In Vitro and In Vivo Studies of the Trypanocidal Effect of Novel Quinolines.

Therapies for human African trypanosomiasis and Chagas disease, caused by Trypanosoma brucei and Trypanosoma cruzi, respectively, are limited, providing minimal therapeutic options for the millions of individuals living in very poor communities. Here the effects of 10 novel quinolines are evaluated in silico and by phenotypic studies using in vitro and in vivo models. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties revealed that most molecules did not infringe on Lipinski's rules, which is a prediction of good oral absorption. These quinolines showed high probabilities of Caco2 permeability and human intestinal absorption and low probabilities of mutagenicity and of hERG1 inhibition. In vitro screens against bloodstream forms of T. cruzi demonstrated that all quinolines were more active than the reference drug (benznidazole [Bz]), except for DB2171 and DB2192, with five (DB2187, DB2131, DB2186, DB2191, and DB2217) displaying 50% effective concentrations (EC50s) of <3 µM (4-fold lower than that of Bz). Nine quinolines were more effective than Bz (2.7 µM) against amastigotes, showing EC50s ranging from 0.6 to 0.1 µM. All quinolines were also highly active in vitro against African trypanosomes, showing EC50s of ≤0.25 µM. The most potent and highly selective candidates for each parasite species were tested in in vivo models. Results for DB2186 were promising in mice with T. cruzi and T. brucei infections, reaching a 70% reduction of the parasitemia load for T. cruzi, and it cured 2 out of 4 mice infected with T. brucei DB2217 was also active in vivo and cured all 4 mice (100% cure rate) with T. brucei infection.

Antitrypanosomal Activity of Sterol 14α-Demethylase (CYP51) Inhibitors VNI and VFV in the Swiss Mouse Models of Chagas Disease Induced by the Trypanosoma cruzi Y Strain.

Chagas disease is a life-threatening infection caused by a variety of genetically diverse strains of the protozoan parasite Trypanosoma cruzi The current treatment (benznidazole and nifurtimox) is unsatisfactory, and potential alternatives include inhibitors of sterol 14α-demethylase (CYP51), the cytochrome P450 enzyme essential for the biosynthesis of sterols in eukaryotes and the major target of clinical and agricultural antifungals. Here we performed a comparative investigation of two protozoon-specific CYP51 inhibitors, VNI and its CYP51 structure-based derivative VFV, in the murine models of infection caused by the Y strain of T. cruzi The effects of different treatment regimens and drug delivery vehicles were evaluated in animals of both genders, with benznidazole serving as the reference drug. Regardless of the treatment scheme or delivery vehicle, VFV was more potent in both genders, causing a >99.7% peak parasitemia reduction, while the VNI values varied from 91 to 100%. Treatments with VNI and VFV resulted in 100% animal survival and 0% natural relapse after the end of therapy, though, except for the 120-day treatment schemes with VFV, relapses after three cycles of immunosuppression were observed in each animal group, and quantitative PCR analysis revealed a very light parasite load in the blood samples (sometimes below or near the detection limit, which was 1.5 parasite equivalents/ml). Our studies support further investigations of this class of compounds, including their testing against other T. cruzi strains and in combination with other drugs.

In Vitro and In Vivo Trypanosomicidal Action of Novel Arylimidamides against Trypanosoma cruzi.

Arylimidamides (AIAs) have been shown to have considerable biological activity against intracellular pathogens, includingTrypanosoma cruzi, which causes Chagas disease. In the present study, the activities of 12 novel bis-AIAs and 2 mono-AIAs against different strains ofT. cruziin vitroandin vivowere analyzed. The most active wasm-terphenyl bis-AIA (35DAP073), which had a 50% effective concentration (EC50) of 0.5 µM for trypomastigotes (Y strain), which made it 26-fold more effective than benznidazole (Bz; 13 µM). It was also active against the Colombiana strain (EC50= 3.8 µM). Analysis of the activity against intracellular forms of the Tulahuen strain showed that this bis-AIA (EC50= 0.04 µM) was about 100-fold more active than Bz (2 µM). The trypanocidal effect was dissociated from the ability to trigger intracellular lipid bodies within host cells, detected by oil red labeling. Both an active compound (35DAP073) and an inactive compound (26SMB060) displayed similar activation profiles. Due to their high selectivity indexes, two AIAs (35DAP073 and 35DAP081) were moved toin vivostudies, but because of the results of acute toxicity assays, 35DAP081 was excluded from the subsequent tests. The findings obtained with 35DAP073 treatment of infections caused by the Y strain revealed that 2 days of therapy induced a dose-dependent action, leading to 96 to 46% reductions in the level of parasitemia. However, the administration of 10 daily doses in animals infected with the Colombiana strain resulted in toxicity, preventing longer periods of treatment. The activity of the combination of 0.5 mg/kg of body weight/day 35DAP073 with 100 mg/kg/day Bz for 10 consecutive days was then assayed. Treatment with the combination resulted in the suppression of parasitemia, the elimination of neurological toxic effects, and survival of 100% of the animals. Quantitative PCR showed a considerable reduction in the parasite load (60%) compared to that achieved with Bz or the amidine alone. Our results support further investigations of this class with the aim of developing novel alternatives for the treatment of Chagas disease.

