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Objective: To report a case of a gastrointestinal stromal tumor presenting as a recurrent tongue mass Methods: Study Design: Case Report Setting: Tertiary Public Hospital Participant: One patient Results: A 40-year-old Filipino soldier from Camarines Sur presented with a recurrent tongue mass two years after excision of a progressively-enlarging, firm, fixed, non-tender mass at the left posterior third of the tongue diagnosed as a Schwannoma. Repeat surgery involved partial glossectomy via midline mandibular swing. Final histopathologic report after immunohistochemical studies for CD117, SMA, and S100 was extraintestinal gastrointestinal stromal tumor of the tongue. The patient was started on the c-kit tyrosine kinase inhibitor Imatinib with no recurrence 10 months post treatment. Conclusion: Gastrointestinal stromal tumor may be considered when presented with a recurrent tongue mass despite complete surgical resection. Surgical removal is curative for most lesions. Post-operative chemotherapy with the use of Imatinib is valuable. Keywords: recurrent tongue mass, gastrointestinal stromal tumor, immunohistochemistry, c-kit tyrosine kinase inhibitor

Objective: The study aimed to evaluate mandibular fractures in a tertiary military hospital, to determine the age group in which injury occurred most often, to examine the various mechanisms of injury, to determine the anatomical part of the mandible most frequently affected and to determine if there were significant relationships between the various mechanisms of injury and the different fracture sites. Methods: Design: Cross-sectional retrospective study Setting: Tertiary Public Military Hospital Patients: Medical records of 328 active military personnel and their dependents, treated for mandibular fracture at the Department of Otorhinolaryngology – Head and Neck Surgery, Armed Forces of the Philippines Medical Center from January 1999 – December 2009 were retrospectively reviewed for data regarding sex, age, various mechanisms of injury and fractured anatomical part of the mandible. The number of fractures per site according to mechanism of injury was tabulated and prevalence ratios (95% confidence intervals) and p values were computed for the different fracture sites among the various mechanisms of injury. The probability or risk of sustaining fractures in these sites based on mechanism of injury was then computed. Results: The most fractured anatomical part of the mandible was the body (28%), followed by the parasymphysis (24%), angle (17%), symphysis (12%), ramus (8%), condyle (7%), alveolar ridge (3%) and coronoid (1%). There were associated injuries in 54% of those with mandibular fractures. In these patients, zygomaticomaxillary complex fractures occurred in 25%, head and neck abrasions and lacerations in 30%, head injuries in 28%, ocular injuries in 10%, nasal fractures in 8% and cervical spine fractures in 5%. Other injuries present were extremity trauma in 60%, thoracic trauma in 5% and abdominal trauma in 3%. Males dominated with a ratio of 99:1. Males 21 to 30 years of age sustained the most mandible fractures. Most fractures were caused by vehicular accidents (60%), followed by gunshot wounds (31%), falls (4%), violent assault (4%) and sports activities (1%). Alcohol was a contributing factor at the time of injury in 20.6% of fractures. All cases were treated by open reduction and internal fixation with plating or wiring. Conclusion: The body was the most commonly fractured anatomic region of the mandible in this series. There appeared to be a statistically significant relationship between violent assault and fractures of the ramus, but not between the other mechanisms of injury and the site of fracture. Its prevalence ratio of 3.32 (95% confidence interval: 1.13; 9.74, p value 0.039) suggests that the prevalence of fractures of the ramus among those exposed to violent assault was 3 times higher than those who were not. Keywords: mandibular fractures, etiology, maxillofacial injuries, trauma

Objectives: To present a case of pleomorphic adenoma arising from heterotopic salivary gland tissue in a supraclavicular lymph node. Design: Case report Setting: Tertiary government hospital Patient: One Result: A 38-year-old female consulted with a six month history of an enlarging right supraclavicular mass. Fine needle aspiration biopsy was negative for malignant cells. No primary tumor could be demonstrated elsewhere. Excision biopsy was performed and final histopathology revealed pleomorphic adenoma surrounded by a normal lymph node. Atypical tumor cells and mitoses were not found. Conclusion: Criteria were met for diagnosing heterotopic salivary gland in a supraclavicular lymph node which subsequently developed into pleomorphic adenoma The rare location of the lesion as well as the unusual histopathological result of pleomorphic adenoma arising from a lymph node merited submission of this case report. Key words: heteroplasia, heterotopic, cancer, metastasis, tumor

