KSR1 regulates BRCA1 degradation and inhibits breast cancer growth.

Kinase suppressor of Ras-1 (KSR1) facilitates signal transduction in Ras-dependent cancers, including pancreatic and lung carcinomas but its role in breast cancer has not been well studied. Here, we demonstrate for the first time it functions as a tumor suppressor in breast cancer in contrast to data in other tumors. Breast cancer patients (n>1000) with high KSR1 showed better disease-free and overall survival, results also supported by Oncomine analyses, microarray data (n=2878) and genomic data from paired tumor and cell-free DNA samples revealing loss of heterozygosity. KSR1 expression is associated with high breast cancer 1, early onset (BRCA1), high BRCA1-associated ring domain 1 (BARD1) and checkpoint kinase 1 (Chk1) levels. Phospho-profiling of major components of the canonical Ras-RAF-mitogen-activated protein kinases pathway showed no significant changes after KSR1 overexpression or silencing. Moreover, KSR1 stably transfected cells formed fewer and smaller size colonies compared to the parental ones, while in vivo mouse model also demonstrated that the growth of xenograft tumors overexpressing KSR1 was inhibited. The tumor suppressive action of KSR1 is BRCA1 dependent shown by 3D-matrigel and soft agar assays. KSR1 stabilizes BRCA1 protein levels by reducing BRCA1 ubiquitination through increasing BARD1 abundance. These data link these proteins in a continuum with clinical relevance and position KSR1 in the major oncoprotein pathways in breast tumorigenesis.

The regulatory roles of phosphatases in cancer.

The relevance of potentially reversible post-translational modifications required for controlling cellular processes in cancer is one of the most thriving arenas of cellular and molecular biology. Any alteration in the balanced equilibrium between kinases and phosphatases may result in development and progression of various diseases, including different types of cancer, though phosphatases are relatively under-studied. Loss of phosphatases such as PTEN (phosphatase and tensin homologue deleted on chromosome 10), a known tumour suppressor, across tumour types lends credence to the development of phosphatidylinositol 3-kinase inhibitors alongside the use of phosphatase expression as a biomarker, though phase 3 trial data are lacking. In this review, we give an updated report on phosphatase dysregulation linked to organ-specific malignancies.

SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.

BACKGROUND: We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown. METHODS: A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer. RESULTS: Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. CONCLUSION: Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity.

LMTK3 is implicated in endocrine resistance via multiple signaling pathways.

Resistance to endocrine therapy in breast cancer is common. With the aim of discovering new molecular targets for breast cancer therapy, we have recently identified LMTK3 as a regulator of the estrogen receptor-alpha (ERα) and wished to understand its role in endocrine resistance. We find that inhibition of LMTK3 in a xenograft tamoxifen (Tam)-resistant (BT474) breast cancer mouse model results in re-sensitization to Tam as demonstrated by a reduction in tumor volume. A whole genome microarray analysis, using a BT474 cell line, reveals genes significantly modulated (positively or negatively) after LMTK3 silencing, including some that are known to be implicated in Tam resistance, notably c-MYC, HSPB8 and SIAH2. We show that LMTK3 is able to increase the levels of HSPB8 at a transcriptional and translational level thereby protecting MCF7 cells from Tam-induced cell death, by reducing autophagy. Finally, high LMTK3 levels at baseline in tumors are predictive for endocrine resistance; therapy does not lead to alteration in levels, whereas in patient's plasma samples, acquired LMTK3 gene amplification (copy number variation) was associated with relapse while receiving Tam. In aggregate, these data support a role for LMTK3 in both innate (intrinsic) and acquired (adaptive) endocrine resistance in breast cancer.

Gene expression in blood is associated with risperidone response in children with autism spectrum disorders.

