Abstract in English:Hepatitis C virus (HCV) is essentially hepatotropic but its manifestations can extend beyond the liver. It can be associated with autoimmune diseases, such as mixed cryoglobulinemia, membranoproliferative glomerulonephritis, autoimmune thyroiditis, and lymphoproliferative disorders. The mechanisms that trigger these manifestations are not completely understood. We describe a 48-year-old man with chronic HCV infection (circulating HCV RNA and moderate hepatitis as indicated by liver biopsy), cryoglobulinemia, and sensory and motor peripheral neuropathy. The diagnosis of multineuropathy was confirmed by clinical examination and electromyographic tests. A nerve biopsy revealed an inflammatory infiltrate in the perineurial space and signs of demyelination and axonal degeneration. The patient had no improvement of neurological symptoms with the use of analgesics and neuro-modulators. He was then treated with interferon-alpha (3 million units subcutaneously, 3 times per week) and ribavirin (500 mg orally, twice a day) for 48 weeks. Six months after the end of therapy, the patient had sustained viral response (negative HCV RNA) and remission of neurological symptoms, but cryoglobulins remained positive. A review of the literature on the pathogenesis and treatment of neurological manifestations associated with HCV infection is presented. This report underscores the need for a thorough evaluation of HCV-infected patients because of the possibility of extrahepatic manifestations. Antiviral treatment with interferon and ribavirin can be effective and should be considered in patients with neurological complications associated with HCV infection.
Abstract in English:The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.
Abstract in English:The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.
Abstract in English:Acute rejection of a transplanted organ is characterized by intense inflammation within the graft. Yet, for many years transplant researchers have overlooked the role of classic mediators of inflammation such as prostaglandins and thromboxane (prostanoids) in alloimmune responses. It has been demonstrated that local production of prostanoids within the allograft is increased during an episode of acute rejection and that these molecules are able to interfere with graft function by modulating vascular tone, capillary permeability, and platelet aggregation. Experimental data also suggest that prostanoids may participate in alloimmune responses by directly modulating T lymphocyte and antigen-presenting cell function. In the present paper, we provide a brief overview of the alloimmune response, of prostanoid biology, and discuss the available evidence for the role of prostaglandin E2 and thromboxane A2 in graft rejection.
Abstract in English:A lectin isolated from the red alga Solieria filiformis was evaluated for its effect on the growth of 8 gram-negative and 3 gram-positive bacteria cultivated in liquid medium (three independent experiments/bacterium). The lectin (500 µg/mL) stimulated the growth of the gram-positive species Bacillus cereus and inhibited the growth of the gram-negative species Serratia marcescens, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus sp, and Pseudomonas aeruginosa at 1000 µg/mL but the lectin (10-1000 µg/mL) had no effect on the growth of the gram-positive bacteria Staphylococcus aureus and B. subtilis, or on the gram-negative bacteria Escherichia coli and Salmonella typhimurium. The purified lectin significantly reduced the cell density of gram-negative bacteria, although no changes in growth phases (log, exponential and of decline) were observed. It is possible that the interaction of S. filiformis lectin with the cell surface receptors of gram-negative bacteria promotes alterations in the flow of nutrients, which would explain the bacteriostatic effect. Growth stimulation of the gram-positive bacterium B. cereus was more marked in the presence of the lectin at a concentration of 1000 µg/mL. The stimulation of the growth of B. cereus was not observed when the lectin was previously incubated with mannan (125 µg/mL), its hapten. Thus, we suggest the involvement of the binding site of the lectin in this effect. The present study reports the first data on the inhibition and stimulation of pathogenic bacterial cells by marine alga lectins.
Abstract in English:Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9% CD34+ cells, 2.6 ± 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.
