A correlative biomarker study and integrative prognostic model in chemotherapy-naïve metastatic castration-resistant prostate cancer treated with enzalutamide.

BACKGROUND: There is a considerable need to incorporate biomarkers of resistance to new antiandrogen agents in the management of castration-resistant prostate cancer (CRPC). METHODS: We conducted a phase II trial of enzalutamide in first-line chemo-naïve asymptomatic or minimally symptomatic mCRPC and analyzed the prognostic value of TMPRSS2-ERG and other biomarkers, including circulating tumor cells (CTCs), androgen receptor splice variant (AR-V7) in CTCs and plasma Androgen Receptor copy number gain (AR-gain). These biomarkers were correlated with treatment response and survival outcomes and developed a clinical-molecular prognostic model using penalized cox-proportional hazard model. This model was validated in an independent cohort. RESULTS: Ninety-eight patients were included. TMPRSS2-ERG fusion gene was detected in 32 patients with no differences observed in efficacy outcomes. CTC detection was associated with worse outcome and AR-V7 in CTCs was associated with increased rate of progression as best response. Plasma AR gain was strongly associated with an adverse outcome, with worse median prostate specific antigen (PSA)-PFS (4.2 vs. 14.7 m; p < 0.0001), rad-PFS (4.5 vs. 27.6 m; p < 0.0001), and OS (12.7 vs. 38.1 m; p < 0.0001). The clinical prognostic model developed in PREVAIL was validated (C-Index 0.70) and the addition of plasma AR (C-Index 0.79; p < 0.001) increased its prognostic ability. We generated a parsimonious model including alkaline phosphatase (ALP); PSA and AR gain (C-index 0.78) that was validated in an independent cohort. CONCLUSIONS: TMPRSS2-ERG detection did not correlate with differential activity of enzalutamide in first-line mCRPC. However, we observed that CTCs and plasma AR gain were the most relevant biomarkers.

Critical Steps for Human Gut Exfoliome RNA Profiling Analysis Using Non-Invasive Stool Samples.

INTRODUCTION: Dietary exposure and drug treatments influence gut cellular pathways and hence growth and potentially even the gut-brain-microbiome axis. Since eukaryotic mRNA presents poly-A sequence that distinguishes them from the prokaryotes mRNA, we could analyze the gene expression of human gut cells using exfoliated gut cells available in stool samples. However, the impact of the critical steps of these non-invasive methods must be analyzed. METHODS: We tested prokaryote contamination in all the steps of different procedures to analyze human exfoliome by microarrays and the influence of the fecal sampling collection process. RESULTS: The least bacterial contamination was found using RNA amplified with oligo dT from the GeneChip 3' IVT Pico Reagent Kit or using RNA purified by both Oligotex® + oligo dT. RNAlater® collection of feces affects the microarray results compared to directly frozen fecal samples, although both methods produce similar cDNA quality. CONCLUSION: This technique is a potential non-invasive diagnostic tool that can be applied to larger studies to quantify intestinal gene expression in humans with non-invasive samples, but samples should always be collected and analyzed under the same procedure.

Deciphering the Role and Signaling Pathways of PKCα in Luminal A Breast Cancer Cells.

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor. Here, we analyzed the importance of PKCα in cellular processes such as proliferation, migration, or apoptosis by inhibiting its gene expression in a luminal A breast cancer cell line (MCF-7). Differential expression analysis and phospho-kinase arrays of PKCα-KD vs. PKCα-WT MCF-7 cells identified an essential set of proteins and oncogenic kinases of the JAK/STAT and PI3K/AKT pathways that were down-regulated, whereas IGF1R, ERK1/2, and p53 were up-regulated. In addition, unexpected genes related to the interferon pathway appeared down-regulated, while PLC, ERBB4, or PDGFA displayed up-regulated. The integration of this information clearly showed us the usefulness of inhibiting a multifunctional kinase-like PKCα in the first step to control the tumor phenotype. Then allowing us to design a possible selection of specific inhibitors for the unexpected up-regulated pathways to further provide a second step of treatment to inhibit the proliferation and migration of MCF-7 cells. The results of this study suggest that PKCα plays an oncogenic role in this type of breast cancer model. In addition, it reveals the signaling mode of PKCα at both gene expression and kinase activation. In this way, a wide range of proteins can implement a new strategy to fine-tune the control of crucial functions in these cells and pave the way for designing targeted cancer therapies.