Children with autism spectrum disorders (ASDs) often have severe behavioral problems. Not all children with these problems respond to atypical antipsychotic medications; therefore, we investigated whether peripheral blood gene expression before treatment with risperidone, an atypical antipsychotic, was associated with improvements in severe behavioral disturbances 8 weeks following risperidone treatment in 42 ASD subjects (age 112.7±51.2 months). Exon expression levels in blood before risperidone treatment were compared with pre-post risperidone change in Aberrant Behavior Checklist-Irritability (ABC-I) scores. Expression of exons within five genes was correlated with change in ABC-I scores across all risperidone-treated subjects: GBP6, RABL5, RNF213, NFKBID and RNF40 (α<0.001). RNF40 is located at 16p11.2, a region implicated in autism and schizophrenia. Thus, these genes expressed before treatment were associated with subsequent clinical response. Future studies will be needed to confirm these results and determine whether this expression profile is associated with risperidone response in other disorders, or alternative antipsychotic response within ASD.

Certification testing as an acute naturalistic stressor for disaster dog handlers.

USA Federal Disaster Canine Teams, consisting of a handler and a dog, are essential for locating survivors following a disaster. Certification, required by the Federal Emergency Management Agency Urban Search and Rescue organization, requires two successful mock searches. Confirmation of the certification testing process as an acute stressor might offer further opportunities to consider stress effects on handlers and dogs in a controlled environment. This study used a pretest-posttest design to evaluate relationships between salivary hormone concentrations (cortisol and testosterone) and subjective stress ratings in handlers and controls, handler assessments of stress in their dogs, and posttest temperature and pulse rate in dogs. Posttest, both subjective stress ratings and salivary cortisol concentration were higher in handlers than controls with both correlated to handlers' assessment of stress in their dogs. Handlers' posttest salivary cortisol concentration was associated with posttest dog pulse and temperature. Posttest cortisol concentration was lower in handlers who were successfully certified compared with those who failed, and was also lower in handlers whose primary occupation was "firefighter". Salivary testosterone concentrations increased from pretest to posttest in handlers but decreased in controls, and higher posttest handler testosterone concentration was negatively associated with posttest dog pulse rate. These findings confirm certification testing as an acute stressor, suggest a relationship between stress and performance moderated by occupation, and demonstrate an interaction between handler stress and dog physiological responses. This certification testing offers a controlled environment for targeted evaluation of effects of an acute naturalistic stressor on disaster dog handlers and dogs.

Corticosteroid effects on blood gene expression in Duchenne muscular dystrophy.

Though Deflazacort and prednisone improve clinical endpoints in Duchenne muscular dystrophy (DMD) patients, Deflazacort produces fewer side effects. As mechanisms of improvement and side effect differences remain unknown, we evaluated effects of corticosteroid administration on gene expression in blood of DMD patients. Whole blood was obtained from 14 children and adolescents with DMD treated with corticosteroids (DMD-STEROID) and 20 DMD children and adolescents naïve to corticosteroids (DMD). The DMD-STEROID group was further subdivided into Deflazacort and prednisone groups. Affymetrix U133 Plus 2.0 expression microarrays were used to evaluate mRNA expression. Expression of 524 probes changed with corticosteroids, including genes in iron trafficking and the chondroitin sulfate biosynthesis pathway. Deflazacort compared with prednisone yielded 508 regulated probes, including many involved in adipose metabolism. These genes and pathways help explain mechanisms of efficacy and side effects of corticosteroids, and could provide new treatment targets for DMD and other neuromuscular disorders.

A proteomic study of serum from children with autism showing differential expression of apolipoproteins and complement proteins.