Abstract in English:Curcumin, a major yellow pigment and active component of turmeric, has multiple anti-cancer properties. However, its molecular targets and mechanisms of action on human colon adenocarcinoma cells are unknown. In the present study, we examined the effects of curcumin on the proliferation of human colon adenocarcinoma HT-29 cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method and confirmed the curcumin-induced apoptosis by morphology and DNA ladder formation. At the same time, p53, phospho-p53 (Ser15), and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, pro-caspase-3, and pro-caspase-9 were determined by Western blot analysis. The colon adenocarcinoma cells were treated with curcumin (0-75 µM) for 0-24 h. We observed that p53 was highly expressed in HT-29 cells and curcumin could up-regulate the serine phosphorylation of p53 in a time- and concentration-dependent manner. An increase in expression of the pro-apoptotic factor Bax and a decrease in expression of the anti-apoptotic factor Bcl-2 were also observed in a time-dependent manner after exposure of 50 µM curcumin, while the expression of the anti-apoptotic factor Bcl-xL was unchanged. Curcumin could also down-regulate the expression of pro-caspase-3 and pro-caspase-9 in a time-dependent manner. These data suggest a possible underlying molecular mechanism whereby curcumin could induce the apoptosis signaling pathway in human HT-29 colon adenocarcinoma cells by p53 activation and by the regulation of apoptosis-related proteins. This property of curcumin suggests that it could have a possible therapeutic potential in colon adenocarcinoma patients.
Abstract in English:The objective of the present investigation was to perform a 14-day time-course study of treatment with salbutamol, a ß2 adrenoceptor agonist, on rat soleus muscle in order to assess fiber type selectivity in the hypertrophic response and fiber type composition. Male Wistar rats were divided into four groups: control (N = 10), treated with salbutamol (N = 30), denervated (N = 30), and treated with salbutamol after denervation (N = 30). Salbutamol was injected intraperitoneally in the rats of the 2nd and 4th groups at a concentration of 0.3 mg/kg twice a day for 2 weeks. The muscles were denervated using the crush method with pean. The animals were sacrificed 3, 6, 9, 12, and 14 days after treatment. Frozen cross-sections of soleus muscle were stained for myosin ATPase, pH 9.4. Cross-sectional area and percent of muscle fibers were analyzed morphometrically by computerized image analysis. Treatment with salbutamol induced hypertrophy of all fiber types and a higher percentage of type II fibers (21%) in the healthy rat soleus muscle. Denervation caused marked atrophy of all fibers and conversion from type I to type II muscle fibers. Denervated muscles treated with salbutamol showed a significantly larger cross-sectional area of type I muscle fibers, 28.2% compared to the denervated untreated muscle. Moreover, the number of type I fibers was increased. These results indicate that administration of salbutamol is able to induce changes in cross-sectional area and fiber type distribution in the early phase of treatment. Since denervation-induced atrophy and conversion from type I to type II fibers were improved by salbutamol treatment we propose that salbutamol, like other ß2 adrenoceptor agonists, may have a therapeutic potential in improving the condition of skeletal muscle after denervation.
Abstract in English:Mice selected on the basis of an acute inflammatory response (AIR) can provide information about the immunopathological mechanisms of glomerulonephritis. We studied the differences between mice selected for a maximal AIR (AIRmax that attract more polymorphonuclear cells to the site of injury) or a minimal AIR (AIRmin that attract more mononuclear cells) in an experimental model of IgA nephropathy in order to investigate the effect of genetic background on glomerular disease progression and the participation of the monocyte chemoattractant protein-1 (MCP-1) chemokine. IgA nephropathy was induced by intraperitoneal ovalbumin injection and bile duct ligation in AIRmax and AIRmin mice. Histological changes, urinary protein/creatinine ratio, serum IgA levels, immunofluorescence for IgA, IgG and complement C3 fraction, immunohistochemistry for macrophages and MCP-1, and MCP-1 levels in macerated kidney were determined. Mesangial IgA deposition was seen only in AIRmin mice, which presented more renal lesions. Increased serum IgA levels (1.5 ± 0.4 vs 0.3 ± 0.1 mg/mL, P < 0.001), high glomerular MCP-1 expression and decreased monocyte/macrophage infiltration in the interstitial area (0.3 ± 0.3 vs 1.1 ± 0.9 macrophages/field, P < 0.05) were detected in AIRmin mice compared to AIRmax mice. No glomerular monocyte/macrophage infiltration was detected in either strain. In spite of the absence of IgA deposition, AIRmax mice presented discrete or absent mesangial proliferation. The study showed that there are differences between mice selected for AIRmax and AIRmin with respect to serum IgA levels, histological damage and MCP-1 chemokine production after ovalbumin injection in combination with bile duct ligation.