Antibody Response to SARS-CoV-2 is Associated with Long-term Clinical Outcome in Patients with COVID-19: a Longitudinal Study.

BACKGROUND: The relationship of host immune response and viral replication with health outcomes in patients with COVID-19 remains to be defined. We aimed to characterize the medium and long-term clinical, virological, and serological outcomes after hospitalization for COVID-19, and to identify predictors of long-COVID. METHODS: Prospective, longitudinal study conducted in COVID-19 patients confirmed by RT-PCR. Serial blood and nasopharyngeal samples (NPS) were obtained for measuring SARS-CoV-2 RNA and S-IgG/N-IgG antibodies during hospital stay, and at 1, 2, and 6 months post-discharge. Genome sequencing was performed where appropriate. Patients filled out a COVID-19 symptom questionnaire (CSQ) at 2-month and 6-month visits, and those with highest scores were characterized. RESULTS: Of 146 patients (60% male, median age 64 years) followed-up, 20.6% required hospital readmission and 5.5% died. At 2 months and 6 months, 9.6% and 7.8% patients, respectively, reported moderate/severe persistent symptoms. SARS-CoV-2 RT-PCR was positive in NPS in 11.8% (median Ct = 38) and 3% (median Ct = 36) patients at 2 months and 6 months, respectively, but no reinfections were demonstrated. Antibody titers gradually waned, with seroreversion occurring at 6 months in 27 (27.6%) patients for N-IgG and in 6 (6%) for S-IgG. Adjusted 2-month predictors of the highest CSQ scores (OR [95%CI]) were lower peak S-IgG (0.80 [0.66-0.94]) and higher WHO severity score (2.57 [1.20-5.86]); 6-month predictors were lower peak S-IgG (0.89 [0.79-0.99]) and female sex (2.41 [1.20-4.82]); no association was found with prolonged viral RNA shedding. CONCLUSIONS: Long-COVID is associated with weak anti-SARS-CoV-2 antibody response, severity of illness, and female gender. Late clinical events and persistent symptoms in the medium and long term occur in a significant proportion of patients hospitalized for COVID-19.

Effects of the oral administration of glycosaminoglycans with or without native type II collagen on the articular cartilage transcriptome in an osteoarthritic-induced rabbit model.

BACKGROUND: In a previous study, the 84-day administration of glycosaminoglycans (GAGs), with or without native collagen type II (NC), in an osteoarthritis (OA)-induced rabbit model slowed down OA progression, improved several micro- and macroscopic parameters and magnetic resonance imaging (MRI) biomarkers in cartilage, and increased hyaluronic acid levels in synovial fluid. To elucidate the potential underlying mechanisms, a transcriptomics approach was conducted using medial femoral condyle and trochlea samples. RESULTS: The administration of chondroitin sulfate (CS), glucosamine hydrochloride (GlHCl), and hyaluronic acid (HA), with (CGH-NC) or without (CGH) NC, strongly modulated several genes involved in chondrocyte extracellular matrix (ECM) remodeling and homeostasis when compared to non-treated rabbits (CTR group). Notably, both treatments shared the main mechanism of action, which was related to ECM modulation through the down-regulation of genes encoding proteolytic enzymes, such as ADAM metallopeptidase with thrombospondin type 1 motif, 9 (Adamts9), and the overexpression of genes with a relevant role in the synthesis of ECM components, such as aggrecan (Acan) in both CGH-NC and CGH groups, and fibronectin 1 (Fn1) and collagen type II, alpha 1 (Col2A1) in the CGH group. Furthermore, there was a significant modulation at the gene expression level of the mTOR signaling pathway, which is associated with the regulation of the synthesis of ECM proteolytic enzymes, only in CGH-NC-supplemented rabbits. This modulation could account for the better outcomes concerning the microscopic and macroscopic evaluations reported in these animals. CONCLUSIONS: In conclusion, the expression of key genes involved in chondrocyte ECM remodeling and homeostasis was significantly modulated in rabbits in response to both CGH and CGH-NC treatments, which would partly explain the mechanisms by which these therapies exert beneficial effects against OA.

Prognostic Implications of Blood Immune-Cell Composition in Metastatic Castration-Resistant Prostate Cancer.