Since it has currently became essential to design more efficient and robust alternative techniques to solve hard optimisation problems in industry or science, and of easy use for practitioners, here a new way of developing simple Artificial Intelligence based Evolutionary Algorithms will be introduced. Our evolutionary computational implementation is a new idea in optimisation. Any evolutionary operators and their associated parameters from well-established evolutionary methods can be considered in such a way that the entire algorithm or intelligent agent-based software performs with very high efficiency without a prior need to investigate which method will be the best for a given optimisation problem.The implementation presented, named Flexible Evolution (FE), has capacity to adapt the operators, the parameters and the algorithm to the circumstances faced at each step of every optimisation run and is able to take into account lessons learned by different research works in the adaptation of operators and parameters. The FE uses Artificial Intelligence concepts to manage internal procedures to adopt decisions and correct the wrong ones. Our aim in this paper will be to give the keys to design these types of procedures, and more specifically, to find the way of achieving an optimum performance of the operators involved in the search, in our case by means of a function included in our algorithm called Sampling Engine. An early implementation has been already developed and tested in our previous works [66–68], so in this paper, new results of a second software implementation are presented comparing the results with those obtained by other methods, using well-known hard test functions. [ABSTRACT FROM AUTHOR]

Peripheral artery disease (PAD) affects millions of people worldwide, presenting with varying symptom severity, including chronic total occlusion of arteries, and occasionally, limb amputation. There are various interventions, such as atherectomy and the use of drug-coated balloons and stents, which have been developed to revascularize affected ischemic regions. However, each interventional approach must be individualized due to a patient's unique underlying conditions. Comorbid conditions, especially diabetes, play a significant role in PAD, as poorly controlled diabetes can accelerate PAD progression. For this reason, an early and accurate diagnosis of PAD is crucial, especially when symptoms may present dissimilar to classic PAD symptoms, often leading to misdiagnosis. The presented cases highlight the tailored interventions to revascularize arteries in patients with diabetic foot wounds utilizing catheters, stents, guidewires, and balloons, made possible after early angiogram. These interventions have been promising in treating PAD patients, and highlight the need for early diagnosis and timely and customized interventions to prevent limb amputation and mitigate potential complications., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Tran et al.)

Penetrating brain injury (PBI) is a subtype of traumatic brain injury (TBI) that has been steadily increasing in prevalence and causing significant mortality in trauma patients. In an emergent setting, it is important to determine the mechanism of injury and decide whether a PBI or a blunt TBI has occurred in order to guide diagnostic imaging and subsequent treatment. In cases where a PBI has been likely or has occurred, it is important to initiate treatment expeditiously as rapid interventions have been shown to lead to better outcomes. However, in cases of unwitnessed pediatric trauma, it can be difficult to ascertain the specific method of injury due to a lack of reliable sources. In such cases of unwitnessed trauma, PBI should be included in the differential of any orbitocranial injury. In this series, we present two cases of unwitnessed pediatric orbitocranial injury that highlight the importance of gathering a detailed history, obtaining appropriate imaging studies, and using physician intuition., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic commonly called COVID-19 brought new changes to healthcare delivery in the US. The purpose of this study is to identify the impact of COVID-19 on the delivery of acute surgical care for patients at a Level 1 trauma center during the lockdown period of the pandemic from March 13-May 1 2020., Methods: All trauma admission to the University Medical Center Level 1 Trauma Center from March 13 to May 13, 2020, were retrospectively abstracted and compared to the same period during 2019. Analysis focused on the lockdown period of March 13-May 1, 2020, and compared to the same dates in 2019. Abstracted data included demographics, care timeframes, length of stay, and mortality. The data were analyzed using Chi-Square, Fisher Exact, and the Mann-Whitney U test., Results: A total of 305 (2019) vs. 220 (2020) procedures were analyzed. No significant differences were seen in mean BMI, Injury Severity Score, American Society of Anesthesia Score, and Charlson Comorbidity Index between the two groups. Diagnosis time, interval to surgery, anesthesia time, surgical preparation time, operation time, transit time, mean hospital stay, and mortality were similar., Conclusion: The results of this study demonstrate that the lockdown period of the COVID-19 pandemic did not significantly affect the trauma surgery service line, aside from case volume, at a Level 1 trauma center in West Texas during the lockdown period. Despite changes to healthcare delivery during the pandemic, care of surgical patients was conserved as timely and of high quality., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier Ltd.)