Different Therapeutic Outcomes of Benznidazole and VNI Treatments in Different Genders in Mouse Experimental Models of Trypanosoma cruzi Infection.

The lack of translation between preclinical assays and clinical trials for novel therapies for Chagas disease (CD) indicates a need for more feasible and standardized protocols and experimental models. Here, we investigated the effects of treatment with benznidazole (Bz) and with the potent experimental T. cruzi CYP51 inhibitor VNI in mouse models of Chagas disease by using different animal genders and parasite strains and employing distinct types of therapeutic schemes. Our findings confirm that female mice are less vulnerable to the infection than males, show that male models are less susceptible to treatment with both Bz and VNI, and thus suggest that male models are much more suitable for selection of the most promising antichagasic agents. Additionally, we have found that preventive protocols (compound given at 1 dpi) result in higher treatment success rates, which also should be avoided during advanced steps of in vivo trials of novel anti-T. cruzi drug candidates. Another consideration is the relevance of immunosuppression methods in order to verify the therapeutic profile of novel compounds, besides the usefulness of molecular diagnostic tools (quantitative PCR) to ascertain compound efficacy in experimental animals. Our study aims to contribute to the development of more reliable methods and decision gates for in vivo assays of novel antiparasitic compounds in order to move them from preclinical to clinical trials for CD.

Profile of skin barrier proteins (filaggrin, claudins 1 and 4) and Th1/Th2/Th17 cytokines in adults with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) in adults and profile of skin barrier proteins and inflammatory cytokines. OBJECTIVE: Evaluation of the expression of skin barrier proteins such as filaggrin, claudins 1 and 4 and of circulating inflammatory cytokines (Th1/Th2/Th17) in adults with AD. METHODS: Thirty-three adult patients with AD diagnosed according to the Hanifin & Rajkacriteria, and 25 healthy controls were enrolled in the study. AD severity was measured by Eczema Area and Severity Index (EASI). Laboratory assays included immunohistochemistry analysis of skin barrier proteins, such as filaggrin, claudins 1 and 4 and interleukin-17 (IL-17) from skin samples and determination of circulating cytokine levels (IL-2, 4, 5, 6, 10, 17A, TNF and IFN-γ) by flow cytometry (Cytometric Bead Array). RESULTS: We observed a reduced expression of filaggrin and claudin 1 in lesional skin of AD patients, when compared to controls. There was an inverse correlation of filaggrin expression and disease severity. In addition, IL-17 expression was enhanced in AD patients. Similarly, higher levels of inflammatory cytokines (IL-2, 5, 6, 10, 17A and IFN-γ) were found in AD patients. CONCLUSION: Our data reinforce the role of an altered skin barrier in the pathogenesis of AD. Our results show not only reduced expression of filaggrin and claudin 1 in lesional atopic skin but also inverse correlation of filaggrin expression and disease severity. Moreover, elevation of in situ IL-17 and of circulating interleukin levels in AD emphasize the systemic, inflammatory profile of this defective skin barrier dermatosis.

Rapid assessment of transient ischaemic attack in a hospital with no on-call neurologist.

