The bitter side of the nightshades: Genomics drives discovery in Solanaceae steroidal alkaloid metabolism.

Steroidal alkaloids (SAs) and their glycosylated forms (SGAs) are toxic compounds largely produced by members of the Solanaceae and Liliaceae plant families. This class of specialized metabolites serves as a chemical barrier against a broad range of pest and pathogens. In humans and animals, SAs are considered anti-nutritional factors because they affect the digestion and absorption of nutrients from food and might even cause poisoning. In spite of the first report on SAs nearly 200 years ago, much of the molecular basis of their biosynthesis and regulation remains unknown. Aspects concerning chemical structures and biological activities of SAs have been reviewed extensively elsewhere; therefore, in this review the latest insights to the elucidation of the SAs biosynthetic pathway are highlighted. Recently, co-expression analysis combined with metabolic profiling revealed metabolic gene clusters in tomato and potato that contain core genes required for production of the prominent SGAs in these two species. Elaborating the knowledge regarding the SAs biosynthetic pathway, the subcellular transport of these molecules, as well as the identification of regulatory and signaling factors associated with SA metabolism will likely advance understanding of chemical defense mechanisms in Solanaceae and Liliaceae plants. It will also provide the means to develop, through classical breeding or genetic engineering, crops with modified levels of anti-nutritional SAs.

Manipulating duckweed through genome duplication.

Significant inter- and intraspecific genetic variation exists in duckweed, thus the potential for genome plasticity and manipulation is high. Polyploidy is recognised as a major mechanism of adaptation and speciation in plants. We produced several genome-duplicated lines of Landoltia punctata (Spirodela oligorrhiza) from both whole plants and regenerating explants using a colchicine-based cocktail. These lines stably maintained an enlarged frond and root morphology. DNA ploidy levels determined by florescence-activated cell sorting indicated genome duplication. Line A4 was analysed after 75 biomass doublings. Frond area, fresh and dry weights, rhizoid number and length were significantly increased versus wild type, while the growth rate was unchanged. This resulted in accumulation of biomass 17-20% faster in the A4 plants. We sought to determine if specific differences in gene products are found in the genome duplicated lines. Non-targeted ultra performance LC-quadrupole time of flight mass spectrometry was employed to compare some of the lines and the wild type to seek identification of up-regulated metabolites. We putatively identified differential metabolites in Line A65 as caffeoyl hexoses. The combination of directed genome duplication and metabolic profiling might offer a path for producing stable gene expression, leading to altered production of secondary metabolites.

Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes.

Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants--as, for example, potato--are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

Asymmetric adaptation to indolic and aliphatic glucosinolates in the B and Q sibling species of Bemisia tabaci (Hemiptera: Aleyrodidae).

The role glucosinolates play in defending plants against phloem feeders such as aphids and whiteflies is currently not clear as these herbivores may avoid bringing glucosinolates from the phloem sap into contact with myrosinase enzymes. Here, we investigated the effects of high levels of aliphatic and indolic glucosinolates on life history traits and detoxification gene expression in two sibling species, B and Q, of the whitefly Bemisia tabaci. High levels of aliphatic glucosinolates decreased the average oviposition rate of both species and reduced the survival and developmental rate of Q nymphs. High levels of indolic glucosinolates decreased the oviposition rate and survival of nymphal stages of the B species and the developmental rate of both species. Molecular analyses revealed two major asymmetries between the B and Q species. First, specific GST genes (BtGST1 and BtGST2) were significantly induced during exposure to indolic glucosinolates only in Q. This may reflect the genes putative involvement in indolic glucosinolates detoxification and explain the species' good performance on plants accumulating indolic glucosinolates. Second, the constitutive expression of eight of the 10 detoxification genes analysed was higher in the Q species than in the B species. Interestingly, four of these genes were induced in B in response to high levels of glucosinolates. It seems, therefore, that the B and Q species differ in their 'optimal defence strategy'. B utilizes inducible defences that are profitable if the probability of experiencing the stress is small and its severity is low, while Q invests significant resources in being always 'ready' for a challenge.

