Evidence for a Second Type of Resistance against Cydia pomonella Granulovirus in Field Populations of Codling Moths.

Cydia pomonella granulovirus (CpGV) is an important biocontrol agent for the codling moth (CM) in organic and integrated apple production worldwide. Previously, Z chromosome-linked dominant resistance in at least 38 CM field populations in Europe was reported, threatening organic apple production. Growers responded by switching to a different resistance-breaking isolate of CpGV that could control these populations. Here, we report a nonuniform response of different CM field populations to CpGV isolates from CpGV genome groups A to E. Even more strikingly, one field population, NRW-WE, was resistant to all known CpGV genome groups except group B. Single-pair crossing experiments with a susceptible strain, followed by resistance testing of the F1 offspring, clearly indicated cross-resistance to CpGV isolates that had been considered to be resistance breaking. This finding provides clear evidence of a second, broader type of CpGV resistance with a novel mode of inheritance that cannot be fully explained by Z-linkage of resistance. IMPORTANCE: CpGV is registered and used in virtually all commercial apple growing areas worldwide and is therefore the most widely used baculovirus biocontrol agent. Recently, resistance to CpGV products was reported in different countries in Europe, threatening organic growers who rely almost exclusively on CpGV products. This resistance appeared to be targeted against a 24-bp repeat in the pe38 gene in isolate CpGV-M of genome group A, which had been used commercially for many years. On the other hand, resistance could be broken by CpGV isolates from CpGV genome groups B to E. Here, we report clear evidence of a second type of field resistance that is also directed against resistance-breaking isolates of CpGV genome groups C, D, and E and which appears not to be targeted against CpGV pe38 Therefore, we propose to differentiate between type I resistance, which is targeted against pe38 of CpGV genome group A, and a novel type II resistance with an unknown molecular target. This finding stresses the need for further adoption of resistance management strategies for CpGV, since growers cannot rely solely on the use of resistance-breaking CpGV isolates.

A Third Type of Resistance to Cydia pomonella Granulovirus in Codling Moths Shows a Mixed Z-Linked and Autosomal Inheritance Pattern.

Different isolates of Cydia pomonella granulovirus (CpGV) are used worldwide to control codling moth larvae (Cydia pomonella) in pome fruit production. Two types of dominantly inherited field resistance of C. pomonella to CpGV have been recently identified: Z-chromosomal type I resistance and autosomal type II resistance. In the present study, a CpGV-resistant C. pomonella field population (termed SA-GO) from northeastern Germany was investigated. SA-GO individuals showed cross-resistance to CpGV isolates of genome group A (CpGV-M) and genome group E (CpGV-S), whereas genome group B (CpGV-E2) was still infective. Crossing experiments between individuals of SA-GO and the susceptible C. pomonella strain CpS indicated the presence of a dominant autosomal inheritance factor. By single-pair inbreeding of SA-GO individuals for two generations, the genetically more homogenous strain CpRGO was generated. Resistance testing of CpRGO neonates with different CpGV isolates revealed that isolate CpGV-E2 and isolates CpGV-I07 and -I12 were resistance breaking. When progeny of hybrid crosses and backcrosses between individuals of resistant strain CpRGO and susceptible strain CpS were infected with CpGV-M and CpGV-S, resistance to CpGV-S appeared to be autosomal and dominant for larval survivorship but recessive when success of pupation of the hybrids was considered. Inheritance of resistance to CpGV-M, however, is proposed to be both autosomal and Z linked, since Z linkage of resistance was needed for pupation. Hence, we propose a further type III resistance to CpGV in C. pomonella, which differs from type I and type II resistance in its mode of inheritance and response to CpGV isolates from different genome groups.IMPORTANCE The baculovirus Cydia pomonella granulovirus (CpGV) is registered and applied as a biocontrol agent in nearly all pome fruit-growing countries worldwide to control codling moth caterpillars in an environmentally friendly manner. It is therefore the most widely used commercial baculovirus biocontrol agent. Since 2005, field resistance of codling moth to CpGV products has been observed in more than 40 field plantations in Europe, threatening organic and integrated apple production. Knowledge of the inheritance and mechanism(s) of resistance is indispensable for the understanding of host response to baculovirus infection on the population level and the coevolutionary arms race between virus and host, as well as for the development of appropriate resistance management strategies. Here, we report a codling moth field population with a new type of resistance, which appears to follow a highly complex inheritance in regard to different CpGV isolates.