BACKGROUND: Risk of stroke soon after a transient ischaemic attack (TIA) is high. Urgent care can reduce this risk. Our aim is to describe and evaluate the efficacy of rapid assessment of TIA patients in a hospital without a neurologist available 24 hours a day. METHODS: In February 2007, we set up a protocol of rapid management of patients with symptoms consistent with acute TIA, with the aim of prioritising urgent care and reducing hospital admissions, without increasing risk of recurrences. We analyse our results since the protocol was implemented with particular focus on the analysis of delay in neurological and neurovascular assessment, percentage and reasons for hospitalisation, and stroke recurrence rates after 3 months. RESULTS: Four hundred and eleven patients were studied, with a final diagnosis of TIA in 282 (68.6%). Among other diagnoses, the most frequent were a vasovagal reaction (5.6%) and confusional syndrome (4.6%). Delay between emergency arrival and neurovascular assessment was <24h in 82% of the cases, and <48 h in 93%. After neurological evaluation, 28.7% of the patients were immediately admitted to hospital (most common causes: severe stenosis of a large artery and crescendo TIA). The incidence of ischaemic stroke in TIA patients was 3.55% after 3 months and 70% of them suffered the recurrence within the first week after the initial TIA. CONCLUSIONS: In a hospital without a neurologist available 24 hours a day, early assessment and management of TIA patients can be carried out in accordance with the guidelines, and may avoid hospitalisation in most cases without increasing recurrence rates.

In vitro and in vivo studies of the biological activity of novel arylimidamides against Trypanosoma cruzi.

Fifteen novel arylimidamides (AIAs) (6 bis-amidino and 9 mono-amidino analogues) were assayed against Trypanosoma cruzi in vitro and in vivo. All the bis-AIAs were more effective than the mono-AIAs, and two analogues, DB1967 and DB1989, were further evaluated in vivo. Although both of them reduced parasitemia, protection against mortality was not achieved. Our results show that the number of amidino-terminal units affects the efficacy of arylimidamides against T. cruzi.

Deletion of MAP2K2/MEK2: a novel mechanism for a RASopathy?

RASopathies are a class of genetic syndromes caused by germline mutations in genes encoding Ras/mitogen-activated protein kinase (Ras/MAPK) pathway components. Cardio-facio-cutaneous (CFC) syndrome is a RASopathy characterized by distinctive craniofacial features, skin and hair abnormalities, and congenital heart defects caused by activating mutations of BRAF, MEK1, MEK2, and KRAS. We define the phenotype of seven patients with de novo deletions of chromosome 19p13.3 including MEK2; they present with a distinct phenotype but have overlapping features with CFC syndrome. Phenotypic features of all seven patients include tall forehead, thick nasal tip, underdeveloped cheekbones, long midface, sinuous upper vermilion border, tall chin, angular jaw, and facial asymmetry. Patients also have developmental delay, hypotonia, heart abnormalities, failure to thrive, obstructive sleep apnea, gastroesophageal reflux and integument abnormalities. Analysis of epidermal growth factor-stimulated fibroblasts revealed that P-MEK1/2 was ∼50% less abundant in cells carrying the MEK2 deletion compared to the control. Significant differences in total MEK2 and Sprouty1 abundance were also observed. Our cohort of seven individuals with MEK2 deletions has overlapping features associated with RASopathies. This is the first report suggesting that, in addition to activating mutations, MEK2 haploinsufficiency can lead to dysregulation of the MAPK pathway.

Age- and gender-related changes in glucose homeostasis in glucocorticoid-treated rats.

The disruption to glucose homeostasis upon glucocorticoid (GC) treatment in adult male rats has not been fully characterized in older rats or in females. Thus, we evaluated the age- and gender-related changes in glucose homeostasis in GC-treated rats. We injected male and female rats at 3 months and 12 months of age with either dexamethasone (1.0 mg/kg body mass, intraperitoneally) or saline, daily for 5 days. All of the GC-treated rats had decreased body mass and food intake, and adrenal hypotrophy. Increased glycemia was observed in all of the GC-treated groups and only the 3-month-old female rats were not glucose intolerant. Dexamethasone treatment resulted in hyperinsulinemia and hypertriacylglyceridemia in all of the GC-treated rats. The glucose-stimulated insulin secretion (GSIS) was higher in all of the dexamethasone-treated animals, but it was less pronounced in the older animals. The ß-cell mass was increased in the younger male rats treated with dexamethasone. We conclude that dexamethasone treatment induces glucose intolerance in both the 3- and 12-month-old male rats as well as hyperinsulinemia and augmented GSIS. Three-month-old female rats are protected from glucose intolerance caused by GC, whereas 12-month-old female rats developed the same complications that were present in 3- and 12-month-old male rats.

Myoclonus-dystonia and Silver-Russell syndrome resulting from maternal uniparental disomy of chromosome 7.