Systemic lupus erythematosus (SLE) is an autoimmune disease with varying dermatological findings. We review a unique presentation of SLE with a literature review. A previously healthy early adolescent female presented with painful, oral mucosal bullae filled with sanguineous fluid. She endorsed a tender right knee, but examination revealed no additional abnormalities. CBC demonstrated severe pancytopaenia. Further workup, including Coombs positive RBCs and positive ANA, anti-Smith, and anti-dsDNA antibodies, confirmed Lupus as the aetiology of this patient's presentation. A form of blistering SLE and resultant thrombocytopaenia was likely responsible for the patient's oral manifestations. After receiving 60 g intravenous immunoglobulin and 3 days high-dose pulse corticosteroids, her dermatological symptoms resolved. Although cases of blistering SLE with mucosal bullae have been described in the literature, this is the first documented case of haemorrhagic mucosal bullae as the presenting symptom of thrombocytopaenia in SLE in a paediatric patient., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Extracellular vesicles (EVs) are released by nearly every cell type and have attracted much attention for their ability to transfer protein and diverse RNA species from donor to recipient cells. Much attention has been given so far to the features of EV short RNAs such as miRNAs. However, while the presence of mRNA and long noncoding RNA (lncRNA) transcripts in EVs has also been reported by multiple different groups, the properties and function of these longer transcripts have been less thoroughly explored than EV miRNA. Additionally, the impact of EV export on the transcriptome of exporting cells has remained almost completely unexamined. Here, we globally investigate mRNA and lncRNA transcripts in endothelial EVs in multiple different conditions., Results: In basal conditions, long RNA transcripts enriched in EVs have longer than average half-lives and distinctive stability-related sequence and structure characteristics including shorter transcript length, higher exon density, and fewer 3' UTR A/U-rich elements. EV-enriched long RNA transcripts are also enriched in HNRNPA2B1 binding motifs and are impacted by HNRNPA2B1 depletion, implicating this RNA-binding protein in the sorting of long RNA to EVs. After signaling-dependent modification of the cellular transcriptome, we observed that, unexpectedly, the rate of EV enrichment relative to cells was altered for many mRNA and lncRNA transcripts. This change in EV enrichment was negatively correlated with intracellular abundance, with transcripts whose export to EVs increased showing decreased abundance in cells and vice versa. Correspondingly, after treatment with inhibitors of EV secretion, levels of mRNA and lncRNA transcripts that are normally highly exported to EVs increased in cells, indicating a measurable impact of EV export on the long RNA transcriptome of the exporting cells. Compounds with different mechanisms of inhibition of EV secretion affected the cellular transcriptome differently, suggesting the existence of multiple EV subtypes with different long RNA profiles., Conclusions: We present evidence for an impact of EV physiology on the characteristics of EV-producing cell transcriptomes. Our work suggests a new paradigm in which the sorting and packaging of transcripts into EVs participate, together with transcription and RNA decay, in controlling RNA homeostasis and shape the cellular long RNA abundance profile., (© 2022. The Author(s).)

Viral infections are known to hijack the transcription and translation of the host cell. However, the extent to which viral proteins coordinate these perturbations remains unclear. Here we used a model system, the human T-cell leukemia virus type 1 (HTLV-1), and systematically analyzed the transcriptome and interactome of key effectors oncoviral proteins Tax and HBZ. We showed that Tax and HBZ target distinct but also common transcription factors. Unexpectedly, we also uncovered a large set of interactions with RNA-binding proteins, including the U2 auxiliary factor large subunit (U2AF2), a key cellular regulator of pre-mRNA splicing. We discovered that Tax and HBZ perturb the splicing landscape by altering cassette exons in opposing manners, with Tax inducing exon inclusion while HBZ induces exon exclusion. Among Tax- and HBZ-dependent splicing changes, we identify events that are also altered in Adult T cell leukemia/lymphoma (ATLL) samples from two independent patient cohorts, and in well-known cancer census genes. Our interactome mapping approach, applicable to other viral oncogenes, has identified spliceosome perturbation as a novel mechanism coordinated by Tax and HBZ to reprogram the transcriptome., Competing Interests: The authors have declared that no competing interests exist.