Prevalence and fate of Giardia cysts in wastewater treatment plants.

The present study was conducted to review factors affecting the prevalence and concentration of Giardia in raw wastewater. The removal and inactivation efficiency of Giardia by wastewater treatment technologies was also reviewed. Data published for the prevalence of Giardia in wastewater and the removal by wastewater treatment plants was reviewed. Giardia cysts are highly prevalent in wastewater in various parts of the world, which may reflect the infection rate in the population. In 23 of 30 (76.6%) studies, all of the tested raw wastewater samples were positive for Giardia cysts at concentrations ranging from 0.23 to 100 000 cysts l(-1). The concentration of Giardia in raw wastewater was not affected by the geographical region or the socio-economic status of the community. Discharge of raw wastewater or the application of raw wastewater for irrigation may result in Giardia transmission. Activated sludge treatment resulted in a one to two orders of magnitude reduction in Giardia, whereas a stabilization pond with a high retention time removed up to 100% of the cysts from wastewater. High-rate sand filtration, ultrafiltration and UV disinfection were reported as the most efficient wastewater treatment methods for removal and disinfection of Giardia cysts. Wastewater treatment may not totally prevent the environmental transmission of Giardia cysts. The reviewed data show that a combination of wastewater treatment methods may results in efficient removal of Giardia cysts and prevent their environmental transmission.

Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.

Comparison of two treatments for the removal of selected organic micropollutants and bulk organic matter: conventional activated sludge followed by ultrafiltration versus membrane bioreactor.

The potential of membrane bioreactor (MBR) systems to remove organic micropollutants was investigated at different scales, operational conditions, and locations. The effluent quality of the MBR system was compared with that of a plant combining conventional activated sludge (CAS) followed by ultrafiltration (UF). The MBR and CAS-UF systems were operated and tested in parallel. An MBR pilot plant in Israel was operated for over a year at a mixed liquor suspended solids (MLSS) range of 2.8-10.6 g/L. The MBR achieved removal rates comparable to those of a CAS-UF plant at the Tel-Aviv wastewater treatment plant (WWTP) for macrolide antibiotics such as roxythromycin, clarithromycin, and erythromycin and slightly higher removal rates than the CAS-UF for sulfonamides. A laboratory scale MBR unit in Berlin - at an MLSS of 6-9 g/L - showed better removal rates for macrolide antibiotics, trimethoprim, and 5-tolyltriazole compared to the CAS process of the Ruhleben sewage treatment plant (STP) in Berlin when both were fed with identical quality raw wastewater. The Berlin CAS exhibited significantly better benzotriazole removal and slightly better sulfamethoxazole and 4-tolyltriazole removal than its MBR counterpart. Pilot MBR tests (MLSS of 12 g/L) in Aachen, Germany, showed that operating flux significantly affected the resulting membrane fouling rate, but the removal rates of dissolved organic matter and of bisphenol A were not affected.

Complication of laparoscopic detorsion of adnexal mass.

Detorsion of an ischemic adnexal mass has recently been advocated for most cases of twisted adnexa. Usually, the affected ovary regains some or all of its vitality and function. However, when the ovary is completely necrotic, it may form an abscess if it contains tissue components that cannot be eliminated by the peritoneal immune system. We report a case of pelvic abscess formation in a detorsed ovary that previously contained an unsuspected dermoid cyst. We call for an extensive inspection of the detorsed ovary before ending the laparoscopic operation, and if it remains necrotic and is suspected of containing a dermoid cyst, it should be removed promptly.

UV disinfection of wastewater effluents for unrestricted irrigation.

