditions. The crystal structures of 3 and 4 were determined by single-crystal X-ray diffraction. Following activation by KOBu-t in isopropanol, compounds 2–4 catalyzed the enantioselective transfer hydrogenation of acetophenone with i-PrOH as the hydrogen source as well as the direct hydrogenation of the ketone by H₂ in low to moderate e.e. (up to 67%).     

Mono(p-tolyl)platinum(II) and bis(p-tolyl)platinum(II) complexes of diethylsulfide as reagents for organoplatinum synthesis. Structures of [Pt(p-Tol)2(μ-SEt2)]2 and PtCl(p-Tol)(bpy) (bpy=2,2′-bipyridine)

The complex trans-PtCl(p-Tol)(SEt₂)₂ is obtained from the reaction of [Pt(p-Tol)₂(SEt₂)]₂ with PtCl₂(SEt₂)₂ and SEt₂ in mole ratio 1:2:2. The mono(p-tolyl)platinum(II) and bis(p-tolyl)platinum(II) complexes of diethylsulfide react with 2,2′-bipyridine to form the complexes PtX(p-Tol)(bpy) (X=p-Tol, Cl) and are useful reagents for organoplatinum chemistry. X-ray crystal structures are presented for square planar PtCl(p-Tol)(bpy) and the centrosymmetric dimer [Pt(p-Tol)₂(μ-SEt₂)]₂.     

Evidence for a third,Ir-associated histocompatibility region in theH-2 complex of the mouse

Skin grafts transplanted from B10.HTT donors onto (A.TL × B10)F₁ recipients are rapidly rejected despite the fact that the B10.HTT and A.TL strains should be carrying the sameH-2 chromosomes and that both the donor and the recipient contain the B10 genome. The rejection is accompanied by a production of cytotoxic antibodies against antigens controlled by theIr region of theH-2 complex. These unexpected findings are interpreted as evidence for a third histocompatibility locus in theH-2 complex,H-2I, located in theIr region close toH-2K. The B10.HTT and A.TL strains are postulated to differ at this hypothetical locus, and the difference between the two strains is explained as resulting from a crossing over between theH-2 ^t1 andH-2 ^s chromosomes in the early history of the B10.HTT strain. TheH-2 genotypes of the B10.HTT and A.TL strains are assumed to beH-2K ^s Ir ^s / ^k Ss ^k H-2D ^d andH-2K ^s Ir ^k Ss ^k H-2D ^d , respectively. Thus, theH-2 chromosomes of the two strains differ only in a portion of theIr region, including theH-2I locus. The B10.HTT(H-2 ^tt) and B10.S(7R)(H-2 ^th) strains differ in a relatively minor histocompatibility locus, possibly residing in theTla region outside of theH-2 complex.