The prognosis for patients with metastatic castration-resistant prostate cancer (mCRPC) varies, being influenced by blood-related factors such as transcriptional profiling and immune cell ratios. We aimed to address the contribution of distinct whole blood immune cell components to the prognosis of these patients. This study analyzed pre-treatment blood samples from 152 chemotherapy-naive mCRPC patients participating in a phase 2 clinical trial (NCT02288936) and a validation cohort. We used CIBERSORT-X to quantify 22 immune cell types and assessed their prognostic significance using Kaplan-Meier and Cox regression analyses. Reduced CD8 T-cell proportions and elevated monocyte levels were substantially connected with a worse survival. High monocyte counts correlated with a median survival of 32.2 months versus 40.3 months for lower counts (HR: 1.96, 95% CI 1.11-3.45). Low CD8 T-cell levels were associated with a median survival of 31.8 months compared to 40.3 months for higher levels (HR: 1.97, 95% CI 1.11-3.5). These findings were consistent in both the trial and validation cohorts. Multivariate analysis further confirmed the independent prognostic value of CD8 T-cell counts. This study highlights the prognostic implications of specific blood immune cells, suggesting they could serve as biomarkers in mCRPC patient management and should be further explored in clinical trials.

Platelet transcriptome analysis in patients with germline RUNX1 mutations.

BACKGROUND: Germline mutations in RUNX1 can cause a familial platelet disorder that may lead to acute myeloid leukemia, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet dysfunction, and a high risk of developing acute myeloid leukemia or myelodysplastic syndrome. Discerning the pathogenicity of novel RUNX1 variants is critical for patient management. OBJECTIVES: To extend the characterization of RUNX1 variants and evaluate their effects by transcriptome analysis. METHODS: Three unrelated patients with long-standing thrombocytopenia carrying heterozygous RUNX1 variants were included: P1, who is a subject with recent development of myelodysplastic syndrome, with c.802 C>T[p.Gln268∗] de novo; P2 with c.586A>G[p.Thr196Ala], a variant that segregates with thrombocytopenia and myeloid neoplasia in the family; and P3 with c.476A>G[p.Asn159Ser], which did not segregate with thrombocytopenia or neoplasia. Baseline platelet evaluations were performed. Ultrapure platelets were prepared for platelet transcriptome analysis. RESULTS: In P1 and P2, but not in P3, transcriptome analysis confirmed aberrant expression of genes recognized as RUNX1 targets. Data allowed grouping patients by distinct gene expression profiles, which were partitioned with clinical parameters. Functional studies and platelet mRNA expression identified alterations in the actin cytoskeleton, downregulation of GFI1B, defective GPVI downstream signaling, and reduction of alpha granule proteins, such as thrombospondin-1, as features likely implicated in thrombocytopenia and platelet dysfunction. CONCLUSION: Platelet phenotype, familial segregation, and platelet transcriptomics support the pathogenicity of RUNX1 variants p.Gln268∗ and p.Thr196Ala, but not p.Asn159Ser. This study is an additional proof of concept that platelet RNA analysis could be a tool to help classify pathogenic RUNX1 variants and identify novel RUNX1 targets.

Whole Sequencing and Detailed Analysis of SARS-CoV-2 Genomes in Southeast Spain: Identification of Recurrent Mutations in the 20E (EU1) Variant with Some Clinical Implications.

During the COVID-19 pandemic caused by SARS-CoV-2, new waves have been associated with new variants and have the potential to escape vaccinations. Therefore, it is useful to conduct retrospective genomic surveillance research. Herein, we present a detailed analysis of 88 SARS-CoV-2 genomes belonging to samples taken from COVID-19 patients from October 2020 to April 2021 at the "Reina Sofía" Hospital (Murcia, Spain) focused to variant appeared later. The results at the mentioned stage show the turning point since the 20E (EU1) variant was still prevalent (71.6%), but Alpha was bursting to 14.8%. Concern mutations have been found in 5 genomes classified as 20E (EU1), which were not characteristic of this still little evolved variant. Most of those mutations are found in the spike protein, namely Δ69-70, E484K, Q675H and P681H. However, a relevant deletion in ORF1a at positions 3675-3677 was also identified. These mutations have been reported in many later SARS-CoV-2 lineages, including Omicron. Taken together, our data suggest that preferential emergence mutations could already be present in the early converging evolution. Aside from this, the molecular information has been contrasted with clinical data. Statistical analyses suggest that the correlation between age and severity criteria is significantly higher in the viral samples with more accumulated changes.

Anti-Tumor Functions of Prelatent Antithrombin on Glioblastoma Multiforme Cells.