Modern methods that use systematic, quantitative and unbiased approaches are making it possible to discover proteins altered by a disease. To identify proteins that might be differentially expressed in autism, serum proteins from blood were subjected to trypsin digestion followed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) on time-of-flight (TOF) instruments to identify differentially expressed peptides. Children with autism 4-6 years of age (n=69) were compared to typically developing children (n=35) with similar age and gender distributions. A total of 6348 peptide components were quantified. Of these, five peptide components corresponding to four known proteins had an effect size >0.99 with a P<0.05 and a Mascot identification score of 30 or greater for autism compared to controls. The four proteins were: Apolipoprotein (apo) B-100, Complement Factor H Related Protein (FHR1), Complement C1q and Fibronectin 1 (FN1). In addition, apo B-100 and apo A-IV were higher in children with high compared to low functioning autism. Apos are involved in the transport of lipids, cholesterol and vitamin E. The complement system is involved in the lysis and removal of infectious organisms in blood, and may be involved in cellular apoptosis in brain. Despite limitations of the study, including the low fold changes and variable detection rates for the peptide components, the data support possible differences of circulating proteins in autism, and should help stimulate the continued search for causes and treatments of autism by examining peripheral blood.

Raised plasma concentration and ex vivo production of inflammatory chemokines in patients with systemic lupus erythematosus.

BACKGROUND: Chemokines are involved in leucocyte chemotaxis. Infiltrating leucocytes play an important role of tissue injury in systemic lupus erythematosus (SLE). OBJECTIVE: To investigate the role of inflammatory chemokines and their association with interleukin 18 (IL18) in SLE pathogenesis and disease activity. METHODS: Plasma concentrations and ex vivo peripheral blood mononuclear cell production of inflammatory chemokines IP-10, RANTES, MIG, MCP-1, TARC, IL8, and GROalpha, and proinflammatory cytokines IL18, IFNgamma, IL2, IL4, and IL10 were assayed in 80 SLE patients with or without renal disease and 40 healthy controls by immunofluorescence flow cytometry and enzyme linked immunosorbent assay. RESULTS: Plasma IP10, RANTES, MIG, MCP-1, GROalpha, and IL18 concentrations in all SLE patients were higher than in controls, and correlated significantly with SLEDAI score (all p<0.05). In SLE patients without renal disease, IP10, RANTES, MIG, MCP-1, IL8, and IL18 correlated positively with SLEDAI score, while in those with renal derangement, IP10, IL8, IL10, and IL18 correlated with disease activity (all p<0.05). Plasma IL18 concentration correlated positively with IP10, MIG, GROalpha, and IL8 in all SLE patients (all p<0.005). Mitogen induced increases in ex vivo production of IP10, MCP-1, TARC, IFNgamma, IL4, and IL10 were higher in all SLE patients regardless of their difference in disease activity (all p<0.05). Patients with renal disease had an augmented ex vivo release of RANTES. CONCLUSIONS: The correlation of raised plasma concentration and ex vivo production of inflammatory chemokines with disease activity, and their association with IL18, supports the view that chemotaxis of Th1/Th2 lymphocytes and neutrophils is important in SLE pathogenesis.

Aberrant production of soluble costimulatory molecules CTLA-4, CD28, CD80 and CD86 in patients with systemic lupus erythematosus.

OBJECTIVE: The costimulatory interactions of the B7 family molecules CD80 and CD86 on antigen-presenting cells with their T-cell counter-receptors CD28 and CTLA-4 modulate T lymphocyte-mediated immune responses in a reciprocal manner. We investigated the possible aberrant production of soluble (s) forms of the T-cell costimulatory molecules CD80, CD86, CD28 and CTLA-4 in plasma of patients with systemic lupus erythematosus (SLE), an autoimmune disease arising from T-lymphocyte dysregulation. METHODS: Plasma concentration and ex vivo production of soluble costimulatory molecules of 79 SLE patients with or without active disease and 40 sex- and age-matched healthy subjects were measured by enzyme-linked immunosorbent assay. RESULTS: Plasma sCTLA-4, sCD28, sCD80 and sCD86 concentrations of all SLE patients were significantly higher than concentrations in control subjects (all P<0.01). These increases were observed even in patients with inactive disease [SLE Disease Activity Index (SLEDAI) <3]. Plasma sCTLA-4 concentration in all SLE patients correlated significantly with SLEDAI score (r = 0.228, P = 0.043). Upon mitogen treatment of peripheral blood mononuclear cells, the percentage increases in ex vivo production of sCD28 and sCD80 and the percentage decrease in sCTLA-4 release were all significantly smaller in SLE patients with active disease than in healthy subjects (P<0.01, P<0.05 and P<0.0001, respectively). CONCLUSION: The aberrant production of soluble T-cell costimulatory molecules is important in the immunopathogenesis of SLE, which occurs by the dysregulation of T-lymphocyte costimulation. Plasma sCTLA concentration could potentially serve as a surrogate marker of SLE disease activity.