Model order reduction techniques to identify submarining risk in a simplified human body model.

This work investigates linear and non-linear parametric reduced order models (ROM) capable of replacing computationally expensive high-fidelity simulations of human body models (HBM) through a non-intrusive approach. Conventional crash simulation methods pose a computational barrier that restricts profound analyses such as uncertainty quantification, sensitivity analysis, or optimization studies. The non-intrusive framework couples dimensionality reduction techniques with machine learning-based surrogate models that yield a fast responding data-driven black-box model. A comparative study is made between linear and non-linear dimensionality reduction techniques. Both techniques report speed-ups of a few orders of magnitude with an accurate generalization of the design space. These accelerations make ROMs a valuable tool for engineers.

Parameters predicting COVID-19-induced myocardial injury and mortality.

Clinical manifestations of COVID-19 affect many organs, including the heart. Cardiovascular disease is a dominant comorbidity and prognostic factors predicting risk for critical courses are highly needed. Moreover, immunomechanisms underlying COVID-induced myocardial damage are poorly understood. OBJECTIVE: To elucidate prognostic markers to identify patients at risk. RESULTS: Only patients with pericardial effusion (PE) developed a severe disease course, and those who died could be identified by a high CD8/Treg/monocyte ratio. Ten out of 19 COVID-19 patients presented with PE, 7 (78%) of these had elevated APACHE-II mortality risk-score, requiring mechanical ventilation. At admission, PE patients showed signs of systemic and cardiac inflammation in NMR and impaired cardiac function as detected by transthoracic echocardiography (TTE), whereas parameters of myocardial injury e.g. high sensitive troponin-t (hs-TnT) were not yet increased. During the course of disease, hs-TnT rose in 8 of the PE-patients above 16 ng/l, 7 had to undergo ventilatory therapy and 4 of them died. FACS at admission showed in PE patients elevated frequencies of CD3+CD8+ T cells among all CD3+ T-cells, and lower frequencies of Tregs and CD14+HLA-DR+-monocytes. A high CD8/Treg/monocyte ratio predicted a severe disease course in PE patients, and was associated with high serum levels of antiviral cytokines. By contrast, patients without PE and PE patients with a low CD8/Treg/monocyte ratio neither had to be intubated, nor died. CONCLUSIONS: PE predicts cardiac injury in COVID-19 patients. Therefore, TTE should be performed at admission. Immunological parameters for dysfunctional antiviral immunity, such as the CD8/Treg/monocyte ratio used here, supports risk assessment by predicting poor prognosis.

Functional plasticity in the immature striate cortex of the monkey shown by the [14C]deoxyglucose method.

Autoradiographic representation of the local rates of cerebral glucose utilization and local cerebral functional activity by means of the [14C]deoxyglucose technique reveals the existence of the ocular dominance columns in the striate cortex of the monkey in the first day of life. In contrast to the stability of these columns in more mature brain, monocular deprivation for 3 months from the first day of life results in their complete disappearance and a reversion of the autoradiographic pattern to that seen in animals with normal binocular vision. These results are consistent with a reorganization of the representation of the visual fields of the two eyes in the striate cortex and provide additional evidence of the plasticity of the striate cortex of the monkey in early life.

Mapping of functional neural pathways by autoradiographic survey of local metabolic rate with (14C)deoxyglucose.

An enzymatic preparation from human brain converts tryptamine to tryptoline (9H-1,2,3,4-tetrahydropyrido(3,4-b)indole) in the presence of 5-methyltetrahydrofolic acid. Similarly, N-methyltryptamine and 5-hydroxytryptamine yield 1-methyltryptoline and 5-hydroxytryptoline, respectively. Neither in vitro nor in vivo formation of these compounds by human tissues has been described.

Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family.

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500-1,000 nm in length and 50-100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus family.

Overcoming the resistance of codling moth against conventional Cydia pomonella granulovirus (CpGV-M) by a new isolate CpGV-I12.

Recently, codling moth (CM, Cydia pomonella L.) populations with a significantly reduced susceptibility to C. pomonella granulovirus (CpGV) products have been observed in Germany. A novel CpGV isolate, designated CpGV-I12, is able to overcome the CpGV resistance. CpGV-I12 originated from Iran and showed superior efficacy in laboratory bioassays against a resistant CM strain (CpR), which has a 100-fold reduced susceptibility to commercially used isolate CpGV-M. Determination of the median lethal concentration (LC(50)) indicated that CpGV-I12 is nearly as efficient in resistant CpR as CpGV-M in a susceptible CM strain (CpS). Beyond, CpGV-I12 caused superior mortality in CpS. Infection experiments showed that the resistance breaking effect can be observed in all instars of CpR. CpGV-I12 is a promising alternative control agent of CM in orchards where conventional CpGV products fail. In addition, we demonstrate in bioassays with recombinant expressed Cry1Ab that cross-resistance to CpGV and Bacillus thuringiensis products is not likely.

Effects of transgenic corn and Cry1Ab protein on the nematode, Caenorhabditis elegans.

The effects of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) on the nematode, Caenorhabditis elegans, were studied with soil from experimental fields cultivated with transgenic Bt corn (MON810) and with trypsinized Cry1Ab protein expressed in Escherichia coli. The content of Cry1Ab protein was above the detection limit of an ELISA test in only half of the soil samples obtained from transgenic plots, ranging from 0.19 to 1.31 ng g(-1) dry weight. In a laboratory bioassay, C. elegans was exposed to rhizosphere and bulk soil from fields with isogenic or transgenic corn or to solutions of Cry1Ab protein (0, 24, 41, 63, 118, and 200 mg l(-1)) over a period of 96 h, with growth and reproduction serving as the test parameters. Nematode reproduction and growth were significantly reduced in rhizosphere and bulk soil of Bt corn compared with soil from isogenic corn and were significantly correlated with concentrations of the Cry1Ab protein in the soil samples. Moreover, the toxicity of pure Cry1Ab protein to the reproduction and growth of C. elegans was concentration-dependent. As significant inhibition occurred at relatively high concentrations of the Cry1Ab protein (41 mg l(-1)), the effects of the soil samples from Bt corn could not be assigned directly to the toxicity of the Cry1Ab protein. The results demonstrate that bioassays with the nematode, C. elegans, provide a promising tool for monitoring the potential effects of Bt toxins in aqueous medium and soils.

Comparative effects of acute and chronic administration of amphetamine on local cerebral glucose utilization in the conscious rat.

The 2-deoxyglucose method was employed in rats following either acute or chronic administration of d-amphetamine. The drug was given either by a single intravenous and/or repeated daily intraperitoneal injections or by osmotic pumps implanted subcutaneously. Each mode of administration resulted in a specific constellation of metabolic effects. Acute doses of d-amphetamine, 5 mg/kg, stimulated glucose utilization in a number of cerebral structures, particularly the components of the extrapyramidal motor system. No effects were observed in components of the mesolimbic dopaminergic system. Repeated daily doses of 5 mg/kg for 2 weeks had no effects unless the dosage was progressively increased to toxic levels of 15 mg/kg over a 3-week period. Dosage sustained by osmotic pumps (12-15 mg/kg/day for 1 week or 6-7 mg/kg/day for 2 weeks), however, resulted in a selected increase in glucose utilization in the nucleus accumbens, an important component of the mesolimbic system. This finding may be of significance to the mechanism of amphetamine psychosis, which is sometimes regarded as a model of schizophrenia and is considered to be evidence in support of the dopamine hypothesis of the disease.