Antithrombin, the main physiological inhibitor of the coagulation cascade, exerts anti-tumor effects on glioblastoma multiforme cells. Antithrombin has different conformations: native, heparin-activated, prelatent, latent, and cleaved. The prelatent form has an intermediate affinity between latent and native antithrombin, although it is the most antiangiogenic form. Herein, we investigate the effect of this conformation on the tumorigenic processes of glioblastoma multiforme cells. Antithrombin forms were purified by chromatography. Chromogenic/fluorogenic assays were carried out to evaluate enteropeptidase and hepsin inhibition, two serine proteases involved in these processes. Wound healing, Matrigel invasion and BrdU incorporation assays were performed to study migration, invasion and proliferation. E-cadherin, Vimentin, VEGFA, pAKT, STAT3, pSTAT3, and pERK1/2 expression was assessed by Western blot and/or qRT-PCR. Prelatent antithrombin inhibited both enteropeptidase and hepsin, although it was less efficient than the native conformation. Exposure to prelatent antithrombin significantly reduced migration and invasion but not proliferation of U-87 MG, being the conformation most efficient on migration. Prelatent antithrombin down-regulated VEGFA, pSTAT3, and pERK1/2 expression in U-87 MG cells. Our work elucidates that prelatent antithrombin has surprisingly versatile anti-tumor properties in U-87 MG glioblastoma multiforme cells. This associates with resistance pathway activation, the decreased expression of tumorigenic proteins, and increased angiogenesis, postulating the existence of a new, formerly unknown receptor with potential therapeutic implications.

COVID-19-associated encephalitis successfully treated with combination therapy.

BACKGROUND: Acute encephalitis can occur in different viral diseases due to infection of the brain or by an immune mechanism. Severe novel coronavirus disease 2019 (COVID-19) is associated with a major immune inflammatory response with cytokine upregulation including interleukin 6 (IL-6). We report a case presenting with acute encephalitis that was diagnosed as having severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection with hyperinflammatory systemic response and recovered after therapy with immunoglobulins and cytokine blockade. CASE REPORT: A 39-year-old-man was brought to the Emergency Department with drowsiness, mental disorientation, intermittent fever and headache. A brain magnetic resonance imaging showed extensive involvement of the brain including cortical and subcortical right frontal regions, right thalamus, bilateral temporal lobes and cerebral peduncles, with no leptomeningeal enhancement. Cerebrospinal fluid (CSF) showed a leukocyte count of 20/µL (90% lymphocytes), protein level of 198 mg/dL, and glucose of 48 mg/dL. SARS-CoV-2 was detected in nasopharyngeal swabs by reverse-transcriptase-PCR (RT-PCR) but it was negative in the CSF. Remarkable laboratory findings in blood tests included low lymphocyte count and elevated ferritin, IL-6 and D-dimer. He had a complicated clinical course requiring mechanical ventilation. Intravenous immunoglobulins and cytokine blockade with tocilizumab, an IL-6 receptor antagonist, were added considering acute demyelinating encephalomyelitis. The patient made a full recovery, suggesting that it could have been related to host inflammatory response. CONCLUSION: This case report indicates that COVID-19 may present as an encephalitis syndrome mimicking acute demyelinating encephalomyelitis that could be amenable to therapeutic modulation.

The simultaneous production of phosphatidic acid and diacylglycerol is essential for the translocation of protein kinase Cepsilon to the plasma membrane in RBL-2H3 cells.

To evaluate the role of the C2 domain in protein kinase Cepsilon (PKCepsilon) localization and activation after stimulation of the IgE receptor in RBL-2H3 cells, we used a series of mutants located in the phospholipid binding region of the enzyme. The results obtained suggest that the interaction of the C2 domain with the phospholipids in the plasma membrane is essential for anchoring the enzyme in this cellular compartment. Furthermore, the use of specific inhibitors of the different pathways that generate both diacylglycerol and phosphatidic acid has shown that the phosphatidic acid generated via phospholipase D (PLD)-dependent pathway, in addition to the diacylglycerol generated via phosphoinosite-phospholipase C (PLC), are involved in the localization of PKCepsilon in the plasma membrane. Direct stimulation of RBL-2H3 cells with very low concentrations of permeable phosphatidic acid and diacylglycerol exerted a synergistic effect on the plasma membrane localization of PKCepsilon. Moreover, the in vitro kinase assays showed that both phosphatidic acid and diacylglycerol are essential for enzyme activation. Together, these results demonstrate that phosphatidic acid is an important and essential activator of PKCepsilon through the C2 domain and locate this isoenzyme in a new scenario where it acts as a downstream target of PLD.