Wastewater reuse in arid regions is important for the production of a water resource to be utilised for non-potable purposes and to prevent the environmental transmission of disease-causing agents. This study was conducted to evaluate the effect of water quality on the comparative disinfection efficiency of viruses, bacteria and spores by UV irradiation. Furthermore, the microbial quality of effluent produced by coagulation, high rate filtration (HRF) and either UV irradiation or chlorination was determined. Using low pressure collimated beam, a UV dose of 80 mWs/cm2 was needed to achieve a 3-log10 inactivation of either rotavirus SA-11 or coliphage MS2, whereas over 5-log10 inactivation of E. coli was reached with a dose of only 20 mWs/cm2. B. subtilis inactivation was found to be linear up to a dose of 40 mWs/cm2 and then a tailing up to a UV dose of 120 mWs/cm2 was observed. It is worth noting that effluent turbidity of < 5 NTU did not influence the inactivation efficiency of UV irradiation. Operation of a pilot plant to treat secondary effluent by coagulation, HRF and UV disinfection at a UV dose of 80 mWs/cm2 resulted in the production of high quality effluent in compliance with the Israel standards for unrestricted irrigation (< 10 CFU/100 mL faecal coliform and turbidity of < 5 NTU). Sulphite reducing clostridia (SRC) were found to be more resistant than coliphages and F coliform for UV irradiation. The results of this study indicated that UV disinfection is suitable for the production of effluents for unrestricted irrigation of food crops.

Non-isomerizable artificial pigments: implications for the primary light-induced events in bacteriorhodopsin.

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the "critical" C13=C14 bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump-probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck-Condon (FC) state does not involve substantial C13=C14 torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C13=C14 isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.

The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.

Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co-ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3-rutinoside (an anthocyanin) and of quercetin-glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were adversely affected by FaMYB1 overexpression. Two-hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C-terminus of FaMYB1 contains the motif pdLNL(D)/(E)Lxi(G)/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.

Retinal isomerization in bacteriorhodopsin is controlled by specific chromophore-protein interactions. A study with noncovalent artificial pigments.

It has previously been shown that, in mutants lacking the Lys-216 residue, protonated Schiff bases of retinal occupy noncovalently the bacteriorhodopsin (bR) binding site. Moreover, the retinal-Lys-216 covalent bond is not a prerequisite for initiating the photochemical and proton pump activity of the pigment. In the present work, various Schiff bases of aromatic polyene chromophores were incubated with bacterioopsin to give noncovalent pigments that retain the Lys-216 residue in the binding site. It was observed that the pigment's absorption was considerably red-shifted relative to the corresponding protonated Schiff bases (PSB) in solution and was sensitive to Schiff base linkage substitution. Their PSB pK(a) is considerably elevated, similarly to those of related covalently bound pigments. However, the characteristic low-pH purple to blue transition is not observed, but rather a chromophore release from the binding site takes place that is characterized by a pK(a) of approximately 6 (sensitive to the specific complex). It is suggested that, in variance with native bR, in these complexes Asp-85 is protonated and Asp-212 serves as the sole negatively charged counterion. In contrast to the bound analogues, no photocycle could be detected. It is suggested that a specific retinal-protein geometrical arrangement in the binding site is a prerequisite for achieving the selective retinal photoisomerization.

Nonisomerizable non-retinal chromophores initiate light-induced conformational alterations in bacterioopsin.

The photoactivation of retinal proteins is usually interpreted in terms of C=C photoisomerization of the retinal moiety, which triggers appropriate conformational changes in the protein. In this work several dye molecules, characterized by a completely rigid structure in which no double-bond isomerization is possible, were incorporated into the binding site of bacteriorhodopsin (bR). Using a light-induced chemical reaction of a labeled EPR probe, it was observed that specific conformational alterations in the protein are induced following light absorption by the dye molecules occupying the binding site. The exact nature of these changes and their relationship to those occurring in the bR photocycle are still unclear. Nevertheless, their occurrence proves that C=C or C=NH(+) isomerization is not a prerequisite for protein conformational changes in a retinal protein. More generally, we show that conformational changes, leading to changes in reactivity, may be induced in proteins by optical excitation of simple nonisomerizable dyes located in the macromolecular matrix.

Cytosolic aldolase is a ripening related enzyme in strawberry fruits (Fragaria x ananassa).