Myoclonus-dystonia (M-D) is a movement disorder that is often associated with mutations in epsilon-sarcoglycan (SGCE), a maternally imprinted gene at 7q21.3. We report a 24-year-old male with short stature (<5th percentile) and a movement disorder clinically consistent with M-D. Single nucleotide polymorphism (SNP) array did not identify significant copy number changes, but revealed three long continuous stretches of homozygosity on chromosome 7 suggestive of uniparental disomy. Parental SNP arrays confirmed that the proband had maternal uniparental disomy of chromosome 7 (mUPD7) with regions of heterodisomy and isodisomy. mUPD7 is the cause of approximately 5-10% of Silver-Russell syndrome (SRS), a disorder characterized by prenatal and postnatal growth retardation. Although SRS was not suspected in our patient, these findings explain his short stature. SGCE methylation testing showed loss of the unmethylated paternal allele. Our findings provide a unifying diagnosis for his short stature and M-D and help to optimize his medication regimen. In conclusion, we show that M-D is a clinical feature that may be associated with SRS due to mUPD7. Individuals with mUPD7 should be monitored for the development of movement disorders. Conversely, individuals with M-D and short stature should be evaluated for SRS.

Leptospirosis-associated acute kidney injury: penicillin at the late stage is still controversial.

WHAT IS KNOWN AND OBJECTIVE: Some antimicrobial agents are active in vitro against Leptospiras. The use of penicillins at the late stage of leptospirosis is still controversial. We aimed to evaluate the use of penicillin in patients with leptospirosis-associated acute kidney injury (AKI). METHODS: A retrospective study was conducted of patients with leptospirosis admitted to two hospitals in Fortaleza city, Brazil, between 1985 and 2008. AKI was defined according to the RIFLE and AKIN classifications. Patients were divided in two groups according to whether they were treated with a penicillin or not. RESULTS: Two hundred and eighty-seven patients were included, with an average age of 36·8±15·6 years and mostly male (80·8%). One hundred and twelve patients (39%) received a penicillin. Patients treated with a penicillin were younger (32±14 years vs. 39±16 years, P=0·0002) and had a shorter hospital stay (8·4±5·0 vs. 11±7·7 days, P<0·0001). There was no difference in the onset of symptoms before hospital admission between the two groups (6·5±3·0 vs. 7·7±4·7, P=0·33). Systolic blood pressure was lower in the penicillin group (111±21 vs. 119±22 mmHg, P=0·04). AKI, need of dialysis and renal recovery at the time of hospital discharge were more frequent in patients who did not use a penicillin (P<0·05). Mortality was similar in both groups (11·6% vs. 13·7%, P=0·60). CONCLUSION: Treatment of leptospirosis with antibiotics, including the penicillin, remains controversial. The main benefit of using penicillin in the present study was a reduction in the length of hospital stay and fewer complications, such as AKI, but its use was not associated with a decrease in mortality. On balance of risks and benefits, we recommend the use of penicillin in late-stage leptospirosis.

Influence of the halothane gene (HAL) on pork quality in two commercial crossbreeds.

We evaluated the effect of the halothane (HAL) gene on the quality of pork in domestic pigs. Half-carcasses from two different commercial pig (Sus domestica) crossbreeds were analyzed, 46 of which were homozygous dominant (HAL(NN)) and 69 of which were heterozygous (HAL(Nn)) for the halothane gene. The measures included backfat thickness, lean meat percentage, carcass weight, pH 24 h after slaughtering, color, and drip loss; DNA was extracted from the haunch muscle. Swine with the HAL(Nn) genotype had less backfat thickness and higher lean meat percentages than swine with the HAL(NN) genotype. Yet, swine with the HAL(Nn) genotype had lower quality meat than those with the HAL(NN) swine. The pH at 24 h was lower in HAL(Nn) swine. The meat color was paler in HAL(Nn) animals, the drip loss was greater in those animals bearing the n allele, and the amount of intramuscular fat was not related to the halothane genotype. We conclude that bearers of the recessive allele of the halothane gene produce more meat, but with quality parameters that are inferior to those sought by consumers and industry.

Coadministration of sitagliptin or metformin has no major impact on the adverse metabolic outcomes induced by dexamethasone treatment in rats.