Role of the cerebellar fastigial nucleus in the physiological regulation of cerebral blood flow.

Local cerebral blood flow (ICBF) was measured with [14C]iodoantipyrine in conscious, unrestrained rats during electrical stimulation of the fastigial nucleus (FN). Electrode position in the FN was determined by blood pressure (MABP) responses to stimulation under anesthesia. In nine rats in which MABP responses had been variable under anesthesia, bipolar stimulation (50 Hz, 0.5 ms, 1 s on/1 s off) with currents of 30-100 microA after recovery from anesthesia produced stereotypic behavior but little effect on MABP and ICBF. In seven other conscious rats currents could be raised to 75-200 microA without inducing seizures, resulting in sustained MABP elevations during the ICBF measurement and significantly increased ICBF in the sensory-motor (+45%), parietal (+31%), and frontal cortices (+56%) and the caudate-putamen (+27%) above control values (n = 9). Glucose utilization, measured with [14C]deoxyglucose, in rats similarly stimulated was significantly increased in six structures, including some of the above, indicating increases in ICBF due to metabolic activation. Unilateral or bilateral electrolytic lesions of the FN, placed 6-7 days before ICBF measurement, had negligible effects on resting ICBF and on autoregulation in conscious rats. These results fail to support a specific role for the FN in physiological regulation of cerebral blood flow in unanesthetized rats.

Measurement of local cerebral blood flow with [14C]iodoantipyrine in the mouse.

Local cerebral blood flow was measured in the mouse by means of the [14C]iodoantipyrine method. This method has been previously used in the monkey, dog, cat, and rat, but its application to small mammals such as the mouse requires special attention to potential sources of error. The small size of the mouse brain requires special attention to the rapid removal and freezing of the brain to minimize effects of postmortem diffusion of tracer in the tissue. Because of the relatively low diameter/length ratios of the catheters needed for arterial sampling in small animals, substantial errors can occur in the determination of the time course of the [14C]iodoantipyrine concentration in the arterial blood unless corrections for lag time and dead space washout in the catheter are properly applied. Local cerebral blood flow was measured in seven awake mice with appropriate care to minimize these sources of error. The values were found to vary from 48 ml/100 g/min in the corpus callosum to 198 ml/100 g/min in the inferior colliculus. The results demonstrate that the [14C]iodoantipyrine method can be used to measure local cerebral blood flow in the mouse and that the values in that species are, in general, somewhat higher than those in the rat.

Examination of potential mechanisms in the enhancement of cerebral blood flow by hypoglycemia and pharmacological doses of deoxyglucose.

Cerebral blood flow (CBF) rises when the glucose supply to the brain is limited by hypoglycemia or glucose metabolism is inhibited by pharmacological doses of 2-deoxyglucose (DG). The present studies in unanesthetized rats with insulin-induced hypoglycemia show that the increases in CBF, measured with the [14C]iodoantipyrine method, are relatively small until arterial plasma glucose levels fall to 2.5 to 3.0 mM, at which point CBF rises sharply. A direct effect of insulin on CBF was excluded; insulin administered under euglycemic conditions maintained by glucose injections had no effects on CBF. Insulin administration raised plasma lactate levels and decreased plasma K+ and HCO3- concentrations and arterial pH. These could not, however, be related to the increased CBF because insulin under euglycemic conditions had similar effects without affecting CBF; furthermore, the inhibition of brain glucose metabolism with pharmacological doses (200 mg/kg intravenously) of DG increased CBF, just like insulin hypoglycemia, without altering plasma lactate and K+ levels and arterial blood gas tensions and pH. Nitric oxide also does not appear to mediate the increases in CBF. Chronic blockade of nitric oxide synthase activity by twice daily i.p. injections of NG-nitro-L-arginine methyl ester for 4 days or acutely by a single i.v. injection raised arterial blood pressure and lowered CBF in normoglycemic, hypoglycemic, and DG-treated rats but did not significantly reduce the increases in CBF due to insulin-induced hypoglycemia (arterial plasma glucose levels, 2.5-3 mM) or pharmacological doses of deoxyglucose.