Elevation of plasma osteopontin concentration is correlated with disease activity in patients with systemic lupus erythematosus.

BACKGROUND: Osteopontin (OPN) is an extracellular matrix cell adhesion phosphoprotein with immunological activities including stimulation of macrophage chemotaxis, T-helper type 1 lymphocyte response and B-cell antibody synthesis. Overexpression of OPN has been associated with the development of the autoimmune/lymphoproliferative syndrome. METHODS: We measured the plasma concentration and ex vivo production of OPN, and the plasma proinflammatory IL-18 concentration in 54 SLE patients with or without renal impairment (RSLE group and SLE group, respectively) and 26 sex- and age-matched control (NC) subjects using an enzyme-linked immunoabsorbent assay. RESULTS: Plasma OPN concentrations were significantly higher in RSLE and SLE patients than in the NC group (both P<0.001). Increase in OPN concentration correlated positively and significantly with SLEDAI score in all SLE patients (r = 0.308, P = 0.023). The ex vivo production of OPN upon mitogen activation of peripheral blood mononuclear cells was significantly higher in the RSLE and SLE groups than in the NC group (both P<0.001). In RSLE patients, plasma OPN concentration showed a significant positive correlation with proinflammatory cytokine IL-18 concentration (r = 0.404, P = 0.037). CONCLUSION: The above results suggest that the production of OPN is associated with the inflammatory process and SLE development, and may serve as a potential disease marker of SLE.

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome.

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, but its immunopathological mechanisms have not yet been fully elucidated. We investigated changes in plasma T helper (Th) cell cytokines, inflammatory cytokines and chemokines in 20 patients diagnosed with SARS. Cytokine profile of SARS patients showed marked elevation of Th1 cytokine interferon (IFN)-gamma, inflammatory cytokines interleukin (IL)-1, IL-6 and IL-12 for at least 2 weeks after disease onset, but there was no significant elevation of inflammatory cytokine tumour necrosis factor (TNF)-alpha, anti-inflammatory cytokine IL-10, Th1 cytokine IL-2 and Th2 cytokine IL-4. The chemokine profile demonstrated significant elevation of neutrophil chemokine IL-8, monocyte chemoattractant protein-1 (MCP-1), and Th1 chemokine IFN-gamma-inducible protein-10 (IP-10). Corticosteroid reduced significantly IL-8, MCP-1 and IP-10 concentrations from 5 to 8 days after treatment (all P < 0.001). Together, the elevation of Th1 cytokine IFN-gamma, inflammatory cytokines IL-1, IL-6 and IL-12 and chemokines IL-8, MCP-1 and IP-10 confirmed the activation of Th1 cell-mediated immunity and hyperinnate inflammatory response in SARS through the accumulation of monocytes/macrophages and neutrophils.

Electrospray ionisation mass spectrometry: principles and clinical applications.