Persistent hyperestrogenism after precocious puberty in young females with McCune-Albright syndrome.

In this review we report the literature about ovarian function of young women with McCune-Albright Syndrome and describe our personal experience in the follow-up of a small cohort.Collectively, the existing data demonstrate that ovarian hyperfunction with ovarian cysts and hyperestrogenism persists in those women who had precocious puberty. The recording of menstrual cycles and the analysis of gonadotropin and estrogen secretion indicate that, when hypothalamic-pituitary pubertal activation begins, alternating episodes of gonadotropin control and ovarian autonomy can be seen. The persistence of estrogen hypersecretion causes menstrual disturbances and hypofertility. The long term consequences of this condition are hypothesized to be an increased risk of ductal breast cancer, which seems to be higher when growth hormone hypersecretion is also present. Therefore, young MAS women should receive counseling regarding fertility and ongoing surveillance for the development of estrogen related diseases.

Evaluation of post-polymerase chain reaction melting temperature analysis for meat species identification in mixed DNA samples.

Real-time uniplex and duplex polymerase chain reaction (PCR) assays with a SYBR Green I post-PCR melting curve analysis were evaluated for the identification and quantification of bovine, porcine, horse, and wallaroo DNA in food products. Quantitative values were derived from threshold-cycle (C(t)) data obtained from serial dilutions of purified DNA. The limits of detection in uniplex reactions were 0.04 pg for porcine and wallaroo DNA and 0.4 pg for cattle and horse DNA. Species specificity of the PCR products was tested by the identification of peaks in DNA melting curves, measured as the decrease of SYBR Green I fluorescence at the dissociation temperature. The peaks could be distinguished above the background even at the lowest amount of template DNA detected by the C(t) method. The system was also tested in duplex reactions, by use of either single-species DNA or DNA admixtures containing different shares of two species. The minimum proportions of each DNA species allowing the resolution of T(m) peaks in the duplex reactions were 5% (cattle or wallaroo) in cattle/wallaroo mixtures, 5% porcine and 1% horse in porcine/horse mixtures, 60% porcine and 1% wallaroo in porcine/wallaroo mixtures, and 1% cattle and 5% horse in cattle/horse mixtures. A loss in the sensitivity of the method was observed for some DNA combinations in the duplex assay. In contrast, the results obtained from SYBR Green I uniplex and duplex reactions with single-species DNA were largely comparable to those obtained previously with species-specific TaqMan probes, showing the suitability of that simpler experimental approach for large-scale analytical applications.

Bisphosphonate treatment of bone fibrous dysplasia in McCune-Albright syndrome.

One of the main features of McCune-Albright syndrome is bone fibrous dysplasia (BFD) often associated with severe clinical outcomes, such as bone pain, bone deformities and pathological fractures. Medical treatment with bisphosphonates started 15 years ago. Recent trials in pediatric patients with BFD have shown encouraging results. We evaluated long-term efficacy and safety of pamidronate treatment of BFD in children and adolescents with MAS. The drug was administered at 4 month-1 year intervals according to alkaline phosphatase levels. The study included 14 patients (10 females and 4 males between the ages of 5.3 and 18.7 years) with moderate or severe BFD. Follow up lasted 1.9-9 years. Bone pain, fractures, deformities, and bone turnover markers were evaluated before every therapeutic course. The study shows the beneficial effects of long-term bisphosponate treatment on BFD lesions leading to reduced fracture rate and bone pain, and radiological evidence of long bone lesion healing.

McCune-Albright syndrome: persistence of autonomous ovarian hyperfunction during adolescence and early adult age.

Gonadal hyperfunction is the most frequent endocrine dysfunction in females with McCune-Albright syndrome (MAS). Peripheral precocious puberty is usually the first MAS manifestation in children, characterized by episodes of hypersecretion of estrogens with a consequent reduction in gonadotropin secretion. Little is known about the course of this endocrine disease in adolescence and during young adult life. The aim of this study was to evaluate ovarian function in 10 females with MAS (age 11.4-20.1 years) to detect the persistence of autonomous ovarian hyperfunction throughout and following adolescence, after at least 1 year wash out of any treatment for precocious puberty. LH, FSH, estradiol, prolactin, androgen secretion, ovarian and breast sonography in luteal and follicular phases of some menstrual cycles were evaluated. We demonstrated the persistence of some ovarian autonomy, documented by hyperestrogenism and/or low or absent gonadotropin secretion and/or ovarian cysts.

