The notion that breast cancers can survive in an individual patient in a dormant state only to grow as metastatic disease in the future, is in our view incontrovertibly established. Convincing too is the evidence that surgery to remove the primary tumor often terminates dormancy resulting in accelerated relapses. Accepting that many deaths due to breast cancer might be averted were we to understand the cellular mechanisms underlying escape from dormancy, we have examined the extracellular signals produced by breast cancers derived from women with metastatic breast disease. In this perspective, we explore the role of extracellular nucleotide signaling that we have proposed constitutes a pathological axis from the transformed tumor cell to the endothelium in the service of intravasation, dissemination, extravasation and angiogenesis. A role for the dinucleotide kinase NM23/NDPK (nucleoside diphosphate kinase) secreted by breast tumor cells in the generation of signals that stimulate vascular leakiness, anti-thrombosis, endothelial migration and growth, constitutes a mechanistic basis for escape from latency and offers putative therapeutic targets for breast cancer management not previously appreciated. This imperative may be flawed. Whether reviewed from the historical perspective so thoroughly offered by Retsky and colleagues , or viewin situ may seem heretical, a better understanding of tumor biology might redefine what could be done before, or concurrent with surgical removal that might establish continued suppression of those cells dormant at the time of surgery. The hazard from primary tumor removal to subsequent metastatic disease has been established in patients untreated following surgery [cure” for breast cancer in mouse models of the human disease [While the notion of leaving a primary breast cancer lesion  surgery ,3,4. The disease -7. Such  disease  such thain situ (DCIS) by mammography has exceeded our diagnostic grasp. A large proportion of such cases are false positives  and othe as 20% , their ce as 20% .If breast cancer cells have left the site of the original transformed lesion to reside elsewhere in the body, but not produce disease for many years, then it is critically important to determine what events permit their passage to other sites in the body; why once disseminated they do not continue to grow, only to lie dormant; and what triggers their exit from dormancy; and once this change occurs, what biologic processes transpire that support their growth as metastases?While these events will be the result of disparate pathways guiding the interaction of transformed cells with normal compartments such as capillary beds in tissues such as the lung, we suggest that some of these answers lie in a better understanding of the actions of extracellular NM23/NDPK (nucleoside diphosphate kinase) shed from breast cancer cells. In the following discussion, we will review recent work from our laboratory and that of others that sheds light on these questions and offers a challenge to our current view of tumor biology.2.2.1.While dissemination of breast disease to regional lymphatics has been used both for diagnostic and treatment objectives, it is clear that clinically important breast cancer metastases to which patients succumb, do not establish as a result of the passage of breast cancer cells via lymph. Indeed, perhaps the best proof for this comes from the surgical approaches taken in the past to provide a surgical “cure” for breast cancer . The radIf cells move early and can, but do not always generate metastatic disease years later, then there are but two inescapable possibilities. One is that the phenotype of the cells that constitute the events of dissemination are disparate, if overlapping with those that support metastatic disease  and thusIn our view, an interesting way to approach the question of metastasis is to ask if breast cancer cells once mobile (this too is not obvious), mimic or even enlist the actions of cells that regularly enter the tissue from the blood stream. In simple terms, intravasation and extravasation of breast cancer cells may be the result of their ability to mimic leukocytes that regularly exit the blood stream to fight infection. The effects of VEGFR2 activation that render capillary vessels permeable , particuWhile a detailed examination of the ability of breast tumors to evade immune recognition is beyond the scope of this perspective, the subject has been examined and thought to relate to the ability of cells to immunoedit . Tumors Whatever the precise details of the epithelial to mesenchymal transition (EMT), it appears that these genetic changes are sufficient to constitute invasive characteristics  that per2.2.awd protein  whose expression was thought to be inversely related to metastasis potential [NM23 is a nucleoside diphosphate kinase (NDPK) regenerating ATP (adenosine 5′-triphosphate) levels for intracellular “housekeeping” enzymes by covalently transferring the γ-phosphate from a nucleoside triphosphate (NTP) such as GTP (guanosine 5′-triphosphate), to a nucleoside diphosphate acceptor ) via a ping-pong transphosphorylase mechanism . In thisotential ,34 althootential ,35-37.et al. confirmed that the catalytically inactive H118Y mutant of NDPK-B significantly suppressed the lung metastasis of human melanoma cells in vivo. Because mutations in NM23 genes are rare in cancer [Contradictory evidence regarding NDPK-induced metastasis inhibition has been found in that RNA (ribonucleic acid) levels of H1, and H2 are elevated in aggressive neuroblastoma and colorectal cancers -41. Thern cancer , NM23 man cancer ), but thin vitro studies, extracellular NDPK-B was secreted by MDA-MB-435 breast cancer, colon and pancreas cell lines [in vitro [A pathological role of extracellular NDPK-A and B is suggested in multiple studies of patient serum where NDPK-A is overexpressed as in extracellular fluid from non-Hodgkin lymphoma ,50, neurll lines , while Nll lines  into thell lines ,60, and in vitro .in vitro [in vivo work from our lab [For an NDPK to appear in the blood stream suggests that it would likely act to generate ATP from ADP in the blood stream were a phosphoryl donor present. It is known that nucleotides appear normally in the blood stream  and thatin vitro ,67,68 an our lab  together our lab ,45-47 thin vivo [in vitro [Expression of a cell-surface NDPK activity is known to regulate extracellular nucleotide levels in various cell types -72. A cain vivo . NDPK  to release nitric oxide, prostacyclin and additional ATP [In the blood stream, ATP and ADP act immediately upon their formation as autocrine and paracrine hormones by stimulating endothelial nucleotide receptors  receptor . We now  of VEGF ,78. This of VEGF  to block1/2 phosphorylation which is prevented by ellagic acid, the NM23/NDPK substrate site inhibitor [Endothelial cell VEGFR-2 activation by extracellular NM23/NDPK results in Erknhibitor , and surnhibitor .2.4.1R and VEGF stimulation of VEGFR-2 phosphorylation, while having little effect on VEGF-stimulated Erk1/2 phosphorylation is consistent with the notion that P2Y1R signals to activate VEGFR-2 via Src activation. The ability of VEGF to activate VEGFR-2 Tyr-1175 phosphorylation is, by contrast, a direct effect of the growth factor to bind to and activate its receptor. The contrast in the transactivation of VEGFR-2 by P2Y1R activation via the action of NM23/NDPK versus VEGF activation can be seen in the ability of PP2 to block P2Y1R but not VEGF activation of VEGFR-2 [1/2 confirming that Src does not phosphorylate Erk in human endothelial cells. The notion established by these data is that VEGF antagonists alone may not perform well clinically as angiogenesis supressors since nucleotide activation of VEGFR would be unaffected. Tyrosine kinase inhibitors have demonstrated clinical utility in inhibiting VEGF receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor [The ability of the inhibitor (PP2) of the proto-oncogene non-receptor tyrosine kinase, Src to block both P2Y VEGFR-2 . The Srcreceptor . Howeverreceptor , are astreceptor ,92 and rreceptor .1 receptor is crucial in the activation of tubule formation in endothelial cells [in vitro. Our more recent examination of the mechanisms of the P2Y1 receptor pathway with regard to transactivation of VEGFR-2 Tyr1175, extracellular signal-related kinase (Erk1/2) activation, and functional determination of cell growth and migration, reinforce the biology of the extracellular actions of NM23/NDPK in support of tumor angiogenesis [We have previously shown that the activation of the P2Yal cells  in vitroogenesis . The abiRecent studies from Cynthia Bamdad and colleagues have focused attention on NM23/NDPK and a heretofore unrecognized binding partner, Muc-1. The mucin glycoprotein normally expressed at the apical border of epithelia (MUC1) is overexpressed by breast cancer cells ,95 that 3.The evidence that we need to revisit our understanding of breast cancer without the bias of our assumptions about uncontrolled growth of tumors as a hallmark of the disease is sorely needed. The fact of dormancy demands that we examine tumor biology in the context not just of the properties of tumor cells themselves, but of the environment in which they move in the body and hijack normal mechanisms. It is time too that we lose the dogmatic assumptions of the associations of genes with metastatic potential as causative or singularly instructive to further research. A case in point is NM23/NDPK. Earlier work that has branded this protein in one or more of its forms, as a metastasis suppressor from gene expression studies, completely ignores its extracellular actions and biases the field to other ideas. Indeed, the transactivation of the vascular endothelial growth factor receptor via NM23/NDPK activation of human endothelial purinergic receptors offers a framework for the dramatic ability of the nucleotide pathway to promote cancer cell dissemination and angiogenesis. Studies to link Muc-1, NDPK, inflammatory cells, the