Abstract in English:The present study evaluated the acute effect of the intraperitoneal (ip) administration of a whey protein hydrolysate (WPH) on systolic arterial blood pressure (SBP) and renal sodium handling by conscious spontaneously hypertensive rats (SHR). The ip administration of WPH in a volume of 1 ml dose-dependently lowered the SBP in SHR 2 h after administration at doses of 0.5 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 176.6 ± 4.9 mmHg, N = 8, P = 0.001) and 1.0 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 163.8 ± 5.9 mmHg, N = 8, P = 0.0018). Creatinine clearance decreased significantly (P = 0.0084) in the WPH-treated group (326 ± 67 µL min-1 100 g body weight-1) compared to 0.15 M NaCl-treated (890 ± 26 µL min-1 100 g body weight-1) and captopril-treated (903 ± 72 µL min-1 100 g body weight-1) rats. The ip administration of 1.0 g WPH/kg also decreased fractional sodium excretion to 0.021 ± 0.019% compared to 0.126 ± 0.041 and 0.66 ± 0.015% in 0.15 M NaCl and captopril-treated rats, respectively (P = 0.033). Similarly, the fractional potassium excretion in WPH-treated rats (0.25 ± 0.05%) was significantly lower (P = 0.0063) than in control (0.91 ± 0.15%) and captopril-treated rats (1.24 ± 0.30%), respectively. The present study shows a decreased SBP in SHR after the administration of WPH associated with a rise in tubule sodium reabsorption despite an angiotensin I-converting enzyme (ACE)-inhibiting in vitro activity (IC50 = 0.68 mg/mL). The present findings suggest a pathway involving ACE inhibition but measurements of plasma ACE activity and angiotensin II levels are needed to support this suggestion.
Abstract in English:Nitric oxide (·NO) is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of ·NO and oxidants leads to the generation of nitrogen dioxide (·NO2), a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2) group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of ·NO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of ·NO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished ·NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the ·NO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce ·NO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.
Abstract in English:A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.
Abstract in English:According to the concepts of cognitive neuropsychology, there are two principal routes of reading processing: a lexical route, in which global reading of words occurs and a phonological route, responsible for the conversion of the graphemes into their respective phonemes. In the present study, functional magnetic resonance imaging (fMRI) was used to investigate the patterns of cerebral activation in lexical and phonological reading by 13 healthy women with a formal educational level greater than 11 years. Participants were submitted to a silent reading task containing three types of stimuli: real words (irregular and foreign words), nonwords and illegitimate graphic stimuli. An increased number of activated voxels were identified by fMRI in the word reading (lexical processing) than in the nonword reading (phonological processing) task. In word reading, activation was greater than for nonwords in the following areas: superior, middle and inferior frontal gyri, and bilateral superior temporal gyrus, right cerebellum and the left precentral gyrus, as indicated by fMRI. In the reading of nonwords, the activation was predominant in the right cerebellum and in the left superior temporal gyrus. The results of the present study suggest the existence of differences in the patterns of cerebral activation during lexical and phonological reading, with greater involvement of the right hemisphere in reading words than nonwords.
Abstract in English:It has been demonstrated that exposure to a variety of stressful experiences enhances fearful reactions when behavior is tested in current animal models of anxiety. Until now, no study has examined the neurochemical changes during the test and retest sessions of rats submitted to the elevated plus maze (EPM). The present study uses a new approach (HPLC) by looking at the changes in dopamine and serotonin levels in the prefrontal cortex, amygdala, dorsal hippocampus, and nucleus accumbens in animals upon single or double exposure to the EPM (one-trial tolerance). The study involved two experiments: i) saline or midazolam (0.5 mg/kg) before the first trial, and ii) saline or midazolam before the second trial. For the biochemical analysis a control group injected with saline and not tested in the EPM was included. Stressful stimuli in the EPM were able to elicit one-trial tolerance to midazolam on re-exposure (61.01%). Significant decreases in serotonin contents occurred in the prefrontal cortex (38.74%), amygdala (78.96%), dorsal hippocampus (70.33%), and nucleus accumbens (73.58%) of the animals tested in the EPM (P < 0.05 in all cases in relation to controls not exposed to the EPM). A significant decrease in dopamine content was also observed in the amygdala (54.74%, P < 0.05). These changes were maintained across trials. There was no change in the turnover rates of these monoamines. We suggest that exposure to the EPM causes reduced monoaminergic neurotransmission activity in limbic structures, which appears to underlie the "one-trial tolerance" phenomenon.