AIMS: Glucocorticoids (GC) in excess cause glucose intolerance and dyslipidemia due to their diabetogenic actions. Conceptually, antidiabetic drugs should attenuate these side effects. Thus, we evaluated whether the coadministration of metformin or sitagliptin (or both) with dexamethasone could attenuate GC-induced adverse effects on metabolism. MATERIALS AND METHODS: Adult male rats were treated for 5 consecutive days with dexamethasone (1 mg/kg, body mass (bm), intraperitoneally). Additional groups were coadministered with metformin (300 mg/kg, bm, by oral gavage (og)) or sitagliptin (20 mg/kg, bm, og) or with both compounds in combination. The day after the last treatments, rats were submitted to glucose tolerance tests, pyruvate tolerance test, and euthanized for biometric, biochemical, morphologic, and molecular analyses. KEY FINDINGS: Dexamethasone treatment resulted in reduced body mass and food intake, increased blood glucose and plasma insulin, dyslipidemia, glucose intolerance, pyruvate intolerance, and increased hepatic content of glycogen and fat. Sitagliptin coadministration improved glucose tolerance compared with the control group, an effect paralleled with higher levels of active GLP-1 during an oral GTT. Overall, sitagliptin or metformin coadministration did not prevent any of the dexamethasone-induced metabolic disturbances. SIGNIFICANCE: Coadministration of sitagliptin or metformin result in no major improvement of glucose and lipid metabolism altered by dexamethasone treatment in male adult rats.

Biological, ultrastructural effect and subcellular localization of aromatic diamidines in Trypanosoma cruzi.

No vaccines or safe chemotherapy are available for Chagas disease. Pentamidine and related di-cations are DNA minor groove-binders with broad-spectrum anti-protozoal activity. Therefore our aim was to evaluate the in vitro efficacy of di-cationic compounds - DB1645, DB1582, DB1651, DB1646, DB1670 and DB1627 - against bloodstream trypomastigotes (BT) and intracellular forms of Trypanosoma cruzi. Cellular targets of these compounds in treated parasites were also analysed by fluorescence and transmission electron microscopy (TEM). DB1645, DB1582 and DB1651 were the most active against BT showing IC50 values ranging between 0.15 and 6.9 microm. All compounds displayed low toxicity towards mammalian cells and DB1645, DB1582 and DB1651 were also the most effective against intracellular parasites, with IC50 values ranging between 7.3 and 13.3 microm. All compounds localized in parasite nuclei and kDNA (with greater intensity in the latter structure), and DB1582 and DB1651 also concentrated in non-DNA-containing cytoplasmic organelles possibly acidocalcisomes. TEM revealed alterations in mitochondria and kinetoplasts, as well as important disorganization of microtubules. Our data provide further information regarding the activity of this class of compounds upon T. cruzi which should aid future design and synthesis of agents that could be used for Chagas disease therapy.

Influence of Chinese breeds on pork quality of commercial pig lines.

We compared carcass and meat quality of pigs from the same sire line and two different dam lines, one that included Chinese breeds and one that did not. Line A consisted of 1/4 Landrace, 1/2 Large White, 1/8 Chinese breeds (Meishan, Fengjing, Jiaxing), and 1/8 Large White, Duroc and Pietrain, and line B consisted of 1/2 Large White and 1/2 Pietrain. The animals (N = 144) were slaughtered at a live weight of 108 kg. Backfat thickness, percentage of lean meat, pH 24 h after slaughter, meat color, percentage of drip loss, and percentage of intramuscular fat were measured and compared using analysis of variance in a completely randomized design; the BioEstat 5.0 test was applied for the comparison of means at a significance level of 5% for all analyses. Backfat was significantly lower for line A (12.78 mm) than for line B (15.90 mm). The pH measured 24 h after slaughter was significantly lower in line A (5.68) compared to line B (5.84). Percent lean meat was significantly higher for line A (61.21%) compared to line B (59.72%). Percentage drip loss was significantly higher in line A (2.73%) than in line B (2.23%). Percentage intramuscular fat and meat color were not significantly different between the lines. The inclusion of Chinese breeds produced a higher percentage of lean meat and reduced fat thickness, along with increased heterosis, which are important characteristics for breeding programs.

Percentage depth dose evaluation in heterogeneous media using thermoluminescent dosimetry.