This mini-review provides a general understanding of electrospray ionisation mass spectrometry (ESI-MS) which has become an increasingly important technique in the clinical laboratory for structural study or quantitative measurement of metabolites in a complex biological sample. The first part of the review explains the electrospray ionisation process, design of mass spectrometers with separation capability, characteristics of the mass spectrum, and practical considerations in quantitative analysis. The second part then focuses on some clinical applications. The capability of ESI-tandem-MS in measuring bio-molecules sharing similar molecular structures makes it particularly useful in screening for inborn errors of amino acid, fatty acid, purine, pyrimidine metabolism and diagnosis of galactosaemia and peroxisomal disorders. Electrospray ionisation is also efficient in generating cluster ions for structural elucidation of macromolecules. This has fostered a new and improved approach (vs electrophoresis) for identification and quantification of haemoglobin variants. With the understanding of glycohaemoglobin structure, an IFCC reference method for glycohaemoglobin assay has been established using ESI-MS. It represents a significant advancement for the standardisation of HbA1c in diabetic monitoring. With its other applications such as in therapeutic drug monitoring, ESI-MS will continue to exert an important influence in the future development and organisation of the clinical laboratory service.

Derivation of a prediction rule for posttraumatic organ failure using plasma DNA and other variables.

The early identification of patients at high risk of developing posttraumatic organ failure would allow preventive therapies to be studied. In this study, highly sensitive and specific guidelines for the early prediction of posttraumatic organ failure (OF) and multiple organ dysfunction syndrome (MODS) using cell-free (plasma) DNA and other predictors of posttraumatic complications were derived. As plasma DNA increases after injury and may be used to predict acute lung injury (ALI), we hypothesized that in combination with other predictors it would predict the later development of OF and MODS. Eighty-three patients (69 males; median age, 36 years) were studied as a consequence of major trauma within 3.5 hours of injury (median time to sampling and assessment, 60 min). Plasma DNA was measured using a real-time, quantitative, polymerase chain reaction assay for the beta-globin gene. OF and MODS occurred in 20/83 (24%) and 9/79 (11%) cases, respectively. At selected cutoff points, the sensitivity of plasma DNA for predicting OF and MODS ranged from 50% to 100%, specificity ranged from 74% to 95%, and the likelihood ratio ranged from 3.89 to 10.50. Other variables studied included serum albumin, creatine kinase, aspartate transaminase, lactate dehydrogenase, leukocyte count, hematocrit, injury severity score, maximal abbreviated injury score, and shock index. Using a classification and regression tree, plasma DNA and aspartate transaminase at optimal cutoffs predicted OF and MODS with an overall correct classification of 93% and 87%, respectively.

Effect of low molecular weight heparin on bone metabolism and hyperlipidemia in patients on maintenance hemodialysis.

The effect of low molecular weight heparin (LMWH) on serum lipid profile in hemodialysis remains controversial and its effect on bone metabolism has not been studied. A crossover study was conducted in 40 patients on stable hemodialysis using unfractionated heparin (UFH) for more than 24 months. These patients were then treated with a LMWH (nadroparin-Ca) for 8 months during hemodialysis and subsequently switched back to UFH for 12 months. Serum lipid profile, biochemical markers for bone metabolism, and bone densitometry (BMD) were monitored at four-month intervals while all medications remained unchanged. Cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), lipoprotein(a) (Lp(a)), apolipoprotein B (Apo B) were raised in 35%, 29%, 12%, 24% and 24% of patients respectively. High-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A1 (Apo A-1) were reduced in 47% and 9% of patients. Bone-specific alkaline phosphatase (BALP) and intact osteocalcin (OSC), both reflecting osteoblastic activity, were raised in 65% and 94% of patients. Tartrate-resistant acid phosphatase (TRACP) reflecting osteoclastic activity and parathyroid hormone (PTH) were elevated in 35% and 88% of patients. Following LMWH treatment, TC, Tg, Lp(a) and Apo B were reduced by 7%, 30%, 21% and 10% respectively (p<0.05 or <0.01) while Apo A-1 were raised by 7% (p<0.01). Simultaneously, TRACP was reduced by 13% (p<0.05). These biochemical changes were detected soon after 4 months of LMWH administration. Although BMD values in our patients were lower than those of age-matched normal subjects, significant changes were not observed with LMWH treatment. After switching back to UFH for hemodialysis, these biochemical indices reverted to previous values during UFH treatment with a significant higher level in TC and Apo B while serum Apo A-1 remained elevated. Our study suggests LMWH may partially alleviate hyperlipidemia and, perhaps, osteoporosis associated with UFH administration in patients on maintenance hemodialysis.