Purpose: The negative correlation between BPH-size and incidence of prostate cancer (PCa) is well-documented in the literature, however the exact mechanism is not well-understood. The present study uses histo-anatomical imaging to study prostate volume in correlation to prostate capsule thickness, and glandular epithelial cell density within the peripheral zone (PZ)., Materials and Methods: Specimens were selected from radical prostatectomies ranging from 20 to 160 mL based on ease of anatomical reconstruction by the slides. A total of 60 patients were selected and underwent quantitative measurements of prostate capsule thickness and glandular epithelial density within the PZ using computer-based imaging software. Pearson's correlation and a stepwise multiple linear regression analysis was conducted to determine the relationship between these measured parameters and the clinical characteristic of these patients., Results: Pearson's correlation analysis revealed a strongly significant, negative correlation between prostate volume and glandular epithelial cell density (r(58)=-0.554, p<0.001), and a strongly significant, positive correlation between prostate volume and average capsule thickness (r(58)=0.462, p<0.001). Results of multiple regression analysis showed that average glandular epithelial cell density added statistically to this prediction (p<0.05)., Conclusions: The results suggest that growth of the transition zone in BPH causes increased fibrosis of the PZ, leading to atrophy and fibrosis of glandular cells. As 80% of PCa originates from the glandular epithelium within the PZ, this observed phenomenon may explain the inverse correlation between BPH and PCa that is well-documented in the literature., Competing Interests: The authors have nothing to disclose., (© The Korean Urological Association, 2021.)

ERG family proteins (ERG, FLI1 and FEV) are a subfamily of ETS transcription factors with key roles in physiology and development. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 regulates both transcription and alternative splicing of pre-messenger RNAs. However, whether wild-type ERG family proteins might regulate splicing is unknown. Here, we show that wild-type ERG proteins associate with spliceosomal components, are found on nascent RNAs, and induce alternative splicing when recruited onto a reporter minigene. Transcriptomic analysis revealed that ERG and FLI1 regulate large numbers of alternative spliced exons (ASEs) enriched with RBFOX2 motifs and co-regulated by this splicing factor. ERG and FLI1 are associated with RBFOX2 via their conserved carboxy-terminal domain, which is present in EWS-FLI1. Accordingly, EWS-FLI1 is also associated with RBFOX2 and regulates ASEs enriched in RBFOX2 motifs. However, in contrast to wild-type ERG and FLI1, EWS-FLI1 often antagonizes RBFOX2 effects on exon inclusion. In particular, EWS-FLI1 reduces RBFOX2 binding to the ADD3 pre-mRNA, thus increasing its long isoform, which represses the mesenchymal phenotype of Ewing sarcoma cells. Our findings reveal a RBFOX2-mediated splicing regulatory function of wild-type ERG family proteins, that is altered in EWS-FLI1 and contributes to the Ewing sarcoma cell phenotype., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Introduction: The inverse relationship between benign prostate hypertrophy and incidence/severity of prostate cancer is well documented in the clinical literature. However, this phenomenon is not well understood. The purpose of this review is to offer an update in the evolving hypothesis of how benign prostate hypertrophy may be protective in prostate cancer., Methods: A literature search was conducted on PubMed limited to articles published within the past 10 years with the search criteria of "interaction" AND "benign prostate hypertrophy" AND "prostate cancer" as well as the key words of this paper., Results: Nine articles from the literature search met inclusion criteria. The articles analyze the prostate on parameters of peripheral zone volume, glandular tissue density and prostate capsule thickness. All 9 articles described peripheral zone atrophy and transition zone hypertrophy in benign prostate hypertrophy patients., Conclusions: As the transition zone grows in benign prostate hypertrophy, volume and glandular density of the peripheral zone as well as the prostate capsule undergo significant changes. The disease processes outlined in this review support the hypothesis that the growing transition zone compresses the peripheral zone against the prostatic capsule causing secondary atrophy, apoptosis, and necrosis of the peripheral zone's glandular tissue. If this hypertrophy-induced disease process of glandular tissue atrophy within the peripheral zone is confirmed in future studies, it will have relevant clinical implications on the diagnosis and treatment of benign prostate hypertrophy and prostate cancer.