Blockade of cerebral blood flow response to insulin-induced hypoglycemia by caffeine and glibenclamide in conscious rats.

The possibility that adenosine and ATP-sensitive potassium channels (KATP) might be involved in the mechanisms of the increases in cerebral blood flow (CBF) that occur in insulin-induced hypoglycemia was examined. Cerebral blood flow was measured by the [14C]iodoantipyrine method in conscious rats during insulin-induced, moderate hypoglycemia (2 to 3 mmol/L glucose in arterial plasma) after intravenous injections of 10 to 20 mg/kg of caffeine, an adenosine receptor antagonist, or intracisternal infusion of 1 to 2 mumol/L glibenclamide, a KATP channel inhibitor. Cerebral blood flow was also measured in corresponding normoglycemic and drug-free control groups. Cerebral blood flow was 51% higher in untreated hypoglycemic than in untreated normoglycemic rats (P < 0.01). Caffeine had a small, statistically insignificant effect on CBF in normoglycemic rats, but reduced the CBF response to hypoglycemia in a dose-dependent manner, i.e., 27% increase with 10 mg/kg and complete elimination with 20 mg/kg. Chemical determinations by HPLC in extracts of freeze-blown brains showed significant increases in the levels of adenosine and its degradation products, inosine and hypoxanthine, during hypoglycemia (P < 0.05). Intracisternal glibenclamide had little effect on CBF in normoglycemia, but, like caffeine, produced dose-dependent reductions in the magnitude of the increases in CBF during hypoglycemia, i.e., +66% with glibenclamide-free artificial CSF administration, +25% with 1 mumol/L glibenclamide, and almost complete blockade (+5%) with 2 mumol/L glibenclamide. These results suggest that adenosine and KATP channels may play a role in the increases in CBF during hypoglycemia.

Identification and sequence analysis of the integration site of transposon TCp3.2 in the genome of Cydia pomonella granulovirus.

Recently, we described the isolation and characterisation of the novel lepidopteran transposon TCp3.2, which was found to be inserted into the genome of a spontaneous mutant of the baculovirus Cydia pomonella granulovirus (CpGV). Transposon TCp3.2, which is a member of the Tcl/mariner superfamily, is an apparently defective copy which became stably integrated into the viral genome (Jehle et al., 1997. J. Mol. Evol., in press). In this study, we located, cloned and sequenced a genomic region of 2.5 kb of CpGV which encompasses the insertion site of TCp3.2. The TCp3.2 was inserted at a TA dinucleotide as it is typical for many Tcl/mariner-like transposons. The TA insertion site was localised within a non-translated region downstream of the homologous gene of baculovirus late expression factor 2 (lef-2). Additionally, three other complete open reading frames (ORF35Ra, ORF35Rb, and ORF36L) with unknown functions were identified. Transposon insertion into intergenic regions of viral genomes may contribute to the genotypic variability of baculoviruses without any phenotypic effect.

Characterization of the ecdysteroid UDP-glucosyltransferase gene of a single nucleocapsid nucleopolyhedrovirus of Buzura suppressaria.

A putative ecdysteroid UDP-glucosyltransferase (egt) gene was identified in the single nucleocapsid nucleopolyhedrovirus of Buzura suppressaria (BusuNPV). This is the first egt gene to be characterized in a SNPV, suggesting that egt genes are prevalent in nucleopolyhedroviruses and possibly in all baculoviruses. The open reading frame (ORF) of the gene is 1539 nucleotides (nt) long, encoding a putative protein (EGT) of 513 amino acids (aa) with a M of 58922. The 5' noncoding region contains three possible TATA boxes. A polyadenylation signal, AATAAA, was found downstream of the translation stop codon. A putative signal peptide of 16 residues was present at the N-terminus of the EGT. The BusuNPV egt gene has a high degree of nucleotide and amino acid sequence homology to multiple nucleocapsid (M) NPV egt genes, the highest being to the Spodoptera exigua MNPV egt. A phylogenetic tree of eleven known EGTs was constructed using maximum parsimony analysis.