Brachydactyly in 14 genetically characterized pseudohypoparathyroidism type Ia patients.

Brachydactyly, classically described as shortening of III, IV, and V metacarpals and I distal phalanx, is the typical and most specific sign of Albright's hereditary osteodystrophy, a peculiar phenotype reported in subjects with pseudohypoparathyroidism type Ia (PHP-Ia) caused by mutations in the GNAS gene, which encodes for the alpha-subunit of the stimulatory G protein (Gsalpha). It has been reported in 70% of PHP subjects from routine radiological examinations, but there are no specific data for hand alterations in genetically characterized PHP-Ia subjects. We evaluated the metacarpophalangeal pattern profile in 14 GNAS-mutated PHP-Ia subjects and determined the prevalence and patterns of left hand bone shortening. To search for genotype/phenotype correlations, we compared metacarpophalangeal pattern profiles in subjects with identical mutations. Shortening below -2 SD score (SDS) was present in at least one bone in each subject, with a prevalence of 100%; however, great variability existed between subjects and between hand bone segments. Between subjects, shortening ranged from -2 to -10.4 SDS and involved 1-19 hand bones (5.3-100%). Between segments, III-IV metacarpals were the most compromised (-10.4 and -10.0 SDS, respectively); V metacarpals and I-IV distal phalanges were the most frequently shortened (85.7%). Overall, bone length median values revealed shortening below -2 SDS in all metacarpals and all distal phalanges, i.e. brachymetacarpia and brachytelephalangy, that cluster together. These segments were shortened in 64.3-85.7% of patients, significantly differing from proximal and middle phalanges, which were shortened in 21.4-50%. Even if these hand alterations were a constant and typical finding in our PHP-Ia population, cluster analysis in subjects with the same genotypes did not generally show a genotype/phenotype correlation. Variability between subjects may be the result of complex interactions between GNAS defects and other genetic or epigenetic factors. In conclusion, hand shortening analysis in 14 genetically characterized patients showed typical brachymetacarpia and brachytelephalangy. Further studies in PHP-Ia subjects without GNAS mutations and in other brachydactyly syndromes will determine whether the pattern described is also specific.

Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.

A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained.

Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction.

Six TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes have been developed for the detection quantitation of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples. Species-specific amplification was achieved by combining only two fluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences, decreasing the cost of the assay significantly. The limits of detection ranged from 0.03 to 0.80 pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1% of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantitation accuracy in samples containing 10-100% of beef or pork DNA was close to 90%. The system is complemented with one additional TaqMan MGB detector based on consensus sequence segments of the nuclear 18S ribosomal RNA gene. A method to evaluate the presence of unknown eukaryotic DNA in a mixture, where data derived from the species-specific detection are compared with the experimental values obtained from the general 18S detector, is presented. This method allows the validation of the quantitative measurements, providing an internal control of the total content of PCR-amplifiable DNA in the sample. The system was tested on DNA mixtures containing different shares of up to four different species and on DNA extracted from processed commercial food samples.

Retinoic acid as a modulator of the activity of protein kinase Calpha.

All-trans-retinoic acid (atRA) is a derivative of vitamin A and possesses antitumor activity. We demonstrate that atRA is able to modulate the activity of protein kinase C alpha (PKCalpha), which is related to tumor development. In vitro, it was found that atRA activated PKCalpha in the presence of Ca(2+) and in the absence of phosphatidylserine, although such activity is considerably inhibited in mutations affecting residues D246 and D248 and also residue N189, all of which are known to be essential for the interaction with Ca(2+) and phosphatidylserine in the C2 domain. It was concluded that atRA substitutes phosphatidylserine although with lower specific activities. However, atRA had a biphasic effect on PKCalpha activity in the presence of activating phospholipids, such as phosphatidylserine and phosphatidylinositol 4,5-bisphosphate, yielding activation at low concentrations but inactivation at higher ones. This second inhibitory characteristic was not shown with K209 and K211 mutations (residues located in the Lys-rich cluster in the C2 domain) in PKCalpha. This interesting effect revealed the importance of phospholipid binding at this site to ensure maximum activity for the wild-type PKCalpha. The C1 domain was not related with the atRA effect on PKCalpha. It was concluded that whereas atRA may activate PKCalpha through the Ca(2+)-phosphatidylserine-binding site of the C2 domain, it may also inhibit the activity of this enzyme when displacing the phospholipid from the Lys-rich cluster also located in the C2 domain.