Introduction: The use of urinary output and vital signs to guide initial burn resuscitation may lead to suboptimal resuscitation. Invasive hemodynamic monitoring may result in over-resuscitation. This study aimed to evaluate the results of a goal-directed burn resuscitation protocol that used standard measures of mean arterial pressure (MAP) and urine output, plus transpulmonary thermodilution (TPTD) and lactate levels to adjust fluid therapy to achieve a minimum level of preload to allow for sufficient vital organ perfusion., Methods: We conducted a three-year prospective cohort study of 132 consecutive critically burned patients. These patients underwent resuscitation guided by MAP (>65 mmHg), urinary output (0.5 to 1 ml/kg), TPTD and lactate levels. Fluid therapy was adjusted to achieve a cardiac index (CI) >2.5 L/minute/m² and an intrathoracic blood volume index (ITBVI) >600 ml/m2, and to optimize lactate levels. Statistical analysis was performed using mixed models. We also used Pearson or Spearman methods and the Mann-Whitney U-test., Results: A total of 98 men and 34 women (mean age, 48 ± 18 years) was studied. The mean total body surface area (TBSA) burned was 35% ± 22%. During the early resuscitation phase, lactate levels were elevated (2.58 ± 2.05 mmol/L) and TPTD showed initial hypovolemia by the CI (2.68 ± 1.06 L/minute/m²) and the ITBVI (709 ± 254 mL/m²). At 24 to 32 hours, the CI and lactic levels were normalized, although the ITBVI remained below the normal range (744 ± 276 ml/m²). The mean fluid rate required to achieve protocol targets in the first 8 hours was 4.05 ml/kg/TBSA burned, which slightly increased in the next 16 hours. Patients with a urine output greater than or less than 0.5 ml/kg/hour did not show differences in heart rate, mean arterial pressure, CI, ITBVI or lactate levels., Conclusions: Initial hypovolemia may be detected by TPTD monitoring during the early resuscitation phase. This hypovolemia might not be reflected by blood pressure and hourly urine output. An adequate CI and tissue perfusion can be achieved with below-normal levels of preload. Early resuscitation guided by lactate levels and below-normal preload volume targets appears safe and avoids unnecessary fluid input.

Injection pain has been associated with veterinary use of the antiemetic maropitant (Cerenia, Pfizer Animal Health). Cerenia is formulated using sulphobutylether-beta-cyclodextrin to bind maropitant and mitigate injection pain. The objective of this study was to determine whether the temperature of Cerenia alters binding between maropitant and sulphobutylether-beta-cyclodextrin and affects injection pain. Binding decreased as temperature increased, and Cerenia-elicited injection pain increased at warmer drug temperatures. These data suggest that the amount of free unbound maropitant increases with temperature and that injection pain increases with temperature in a similar fashion. Clinically, these studies suggest that injection of refrigerated Cerenia may significantly reduce or eliminate pain associated with SC injection of Cerenia.

An in vivo binding assay is characterized for [(3)H]M100907 binding to rat brain, as a measure of 5-HT(2A) receptor occupancy. Dose-response analyses were performed for various 5-HT(2A) antagonist reference agents, providing receptor occupancy ED(50) values in conjunction with plasma and brain concentration levels. Ketanserin and M100907 yielded dose-dependent increases in 5-HT(2A) receptor occupancy with ED(50)s of 0.316 mg/kg and 0.100 mg/kg, respectively. The atypical antipsychotics risperidone, olanzapine, and clozapine dose-dependently inhibited in vivo [(3)H]M100907 binding with ED(50) values of 0.051, 0.144, and 1.17 mg/kg, respectively. In contrast, the typical antipsychotic haloperidol exhibited only 20.1% receptor occupancy at 10 mg/kg despite producing dose-dependent increases in plasma and brain exposure levels. The novel psychopharmacologic agent asenapine dose-dependently occupied 5-HT(2A) receptors in rat brain with an ED(50) of 0.011 mg/kg, demonstrating higher 5-HT(2A) receptor potency compared with the other atypical antipsychotics tested. This enhanced potency was supported by a lower plasma exposure EC(50) of 0.477 ng/ml, compared with risperidone (1.57 ng/ml) and olanzapine (7.81 ng/ml) and was confirmed in time course studies. The validated [(3)H]M100907 rat in vivo binding assay allows for preclinical measurement of 5-HT(2A) receptor occupancy, providing essential data for understanding the pharmacological profile of novel antipsychotic agents. Additionally, the corresponding plasma and brain drug exposure data analyses provides a valuable data set for 5-HT(2A) reference agents by enabling direct comparison with any complementary studies performed in rats, thus providing a foundation for predictive pharmacokinetic/pharmacodynamic models and, importantly, allowing for translation to human receptor occupancy studies using [(11)C]M100907 positron emission tomography.