Two aldolase isoenzymes have been isolated from ripe strawberry fruits (Fragaria x ananassa cv. Camarosa and Elsanta) and partially purified by DEAE anion exchange and Sephacryl size exclusion chromatography. The isoenzymes were identified as class I cytosol and plastid aldolase on the basis of their chromatographic behavior on DEAE-cellulose columns, native molecular weight, pH optimum pattern, Km value for D-fructose-1,6-bisphosphate, tendency to be inactivated by lower pH values and SDS-PAGE subunit determination of 40 and 38 kDa, respectively. Total aldolase activity and distribution of both aldolase isoenzymes was also investigated at different stages of strawberry fruit ripening. Strawberries in the green and white ripening stage showed the same ratio of the two isoenzymes as green leaves with 15 and 8% cytosol aldolase activity, respectively. During strawberry fruit development the overall total aldolase activity decreased until the pink ripening stage and then increased due to a rise of cytosol aldolase yielding up to 75% in red strawberries. A cDNA putatively encoding the cytosolic form of aldolase in strawberry was cloned during the course of this study. Both microarray and RNA gel blot analyses showed that the cytosolic aldolase gene expression is induced during ripening as detected for the cytosolic aldolase enzyme. We suggest that induction of the cytosolic aldolase both at the levels of transcription and translation might be part of a ripening related stress response in the receptacle tissue.

Bacteriorhodpsin experiences light-induced conformational alterations in nonisomerizable C(13)=C(14) pigments. A study with EPR.

The mechanism by which bacteriorhodopsin is activated following light absorption is not completely clear. We have detected protein conformational alterations following light absorption by retinal-based chromophores in the bacteriorhodopsin binding site by monitoring the rate of reduction-oxidation reactions of covalently attached spin labels, using EPR spectroscopy. It was found that the reduction reaction with hydroxylamine is light-catalyzed in the A103C-labeled pigment but not in E74C or M163C. The reaction is light-catalyzed even when isomerization of the C(13)=C(14) bond of the retinal chromophore is prevented. The reverse oxidation reaction with molecular oxygen is effective only in apomembrane derived from the mutant A103C. This reaction is light-accelerated following light absorption of the retinal oxime, which occupies the binding site. The light-induced acceleration is evident also in "locked" bacteriorhodopsin in which isomerization around the C(13)=C(14) bond is prevented. It is evident that the chromophore-protein covalent bond is not a prerequisite for protein response. In contrast to the case of the retinal oxime, a reduced C=N bond A103C-labeled pigment did not exhibit acceleration of the oxidation reaction following light absorption. Acceleration was observed, however, following substitution of the polyene by groups that modify the excited state charge delocalization. It is suggested that protein conformational alterations are induced by charge redistribution along the retinal polyene following light absorption.

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

The application of DNA microarrays in gene expression analysis.

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

Assessment of endometrial receptivity for gestation in patients undergoing in vitro fertilization, using endometrial thickness and the endometrium-myometrium relative echogenicity coefficient.

OBJECTIVE: To evaluate the outcome of in vitro fertilization (IVF) treatment in relation to the sonographic parameters of the endometrium. DESIGN AND METHODS: Seventy-five patients with no uterine pathology (age 31.1 +/- 5.4 years) treated in our IVF clinic for various indications were assessed during 75 cycles in which good-quality (grades 1 and 2) embryos were transferred. Controlled ovarian stimulation was achieved by the long protocol (gonadotropin releasing hormone agonist and gonadotropins). The bilayered endometrial thickness (BET), estradiol, luteinizing hormone and progesterone serum levels were measured in 272 tests. A special computer program was used to measure endometrial echogenicity relative to myometrial echogenicity. The gray-level data were analyzed on the basis of the midsagittal sonographic uterine image. Endometrium-myometrium relative echogenicity coefficient (E/M REC) values were computed and displayed graphically along the anteroposterior axis of the endometrial layers in the upper part of the uterine cavity. The area under the E/M REC curve within the BET limits was defined as the relative echogenicity area (REA) and was used as a measure of endometrial echogenicity. Each cycle was sampled in six time segments representing desensitization, follicular and luteal phases. Assigning the day of ovum pick-up as day 0, the time segments of each cycle were: first, day -20 to day -11; second, day -10 to day -6; third, day -5 to day -2; fourth, day 0; fifth, day +7 to day +14; sixth, day +15 to day +21. RESULTS: A total of 276 embryos were transferred (3.68 +/- 1.01 per cycle), of which 223 were of good quality (2.97 +/- 1.51 per cycle). An intrauterine pregnancy was diagnosed in 29 patients. All patients in this study had a BET of > 5 mm in the third and the fourth time segments. There was no significant difference in BET and REA between pregnant and non-pregnant patients tested in the first to the fifth time segments of the IVF cycles. Both BET and REA measured in the sixth time segment were significantly higher in pregnant compared to non-pregnant patients. CONCLUSIONS: Our results suggest that the proposed sonographic assessment of the endometrium shows no benefit in characterization of uterine receptivity in IVF patients with a reactive endometrium. High BET and REA values can indicate pregnancy during the sixth time segment, when the decidualization of the endometrium is well established.

