Immunohistochemical assessment of protein phosphorylation state: the dream and the reality

The development of phosphorylation state-specific antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks. The extent to which this promise has been fulfilled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable attention for their potential to reveal cell type-specific activation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the efficacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-specific lability, and implementation of methods for standardization

Patient dose reduction during voiding cystourethrography

Voiding cystourethrography (VCUG) is a commonly performed examination in a pediatric uroradiology practice. This article contains suggestions on how the radiation dose to a child from VCUG can be made ‘as low as reasonably achievable–(ALARA). The pediatric radiologist should consider the appropriateness of the clinical indication before performing VCUG and utilize radiation exposure techniques and parameters during VCUG to reduce radiation exposure to a child. The medical physicist and fluoroscope manufacturer can also work together to optimize a pulsed-fluoroscopy unit and further reduce the radiation exposure. Laboratory and clinical research is necessary to investigate methods that reduce radiation exposures during VCUG, and current research is presented here

A Visual Metaphor Describing Neural Dynamics in Schizophrenia

Background: In many scientific disciplines the use of a metaphor as an heuristic aid is not uncommon. A well known example in somatic medicine is the 'defense army metaphor' used to characterize the immune system. In fact, probably a large part of the everyday work of doctors consists of 'translating' scientific and clinical information (i.e. causes of disease, percentage of succes versus risk of side-effects) into information tailored to the needs and capacities of the individual patient. The ability to do so in an effective way is at least partly what makes a clinician a good communicator. Schizophrenia is a severe psychiatric disorder which affects approximately 1% of the population. Over the last two decades a large amount of molecular-biological, imaging and genetic data have been accumulated regarding the biological underpinnings of schizophrenia. However, it remains difficult to understand how the characteristic symptoms of schizophrenia such as hallucinations and delusions are related to disturbances on the molecular-biological level. In general, psychiatry seems to lack a conceptual framework with sufficient explanatory power to link the mental- and molecular-biological domains. Methodology/Principal Findings: Here, we present an essay-like study in which we propose to use visualized concepts stemming from the theory on dynamical complex systems as a 'visual metaphor' to bridge the mental- and molecular-biological domains in schizophrenia. We first describe a computer model of neural information processing; we show how the information processing in this model can be visualized, using concepts from the theory on complex systems. We then describe two computer models which have been used to investigate the primary theory on schizophrenia, the neurodevelopmental model, and show how disturbed information processing in these two computer models can be presented in terms of the visual metaphor previously described. Finally, we describe the effects of dopamine neuromodulation, of which disturbances have been frequently described in schizophrenia, in terms of the same visualized metaphor. Conclusions/Significance: The conceptual framework and metaphor described offers a heuristic tool to understand the relationship between the mental- and molecular-biological domains in an intuitive way. The concepts we present may serve to facilitate communicatio

A three year descriptive study of early onset neonatal sepsis in a refugee population on the Thailand Myanmar border.

BACKGROUND: Each year an estimated four million neonates die, the majority in the first week of life. One of the major causes of death is sepsis. Proving the incidence and aetiology of neonatal sepsis is difficult, particularly in resource poor settings where the majority of the deaths occur. METHODS: We conducted a three year observational study of clinically diagnosed early onset (<7 days of age) neonatal sepsis (EONS) in infants born to mothers following antenatal care at the Shoklo Malaria Research Unit clinic in Maela camp for displaced persons on the Thailand-Myanmar border. Episodes of EONS were identified using a clinical case definition. Conventional and molecular microbiological techniques were employed in order to determine underlying aetiology. RESULTS: From April 2009 until April 2012, 187 infants had clinical signs of EONS, giving an incidence rate of 44.8 per 1000 live births (95% CI 38.7-51.5). One blood culture was positive for Escherichia coli, E. coli was detected in the cerebrospinal fluid specimen in this infant, and in an additional two infants, by PCR. Therefore, the incidence of bacteriologically proven EONS was 0.7 per 1000 live births (95% CI 0.1-2.1). No infants enrolled in study died as a direct result of EONS. CONCLUSION: A low incidence of bacteriologically proven EONS was seen in this study, despite a high incidence of clinically diagnosed EONS. The use of molecular diagnostics and nonspecific markers of infection need to be studied in resource poor settings to improve the diagnosis of EONS and rationalise antibiotic use

Corneal Alternations Induced by Topical Application of Benzalkonium Chloride in Rabbit

Benzalkonium chloride (BAC) is the most common preservative in ophthalmic preparations. Here, we investigated the corneal alternations in rabbits following exposure to BAC. Twenty-four adult male New Zealand albino rabbits were randomly divided into three groups. BAC at 0.01%, 0.05%, or 0.1% was applied twice daily to one eye each of rabbits for 4 days. The contralateral untreated eyes were used as control. Aqueous tear production and fluorescein staining scores of BAC-treated eyes were compared with those of controls. The structure of the central cornea was examined by in vivo confocal microscopy. Expression of mucin-5 subtype AC (MUC5AC) in conjunctiva was detected by immunostainig on cryosections. Corneal barrier function was assessed in terms of permeability to carboxy fluorescein (CF). The distribution and expression of ZO-1, a known marker of tight junction, and reorganization of the perijunctional actomyosin ring (PAMR) were examined by immunofluorescence analysis. Although there were no significant differences between control and BAC-treated eyes in Schirmer scores, corneal fluorescein scores and the number of conjunctival MUC5AC staining cells, in vivo confocal microscopy revealed significant epithelial and stromal defects in all BAC-treated corneas. Moreover, BAC at 0.1% resulted in significant increases in central corneal thickness and endothelial CF permeability, compared with those in control eyes, and endothelial cell damage with dislocation of ZO-1 and disruption of PAMR. Topical application of BAC can quickly impair the whole cornea without occurrence of dry eye. A high concentration of BAC breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR and remodeling of apical junctional complex in vivo