In this study, EDTA and heparin are compared as anticoagulants with respect to their efficiency in preventing clot formation in plasma samples that were subsequently analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A pilot in vivo pharmacokinetic study for the drug chlorpheniramine was conducted in which both EDTA and heparin plasma samples were collected simultaneously. All conditions except the anticoagulant were held constant during the pharmacokinetic study. Bioanalytical results were compared from samples transferred by manual pipette and by an automated liquid handler workstation. The concentration of chlorpheniramine in samples was determined by LC/MS/MS. Results from the analysis of variances (ANOVA) of log-transformed plasma chlorpheniramine concentrations were used to calculate 90% confidence intervals for the ratio least-squares mean values for anticoagulants and for transfer methods. Analytical concentrations of the drug chlorpheniramine were equivalent in heparin- and EDTA-containing plasma. Results suggest that the failure rate for transfer of EDTA plasma (50 micro L by automated workstation or manually) is less than that for heparinized plasma. As a consequence of these results, the vast majority of plasma samples in our laboratories are now collected in EDTA, which allows for use of automated sample transfer resulting in a three-fold timesaving over manual transfer using a single-channel pipette. The ability to use automation has resulted in improved efficiency and cost savings., (Copyright 2003 John Wiley & Sons, Ltd.)

Objective: To develop a quantitative reverse transcriptase-polymerase chain reaction assay for monitoring the prostate-specific antigen (PSA) mRNA., Methods: PSA mRNA is amplified, in parallel, with the mRNA of beta-actin, a housekeeping gene. The ratio of the amplification products obtained reflects the relative amount of PSA mRNA with respect to actin mRNA. During PCR, digoxigenin-dUTP is incorporated in the amplified sequences. The PCR products are analyzed separately by time-resolved immunofluorometric hybridization assays, using specific probes immobilized in microtiter wells. The hybrids are reacted with alkaline phosphatase-labeled anti-digoxigenin antibody. The phosphate ester of fluorosalicylate is used as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb(3+)-EDTA which is measured by time-resolved fluorometry., Results: The hybridization assays for both PSA and actin amplification products show linearity in the range of 1.4-110 pmol/L. The exponential phase of PCR amplification extends up to 200,000 and 100,000 PSA and actin cDNA molecules, respectively. We prepared mixtures containing various numbers of LNCaP cells in one million cells that do not express PSA and used them as samples in the proposed assay. The ratio of the fluorescence values obtained after analysis of PSA and actin amplification products is linearly related to the number of LNCaP cells in the range of 20 to 3000 cells. Reproducibility studies demonstrate %CVs for the fluorescence ratios of 14.7, 11.8, and 12.2 when samples containing 150, 300 and 1600 LNCaP cells were analyzed (n = 4)., Conclusions: A quantitative analytical methodology is provided for monitoring PSA mRNA. The assay is expected to be beneficial in the study of prostate cancer spread.

We developed microtiter well-based bioluminescence hybridization assays using the photoprotein aequorin as a reporter molecule. The target DNA was hybridized simultaneously with a capture probe and a detection probe. The capture probe was immobilized on the wells through digoxigenin/anti-digoxigenin interaction. The detection probe was biotinylated. The hybrids were determined by using aequorin covalently attached to streptavidin or complexes of biotinylated aequorin with streptavidin. The luminescence was then measured in the presence of excess Ca2+. The optimized protocols showed linearity in the range from 5 amol to 10 fmol of target DNA. In combination with reverse transcriptase polymerase chain reaction, the proposed assay was applied to the detection of the mRNA for prostate-specific antigen (PSA). PSA mRNA from a single cell, in the presence of one million cells that do not express PSA, was detected with a signal-to-background ratio of 2.5. Typical CVs obtained were 6%.

Objective: To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity., Methods: The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+ - EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer., Results: The truncated form of the T-cell protein tyrosine phosphatase (TCdeltaC11 PTP) was determined in the range 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay., Conclusions: The two nonisotopic assays should prove beneficial for the determination and study of various PTP.

We have developed a time-resolved fluorometric hybridization assay for detecting prostate-specific antigen (PSA) mRNA amplified by reverse transcription polymerase chain reaction. During PCR, digoxigenin-11-dUTP is incorporated into the amplified product. An oligonucleotide internal to the primers is used as a specific probe, being biotinylated and captured on streptavidin-coated microtiter wells. Denatured PCR product hybridizes with the probe, and the hybrids are detected with an alkaline phosphatase-labeled antidigoxigenin antibody. We used the phosphate ester of fluorosalicylic acid as the substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which we can measure by time-resolved fluorometry. A signal-to-background ratio of 10 was obtained when 160 PSA cDNA molecules were present in the preamplification sample. Also, mRNA corresponding to one LNCaP cell in the presence of 10(6) PSA-negative cells can be detected (signal-to-background ratio of 3.1). Samples containing 100, 1000, and 50,000 LNCaP cells gave CVs of 12.4%, 4.9%, and 6.8%, respectively (n = 10).