Uterus didelphys with unilateral imperforate vagina: spontaneous rupture of the vaginal septum.

A 13-year-old girl with abnormal vaginal bleeding was found to have uterus didelphys with unilateral imperforate vagina. A brief discussion of this rare anomaly and its treatment accompanies the case report.

Preservation of autoregulatory cerebral vasodilator responses to hypotension after inhibition of nitric oxide synthesis.

Effects of inhibition of nitric oxide (NO) synthesis on the cerebrovascular autoregulatory vasodilator response to hypotension were studied in conscious rats. Cerebral blood flow (CBF) was determined with [14C]iodoantipyrine in a saline-treated control group and in three groups following inhibition of NO synthase activity by twice daily intraperitoneal injections of 50 mg/kg of NG-nitro-L-arginine methyl ester (L-NAME) for four days. In the saline-control group (n = 8) and in the L-NAME-treated Group (a) (n = 8) CBF was determined while systemic mean arterial blood pressure (MABP) remained at its resting level (means +/- S.D., 128 +/- 6 and 151 +/- 11 mmHg, respectively). In the other groups CBF was determined after MABP was reduced by blood withdrawal to 118 +/- 9 and 88 +/- 8 mmHg in Groups (b) (n = 8) and (c) (n = 8), respectively. Despite the elevated MABP, global CBF was significantly lower in L-NAME-treated Group (a) than in the saline-controls (P < 0.005), indicating cerebral vasoconstriction resulting from inhibition of NO synthesis. Global CBF was not significantly reduced further in the two groups with hypotension. Local CBF in the hypotensive rats showed no significant reductions below values in L-NAME-treated control rats (Group (a)) in 31 of 32 brain structures; the only exception was in the auditory cortex of the severely hypotensive rats (Group (c)). The autoregulatory mechanism for cerebral vasodilatation to compensate for reduced arterial blood pressure is maintained following inhibition of NO synthesis.

The distribution of alterations in energy metabolism in the rat brain produced by apomorphine.

The effects of the putative dopaminergic agonist, apomorphine (0.15-5 mg/kg, i.v.), on glucose utilization in 43 anatomically discrete regions of the rat brain have been examined by the quantitative autoradiographic 2-deoxyglucose technique. Apomorphine failed to alter the rates of glucose utilization in 25 of these regions (for example, primary auditory areas, regions of white matter, hippocampal areas, nucleus accumbens and caudal regions of the neocortex). Dose-dependent alterations in glucose utilization were observed following apomorphine administration in a number of regions known to contain dopaminergic receptors (viz: caudate nucleus, substantia nigra, amygdala, subthalamic nucleus and anterior cingulate cortex). Moreover, dose-dependent alterations in glucose utilization were produced by apomorphine in a number of regions thought to contain few specific dopaminergic receptors (e.g., cerebellar hemisphere and vermis, lamina VI of rostral neocortical areas, and ventral nucleus of the thalamus). The distribution of alterations in glucose utilization following apomorphine administration are considered to reflect the functional involvement of the region in the overall response to apomorphine, and not simply the topography of dopaminergic receptor mechanisms.

[Determination of phasic flow speeds in the coronary vessels by means of a simple photodensitometric technique (author's transl)]. / Bestimmung der phasischen Flussgeschwindigkeiten im Koronargefässsystem mit einem einfachen fotodensitometrischen Verfahren.

Systolic and diastolic flow rates in coronary arteries were determined from cineangiograms using a photodensitometric measurements system. The front velocity of a bolus of contrast medium was evaluated by two different methods. Measurements in aortocoronary bypass grafts showed that the photodensitometric determination of flow rates overestimated the electromagnetically measured flow by about 20%. Measurements in coronary arteries proved a good reproducibility (r=0.98) and the typical pattern of phasic flow. The velocity of flow in coronary arteries was nearly identical before and after branchings of the vessels (r=0.96).