The purpose of this study is to investigate the influence of lung heterogeneity inside a soft tissue phantom on percentage depth dose (PDD). PDD curves were obtained experimentally using LiF:Mg,Ti (TLD-100) thermoluminescent detectors and applying Eclipse treatment planning system algorithms Batho, modified Batho (M-Batho or BMod), equivalent TAR (E-TAR or EQTAR), and anisotropic analytical algorithm (AAA) for a 15 MV photon beam and field sizes of 1 x 1, 2 x 2, 5 x 5, and 10 x 10 cm 2 . Monte Carlo simulations were performed using the DOSRZnrc user code of EGSnrc. The experimental results agree with Monte Carlo simulations for all irradiation field sizes. Comparisons with Monte Carlo calculations show that the AAA algorithm provides the best simulations of PDD curves for all field sizes investigated. However, even this algorithm cannot accurately predict PDD values in the lung for field sizes of 1 x 1 and 2 x 2 cm 2 . An overdosage in the lung of about 40% and 20% is calculated by the AAA algorithm close to the interface soft tissue/lung for 1 x 1 and 2 x 2 cm 2 field sizes, respectively. It was demonstrated that differences of 100% between Monte Carlo results and the algorithms Batho, modified Batho, and equivalent TAR responses may exist inside the lung region for the 1 x 1 cm 2 field.

Generation of 10 patient-specific induced pluripotent stem cells (iPSCs) to model Pitt-Hopkins Syndrome.

Autosomal dominant mutations in transcription factor 4 (TCF4) are associated with a rare syndromic form of Autism Spectrum Disorder (ASD) called Pitt-Hopkins Syndrome (PTHS). Here, we report the generation of a collection of induced pluripotent stem cells (iPSCs) from 5 patients diagnosed with PTHS and 5 familial controls. These patient-derived iPSCs contain a variety of mutations within the TCF4 gene, possess a normal karyotype and express all the appropriate pluripotent stem cell markers. These novel patient lines will be a useful resource for the research community to study PTHS and the function of TCF4.

Pseudoaminopterin syndrome: clinical report with new characteristics.

Fetuses exposed to aminopterin during the 8th-9th week of development may show aminopterin embryophathy (AE). Surviving children have a specific phenotype that includes unusual face, skull, and skeletal abnormalities. Fraser et al. [Fraser et al. (1987); Clin Genet 32:28-34] described two children with multiple malformations characteristic of the aminopterin syndrome but without history of exposure to aminopterin in the mothers and suggested that this represents a new syndrome, the aminopterin syndrome-like sine aminopterin (ASSA) syndrome. Here we describe a 9-year-old girl, born to unaffected first cousin parents. She has short stature, microcephaly, broad forehead with high hair implantation; sparse and fine hair, areas of alopecia; arched eyebrows with upturned hair, synophris; ocular hypertelorism, epicanthal folds, palpebral ptosis; oligodontia; low-set and small ears with hypoplasia of antihelices; brachydactyly, clinodactyly of both 4th and 5th fingers; hypoplasia of the 4th metacarpal and clinodactyly of the 4th and 5th toes; overlap of the second over the third toe; bilateral hip luxation; patent foramen ovale; left posterior diaphragmatic hernia, absence of spleen and horseshoe kidney. She, her mother and her brother have a karyotype of 46,XX, with an inv(9)(p12q13) polymorphism. Although this patient has some characteristics did not described before in patients with ASSA such as, palpebral ptosis, oligodontia, left posterior diaphragmatic hernia, absence of spleen, and horseshoe kidney, her phenotype strongly suggest she has the pseudoaminopterin syndrome. However, we do not exclude the possibility that this is a different condition not described previously.

Acute bilineal leukemia: a rare disease with poor outcome.

Most cases of acute leukemia can be assigned to the myeloid, B or T lineage. In a few cases, definitive assignment cannot be achieved because blasts express antigens of more than one lineage. A subset of these, referred to as acute bilineal leukemias (aBLLs), is characterized by the presence of more than one population of blasts, each comprising a single lineage. We identified 19 cases of aBLL, including 10 mixed T and myeloid (T-My) and nine mixed B and myeloid (B-My); no mixed B and T cases were identified. Cytogenetic data were available for 16 patients. Three of seven patients with B-My had a t(9;22)(q34q11.2), two had 11q23 translocations and one had del(9). Two of nine patients with T-My had 2p13 translocations; five had other unrelated abnormalities. Of 16 patients with outcome data, only six achieved complete remission and only two remain free of disease 2.5 and 4.5 years after chemotherapy or stem cell transplantation. aBLL is a rare disease that combines B or T and myeloid blasts. Cytogenetic abnormalities of t(9;22) and 11q23 are common in, and may be restricted to, B-My cases, while T-My cases have frequent but generally non-recurring abnormalities. Both types of aBLL are associated with poor outcome.

Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures.

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L-1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.