endothelial cell and its P2Y receptors may further our understanding of tumor cell mediated metastasis.in vivo models since none of this will be useful if it cannot lead to new ways of treating breast cancer. Translation is essential. Breast cancer research cannot afford the luxury of eccentricity. This translation is ongoing now for the actions of NM23/NDPK described here. We have collected evidence at the time of writing that metastasis of MDA-MB-231 cells in immunocompromized SCID mice is significantly lessened  by treating animals with NM23/NDPK inhibitor and an antagonist of the endothelial P2Y receptor. Breast cancer has eluded our efforts to prevent it for centuries. Perhaps it is time to look beyond what has been assumed, or learned from other diseases and be led by the data, no matter how inconvenient that may be for the field.In the end, what matters to women is what we can learn to treat their disease more effectively. What matters in the laboratory is that we can advance our ideas to some reasonable measure of effectiveness in


Anxiety disorders are one of the most psychiatric disorders in children and adolescents that can cause long life functional disability. The first line treatment for this disorder is cognitive behavioral therapy that has primary, secondary and tertiary preventive effect, but is expensive and long time. Today there is some effort to find short term, group, semi-attendance and low cost therapies.Subjects were 42 girls (12- 17 y) with at least one anxiety disorder according to DSM-IV-TR with their parents who were divided into two groups randomly: group A which participated in 8 sessions and group B which participated in 4 sessions and the contents of sessions 3, 4, 6, and 7 were recorded on a CD for them. The tests used in this study were: SCARED, CATS, CAIS-C, CAIS-P, conducted before (T0), just after (T1) and three months after the treatment (T2). The collected data were analyzed by multivariate analysis of covariance test using SPSS software package, version 15.0.There was no significant difference between efficacy of semi-attendance group CBT and attendance group CBT in T0, T1 and T2 according to 4 tests (p = 0.311). The difference between the scores of these tests between T0 andT1 and T0 and T2 was significant in both groups (p < 0.001) but the difference between T1 and T2 was not significant. (p = 0.771).The efficacy of semi-attendance group CBT and attendance group CBT is similar and would sustain after 3 months. Anxiety disorders are among common psychiatric disorders of children and adolescents.610The first article on the efficacy of cognitive behavioral therapy on anxiety of children and adolescents were written over ten years ago.1151126914The combination of this treatment with medication in acute conditions such as school refusal, social isolation and low response to psychotherapy has been reported effective.56Since these disorders are more prevalent among low socioeconomic classes of society,15Nowadays, there are studies being carried out on distance methods through computer or internet programs with optimistic results, although some experts believe that face to face communication is necessary for CBT.1516This study has been designed and conducted to find an effective, harmless, low cost, short term and attractive treatment for adolescents through which the patient plays an active role in controlling his/her signs.This is a randomized clinical trial that was specially conducted for one year from September 2008 to November 2009. The study was carried out on child/adolescent sub-specialty psychiatry clinic in Noor Hospital of Isfahan University of Medical Sciences, Iran. First, forty four adolescent girls aged from 12 to 17 years, who referred to child/adolescent clinic, diagnosed with at least one anxiety disorder based on DSM-IV-TR  by a sub-specialist of pediatric psychiatry, were selected. Then, semi structured interview of ADIS : That is the best self multi dimensional measurement questionnaire with 66 items for children and adolescents’ anxiety disorders18CATS : That is a self-report tool for ages 8-17 years with 40 items to measure automatic thoughts within recent week. Its internal consistency, including total score and score of its four subscales is so high. Its test-retest reliability is acceptable up to 1-3 months and its discriminative validity between depression, anxiety and behavioral disorders is acceptable.23CAIS-C ): That is a self-report scale to investigate the effect of fears and worries on different domains of life.CAIS-P ): That measures anxiety function interference of children in different domains of their parents’ life.1The size of each group was 10 subjects. For the attendance group, there were 8 sessions held weekly.The protocol used in these sessions was taken from BRAVE program of Australian Macquarie University Anxiety Research Unit that is a group cognitive behavioral therapy for adolescents aged 13-17 years with various anxiety disorders.In the semi-attendance group, there were 4 sessions