Functional reentry's influence on intracellular calcium in the LRd membrane equations.

This paper examines relationships between transmembrane potential (Vm), [Ca2+]i dependent membrane ionic currents, and [Ca2+]i handling by the sarcoplasmic reticulum (SR) in a two-dimensional model of cardiac tissue. Luo-Rudy dynamic (LRd) membrane equations were used because they include detailed formulations for triggered SR Ca2+ release dependent on membrane Ca2+ influx (CICR) and for spontaneous SR Ca2+ release following calsequestrin buffer overload (SCR). Reentry's rapid rate (110-ms cycle length) elevated [Ca2+]i and limited CICR, which in turn promoted SCR that occurred at intervals of 320-350 ms, was preferential at sites located inside the functional center, and destabilized the reentrant activation sequence. Although adjustment of LRd parameters for SR Ca2+ modified SCR interval and peak [Ca2+]i in voltage clamp simulations with a command waveform representing Vm time course within the functional center, SCR persisted. Using the same command waveform, SCR also occurred with an alternate SR Ca2+ formulation that represented subcellular details underlying CICR. LRd parameter adjustments to promote CICR and limit SCR in subsequent reentry simulations failed to eliminate SCR completely, as they modulated SCR intervals in a manner consistent with the voltage clamp simulations. Taken together, our findings support a destabilizing influence of functional reentry on [Ca2+]i handling. However, [Ca2+]i instabilities did not always fractionate depolarization wavefronts during reentry. Fractionation depended, in part, upon CICR and SCR parameters in the LRd formulation for SR Ca2+ release.

Infection of a knee prosthesis with Tsukamurella species.

A 69-year-old woman with a history of multiple infections of a postoperative wound from a knee replacement was diagnosed with an infection with Tsukamurella sp. The infection was treated with a course of vancomycin and pipercillin/tazobactam, followed by a course of clarithromycin, ciprofloxacin, and ethambutol. The patient responded well. This represents the first report of a Tsukamurella infection of an artificial joint.

Recognizing and treating syphilis in pregnancy.

The number of primary and secondary syphilis cases in young women rose dramatically in the late 1980s and early 1990s, due to illicit drug use and the exchange of drugs for sex. Of infants born to mothers with primary or secondary syphilis, up to 50% will be premature, stillborn, or die in the neonatal period; further, most of these children are born with congenital disease that may not be apparent for years. While appropriate treatment of the pregnant female can prevent congenital syphilis, the major deterrent has been the inability to effectively identify these women and get them to undergo treatment. In determining a penicillin regimen, the clinician must consider the stage of maternal infection, the length of fetal exposure, and physiologic changes in pregnancy that can affect the pharmacokinetics of antibiotics. Treatment decisions may be further complicated in patients who are allergic to penicillin or infected with HIV. The pathogenesis of congenital syphilis is not completely understood, but placental invasion is the presumed major route. All women should be screened for syphilis with a nontreponemal test (eg, rapid plasma reagin [RPR] or venereal disease research laboratory [VDRL] test) in the first trimester. Those at high risk should be retested at 28 weeks and near delivery. Even with appropriate treatment of syphilis during pregnancy, fetal infection may still occur in up to 14% of cases. Treating syphilis during pregnancy can be difficult due to physiologic changes that can alter drug levels and the risk that drugs will induce uterine contractions or compromise the health of the fetus. While there are added risks and potential complications, treatment regimens parallel those in nonpregnant women.