Abrogation of Junctional Adhesion Molecule-A Expression Induces Cell Apoptosis and Reduces Breast Cancer Progression

Intercellular junctions promote homotypic cell to cell adhesion and transfer intracellular signals which control cell growth and apoptosis. Junctional adhesion molecule-A (JAM-A) is a transmembrane immunoglobulin located at tight junctions of normal epithelial cells of mammary ducts and glands. In the present paper we show that JAM-A acts as a survival factor for mammary carcinoma cells. JAM-A null mice expressing Polyoma Middle T under MMTV promoter develop significantly smaller mammary tumors than JAM-A positive mice. Angiogenesis and inflammatory or immune infiltrate were not statistically modified in absence of JAM-A but tumor cell apoptosis was significantly increased. Tumor cells isolated from JAM-A null mice or 4T1 cells incubated with JAM-A blocking antibodies showed reduced growth and increased apoptosis which paralleled altered junctional architecture and adhesive function. In a breast cancer clinical data set, tissue microarray data show that JAM-A expression correlates with poor prognosis. Gene expression analysis of mouse tumor samples showed a correlation between genes enriched in human G3 tumors and genes over expressed in JAM-A +/+ mammary tumors. Conversely, genes enriched in G1 human tumors correlate with genes overexpressed in JAM-A−/− tumors. We conclude that down regulation of JAM-A reduces tumor aggressive behavior by increasing cell susceptibility to apoptosis. JAM-A may be considered a negative prognostic factor and a potential therapeutic target

Junctional Adhesion Molecule 2 Mediates the Interaction between Hatched Blastocyst and Luminal Epithelium: Induction by Progesterone and LIF

National Basic Research Program of China [2011CB944402]; National Natural Science Foundation of China [30930013, 31071276]Background: Junctional adhesion molecule 2 (Jam2) is a member of the JAM superfamily. JAMs are localized at intercellular contacts and participated in the assembly and maintenance of junctions, and control of cell permeability. Because Jam2 is highly expressed in the luminal epithelium on day 4 of pregnancy, this study was to determine whether Jam2 plays a role in uterine receptivity and blastocyst attachment in mouse uterus. Methodology/Principal Findings: Jam2 is highly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy. Progesterone induces Jam2 expression in ovariectomized mice, which is blocked by progesterone antagonist RU486. Jam2 expression on day 4 of pregnancy is also inhibited by RU486 treatment. Leukemia inhibitory factor (LIF) up-regulates Jam2 protein in isolated luminal epithelium from day 4 uterus, which is blocked by S3I-201, a cell-permeable inhibitor for Stat3 phosphorylation. Under adhesion assay, recombinant Jam2 protein increases the rate of blastocyst adhesion. Both soluble recombinant Jam2 and Jam3 can reverse this process. Conclusion: Jam2 is highly expressed in the luminal epithelium of receptive uterus and up-regulated by progesterone and LIF via tyrosine phosphorylation of Stat3. Jam2 may play a role in the interaction between hatched blastocyst and receptive uterus

Increased α-synuclein phosphorylation and nitration in the aging primate substantia nigra

Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson's disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes

FimL Regulates cAMP Synthesis in Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous bacteria found in diverse ecological niches, is an important cause of acute infections in immunocompromised individuals and chronic infections in patients with Cystic Fibrosis. One signaling molecule required for the coordinate regulation of virulence factors associated with acute infections is 3′, 5′-cyclic adenosine monophosphate, (cAMP), which binds to and activates a catabolite repressor homolog, Vfr. Vfr controls the transcription of many virulence factors, including those associated with Type IV pili (TFP), the Type III secretion system (T3SS), the Type II secretion system, flagellar-mediated motility, and quorum sensing systems. We previously identified FimL, a protein with histidine phosphotransfer-like domains, as a regulator of Vfr-dependent processes, including TFP-dependent motility and T3SS function. In this study, we carried out genetic and physiologic studies to further define the mechanism of action of FimL. Through a genetic screen designed to identify suppressors of FimL, we found a putative cAMP-specific phosphodiesterase (CpdA), suggesting that FimL regulates cAMP levels. Inactivation of CpdA increases cAMP levels and restores TFP-dependent motility and T3SS function to fimL mutants, consistent with in vivo phosphodiesterase activity. By constructing combinations of double and triple mutants in the two adenylate cyclase genes (cyaA and cyaB), fimL, and cpdA, we show that ΔfimL mutants resemble ΔcyaB mutants in TM defects, decreased T3SS transcription, and decreased cAMP levels. Similar to some of the virulence factors that they regulate, we demonstrate that CyaB and FimL are polarly localized. These results reveal new complexities in the regulation of diverse virulence pathways associated with acute P. aeruginosa infections