and the content of other four sessions was given to the patients in form of CDs. The CDs included some films which were taken from sessions 3, 4, 6, and 7 in addition to some diagrams and photos. The CD was developed by the researches and it took one hour for each session.0), just after finishing the program (T1) and three months after the treatment (T 2). The obtained data were analyzed by multivariate analysis of covariance test using SPSS software package, version 15.0.The subjects were followed up through phone calls. The sessions were held for 75 minutes. Adolescents and their parents attended a shared session for 15 minutes, then they had parallel and separate sessions, each for 45 minutes with a therapist and in the last 15 minutes again they were in a session together. The sessions of adolescents and parents were held simultaneously in order to facilitate families’ attendance in treatment and play therapy room was ready to those with small children. The therapists performance were quite identical as they were recorded and re-checked by the researchers. All tests were taken before treatment . Regarding the frequency distribution of various psychiatric disorders, there were significant difference only for panic disorder (p = 0.011) and eating disorder (p = 0.011). The mean ± standard deviation (SD) of the scores in SCARED, CATS, CAIS-C and CAIS-P questionnaire before, just after finishing and three months after treatment in both groups have been presented in Before interventions, scores of four anxiety measurement tools were considered as modifying variables and the scores for differences of changes just after and three months after intervention were considered as dependent variables and therapeutic effect of semi-attendance and attendance group therapies on dependent variables were measured. Age and education level were controlled in data analysis. The assumptions of the fitting model of multivariate analysis of covariance were checked. Box test of equality of covariance matrices and Leven’s test of equality of variances of the four dependent variables were equal across the groups (p > 0.05). Mean scores of each questionnaire based on studied groups in three different times have been presented in figures The results show that these differences of changes just after and three month after intervention did not differ significantly between the two groups (p > 0.05).In overall results show that all four anxiety measurement tools had a significant difference before, just after and three months after intervention (p < 0.001) using ANCOVA Repeated Measure of analysis (details not shown). Post Hoc analysis revealed that the difference was significant between the first and second time and the first and the third time (p < 0.001) but not between the second and the third time (p = 0.771).There was no statistically significant difference between the groups. In semi-attendance group CBT patients, families and therapists spent less time, the adolescents played a more active role in treatment and control of their signs, so it maybe more attractive for them and their parents. Both groups had the same levels of drop out and compliance.CBT has been proven to be effective for treating anxiety disorders in children and adolescents.24252829Nowadays, there are studies being carried out on distance methods through computer or internet programs with optimistic results. Spence et al (2006) studied 72 children and adolescents aged from 7-14 years with anxiety disorders. Attendance group CBT and semi-attendance group CBT through internet had both significantly showed recovery signs compared to waiting list and had permanent therapeutic effect for 12 months. Semi-attendance group CBT was accepted for the families with the lowest sample drop outs.16March et al (2009) surveyed 73 children aged from 7-12 years with anxiety disorders and their parents with distance group CBT through internet and then compared them with waiting list. There was a decrease in signs and an increase in function in six months follow up compared to control and 75% of the subjects became completely symptom free.303239In conclusion, present results demonstrate that both attendance and semi-attendance group CBT are effective in treatment of different kinds of anxiety disorders in adolescent girls, but still further studies and researches in this field are needed to be performed.One of the main limitations to the study was the small sample size. Due to this limitation, the differences may not be shown significantly, so a study with larger sample size is suggested to be conducted. The other limitation in this study was preparation of very attractive CDs for adolescents. Although, Preparation of the CD for the first time seems expensive, but compared to the costs spent on patients’ commutation and the costs of therapist visits, semi-attendance group CBT seems somehow cost effective. Anyhow, it is a rough estimation and more studies focused on cost efficacy of this type of therapy are suggested to be conducted.