Prospective randomized comparison between an open and closed vaginal cuff in abdominal hysterectomy.

OBJECTIVE: To compare an operative and postoperative course of open vaginal cuff hysterectomy and closed vaginal cuff hysterectomy, and to correlate the length of stay, febrile morbidity and the incidence of pelvic fluid collections to the type of surgery. PARTICIPANTS: One-hundred women scheduled for hysterectomy were prospectively randomized into two groups that underwent either a closed or an open vaginal cuff technique. RESULTS: The open vaginal cuff technique took on average 19% more time than the closed vaginal cuff operation (P < 0.05, t-test). The incidence and size of pelvic fluid collections was significantly higher after the closed vaginal cuff hysterectomy than after the open technique (P < 0.01, t-test). However, the postoperative length of stay, febrile morbidity and the rate of complications were similar. CONCLUSIONS: Both techniques of hysterectomy produced a similar postoperative course despite the fact that the closed vaginal cuff technique resulted in a higher incidence of pelvic fluid collections. Therefore considering a shorter operation time for the closed vaginal cuff hysterectomy, this technique seems slightly preferable.

Differences between Twin Umbilical Coiling Indices Correlate to Differences in Twin Weight and Doppler Indices.

> Objective: The aim of our study was to evaluate sonographic and Doppler detectable differences in umbilical coiling index and fetoplacental circulation of discordant twins. Study Design: Doppler blood flow studies in 13 pairs of concordant and 20 pairs of discordant twins were performed from umbilical artery, middle cerebral artery, inferior vena cava, and ductus venosus. Flow studies were compared and correlated with the antenatal sonographic coiling index and the actual umbilical cord length, number of vascular helices, and birth weight. All studies were performed within 72 h before delivery. Pulsatility index (PI) values were calculated for the arteries and preload index (PLI) values for the veins. The umbilical coiling index (CI) was calculated using sonographic longitudinal views of cord vessels from several segments antenatally and by dividing the total number of helices by cord length (cm) postnatally. Discordancy was defined as a more than 20% intrapair actual birth weight difference. For all these index values the intertwin differences (Delta values) were calculated by subtracting the values obtained in the larger twin with those of the smaller twin. Results: The mean +/- SD intertwin difference in umbilical coiling index was 27.4 +/- 10.5% in the antepartum period and 28.9 +/- 10.0% after birth. Regression analysis showed a significant linear trend (r = 0.77, P < 0.001) between intertwin birth weight difference (DeltaBW) and intertwin coiling index difference (DeltaCI). A good correlation was found between DeltaCI and DeltaPLI in the ductus venosus (r = 0.63, P < 0.05), DeltaPLI in the inferior vena cava (r = 0.51, P < 0.005), and DeltaPI in the middle cerebral artery (r = 0.44, P < 0.05). Conclusions: Intertwin difference in antepartum umbilical coiling index can be determined by ultrasound and correlates well with: 1) the actual difference in coiling indices at birth, 2) the intertwin birth weight difference and 3) the intertwin Doppler flow characteristics in the fetal cerebral and venous circulation.