Abstract in English:Depression found in Parkinson disease (PD) usually responds to selective serotonin reuptake inhibitors (SSRIs). Drugs that modify experimental neuroleptic catalepsy (NC) might affect extrapyramidal symptoms in PD. Therefore, the effects of SSRIs on NC were tested in mice, 26-36 g, separated by sex. Catalepsy was induced with haloperidol (H; 1 mg/kg, ip) and measured at 30-min intervals using a bar test. An SSRI (sertraline, ST; paroxetine, PX; fluoxetine) or vehicle (C) was injected ip 30 min before H. Dunnett's test was used for comparison of means. ST (1-5 mg/kg) or PX (1-5 mg/kg) attenuated NC, with a similar inhibition found in both sexes (5 mg/kg, 180 min: ST - males: 124 ± 10 vs 714 ± 15 s in C; females: 116 ± 10 vs 718 ± 6 s in C; PX - males: 106 ± 10 vs 714 ± 14 s in C; females: 102 ± 10 vs 715 ± 14 s in C). At 0.3 mg/kg, neither of these drugs affected NC. Fluoxetine (1-25 mg/kg) also inhibited catalepsy, although the effect was not dose-dependent; no differences were observed between males and females (5 mg/kg, 180 min: males, 185 ± 14 vs 712 ± 14 s in C; females, 169 ± 10 vs 710 ± 19 s in C). For these SSRIs, maximal inhibition of NC was obtained with 5 mg/kg, 180 min after H. These results are consistent with the hypothesis that serotonergic mechanisms modulate nigrostriatal transmission, and suggest that SSRIs are possibly safe in depressive PD patients.
Abstract in English:Leishmaniasis, Chagas' disease and schistosomiasis (bilharzia) are parasitic diseases with wide distribution on the American continent, affecting millions of people. In the present study, biological assays for antiprotozoal and molluscicidal activities were carried out with ethanolic extracts of plant species from the Brazilian part of the Upper Paraná River. Crude extracts were obtained by percolation with absolute ethanol from the leaves of Cayaponia podantha Cogn., Nectandra falcifolia (Nees) Castiglioni and Paullinia elegans Cambess., as well as from the aerial parts of Helicteres gardneriana St. Hil. & Naud. and Melochia arenosa Benth., all belonging to genera used in folk medicine. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose. Anti-leishmanial activity was determined over cultures of promastigote forms of the parasite in Schneider's Drosophila medium. Microscopic countings of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the percentage of growth inhibition. C. podantha and M. arenosa, at a concentration of 10 µg/mL, showed 90.4 ± 11.52 and 88.9 ± 2.20% growth inhibition, respectively, of epimastigote forms of Trypanosoma cruzi, whereas N. falcifolia demonstrated an LD50 of 138.5 µg/mL against promastigote forms of Leishmania (Viannia) braziliensis. Regarding molluscicidal activity, the acute toxicity of the extracts on Biomphalaria glabrata was evaluated by a rapid screening procedure. M. arenosa was 100% lethal to snails at 200 µg/mL and showed an LD50 of 143 µg/mL. Screening of plant extracts represents a continuous effort to find new antiparasitic drugs.
Abstract in English:The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 µg/kg wet organ (4 days after the first dose) to 2.07 µg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.
Abstract in English:Electrocardiograms (ECG) obtained with standard limb leads and augmented unipolar limb leads were recorded from 17 unanesthetized adult sloths. The animals were held in their habitual position in an experimental chair. We determined heart rate and rhythm from the R-R intervals, the amplitude and duration of each wave, and the duration of the segments and intervals of the ECG. The mean electrical axes of P and T waves and QRS complex were calculated on the basis of the amplitude of these waves in leads I, II, III, aV R, aV L, and aV F. The P wave appeared positive in most tracings with low amplitude in lead II, the QRS complex was generally negative in leads aV R, III and aV F, and no arrhythmias were observed. With a mean ± SD heart rate for all recordings of 81 ± 18 bpm, the duration of P and T waves, QRS complex, and PR, QT and RR intervals averaged 0.05 ± 0.02, 0.15 ± 0.05, 0.07 ± 0.02, 0.13 ± 0.02, 0.38 ± 0.04, and 0.74 ± 0.17 s, respectively. The ECG shape had a definite configuration on each lead. The angles of the mean ± SD electrical axes for atrial and ventricular depolarization and ventricular repolarization in the horizontal plane were +34 ± 68º, -35 ± 63º, and -23 ± 68º, respectively. All electrical axes showed great variations and their mean values suggest that, when the sloth is in a seated position, the heart could be displaced by the diaphragm to a semi-horizontal position.