Young-onset colorectal cancer is clinicopathologically different from older-onset colorectal cancer and tends to occur in patients with hereditary germline conditions such as Lynch syndrome and familial adenomatous polyposis.We describe the case of a 44-year-old man with a paternal history of colon polyps, a personal 2-year history of hematochezia, and a diagnosis of rectal cancer. Further clinical evaluation of the patient at our institution determined the cancer to be stage IIIA. The patient underwent genetic counseling and testing, which indicated he was negative for the most common familial cancer syndromes. After treatment with neoadjuvant chemoradiotherapy, surgery, and adjuvant chemotherapy, the patient has done well. We review the hereditary cancer syndromes and genetic tests to consider for patients with early-onset colorectal cancer.This case underscores the importance of following cancer-screening guidelines. Colorectal cancer (CRC) is a common malignancy in North America and Europe; most patients have sporadic disease without a known genetic predisposition to the illness. With the exception of the hereditary germline conditions Lynch syndrome, familial adenomatous polyposis (FAP), and attenuated FAP, which are marked by an early age of onset and familial CRC clustering, most cases of CRC do not develop until age 65 years or older. Still, up to 20% of all cases of CRC arise in persons aged 50 years or younger who do not have Lynch syndrome or FAP.3Lynch syndrome, formerly known as hereditary nonpolyposis colorectal cancer, is caused by a germline mutation in 1 of several DNA mismatch repair (MMR) genes and is the most common single-gene–related cause of hereditary colon cancer in North American and European populations.2One report based on the experience from 2 cancer registries in the United States, the National Program of Cancer Registries and the Surveillance, Epidemiology and End Results studies, emphasized that CRC is of concern for young adults: it is among the top 10 cancers in persons aged 20 to 49 years of all races.A 44-year-old man sought medical care in 2001 for a 2-year history of hematochezia. Colonoscopy performed at an outside institution in September 2001 showed a lesion within the rectum. Biopsy performed at that time confirmed a poorly differentiated, grade 3 adenocarcinoma, which measured 2×1.3×1 cm, with invasion through the muscularis mucosa. He came to our institution in October 2001 for further evaluation.The patient’s family history of cancer was primarily limited to a brother who received a diagnosis of chronic myelogenous leukemia at age 47 years. His father had colon polyps removed in his mid 50s, and a maternal aunt had colon polyps at age 35 years. Distant paternal relatives may have had CRC in old age.The patient had been a smoker for the previous 16 years and had a history of gastroesophageal reflux disease. He had a history of sebaceous cysts on the left scapula and the back of the thighs and a lipoma on the inferior border of the right scapula. He did not have supernumerary teeth.APC gene mutation because he was not suspected to have FAP  by the American Joint Committee on Cancer 6th edition criteria.2). He was treated with 180 cGy in 28 fractions, for a total of 5040 cGy. He tolerated the therapy well, with some mild diarrhea, weight loss, decreased energy, and perianal irritation. The cancer in the rectum showed complete response to therapy.Neoadjuvant chemoradiotherapy was recommended on the basis of the tumor stage and the proximity of the tumor to the prostate. From November through December 2001, the patient was given a continuous infusion of radiosensitizing 5-fluorouracil (5-FU)  was decreased by 10% (to 380 mg/m2). He completed chemotherapy in August 2002.In April 2002 the patient began his first of 4 cycles of adjuvant 5-FU and leucovorin systemic chemotherapy. Prolonged neutropenia and gastrointestinal tract bleeding occurred in the middle of the first cycle of chemotherapy; therefore, the dosage of 5-FU .Although only 15% to 20% of sporadic cancers are MSI-H, 90% of patients who meet the Amsterdam criteria for Lynch syndrome have MSI-H CRC.BRAF V600E mutation testing of tumor DNA can be used to distinguish an MSI-H tumor with absent MLH1 expression caused by hypermethylation of the MLH1 promoter from a tumor caused by a germ-line MLH1 mutation. Tumors with hypermethylation of MLH1 and with the BRAF V600E mutation nearly always represent sporadic CRC not caused by a germline MLH1 mutation and not associated with Lynch syndrome.Lynch syndrome, an autosomal dominant disorder, is the most common hereditary colon cancer syndrome. Mutations in 1 of the MMR genes usually result in truncated or lost protein product. Thus, tumors can be screened for defective DNA MMR by using polymerase chain reaction to test for MSI or IHC to test for loss of MMR protein expression. The results of IHC may then be used to direct germline sequencing toward a specific DNA MMR gene in young-onset cases or in persons with clinical or family history criteria suggestive of Lynch syndrome. Hypermethylation assays and MLH1, MSH2, and MSH6 by IHC. MSI testing was not performed. A previous study from our institution showed that a normal IHC test for MLH1 and MSH2 has a 96.7% positive predictive value for a microsatellite stable/MSI-L phenotype.9In the case presented here, the patient was tested for a familial syndrome because of his relatively young age at presentation (44 years); however, he lacked many of the features of either FAP or Lynch syndrome. The genetic diagnosis of Lynch syndrome requires a germline mutation in 1 of the MMR genes. The patient’s tumor showed normal expression of the MMR genes MYH-associated polyposis (MAP). MAP has a phenotypic overlap with FAP, attenuated FAP, and Lynch syndrome; biallelic carriers have an 80% cumulative lifetime risk of CRC by age 70 years.MYH mutation.13Another hereditary CRC syndrome that can be considered is Although CRC does not usually develop until age 65 years or older, up to 20% of CRC cases will arise in persons 50 years or younger who do not have either of the known hereditary CRC conditions.MYH mutations, regardless of their family history or the number of colon polyps. IHC can be used in clinical practice to test for MAP regardless of the specific MYH mutations.17If testing for MMR is negative, patients with early-onset CRC may be tested for The case presented here highlights that CRC can occur at an age younger than the cancer-screening guidelines suggest for average-risk patients and also shows the importance of using family history to determine the timing of the first CRC screening. Had our patient undergone his first screening colonoscopy at age 40 years as recommended by the American Gastroenterological Association—given that the patient’s father had colon polyps removed before age 60 years—his CRC might have been diagnosed at an earlier stage. Similarly, the patient’s 2-year history of hematochezia warranted a colon examination. We stress the importance of acknowledging and pursuing these symptoms, even in younger patients.


Creatine supplementation can improve performance during high intensity exercise in humans and improve muscle strength in certain myopathies. In this present study, we investigated the direct effects of acute creatine incubation on isolated mouse fast-twitch EDL muscles, and examined how these effects change with fatigue.digitorum longus muscle from mice aged 12–14 weeks was isolated and stimulated with field electrodes to measure force characteristics in 3 different states: (i) before fatigue; (ii) immediately after a fatigue protocol; and (iii) after recovery. These served as the control measurements for the muscle. The muscle was then incubated in a creatine solution and washed. The measurement of force characteristics in the 3 different states was then repeated. In un-fatigued muscle, creatine incubation increased the maximal tetanic force. In fatigued muscle, creatine treatment increased the force produced at all frequencies of stimulation. Incubation also increased the rate of twitch relaxation and twitch contraction in fatigued muscle. During repetitive fatiguing stimulation, creatine-treated muscles took 55.1±9.5% longer than control muscles to lose half of their original force. Measurement of weight changes showed that creatine incubation increased EDL muscle mass by 7%.The extensor 2+ sensitivity of contractile proteins as a result of ionic strength decreases following creatine incubation.Acute creatine application improves force production in isolated fast-twitch EDL muscle, and these improvements are particularly apparent when the muscle is fatigued. One likely mechanism for this improvement is an increase in Ca This Pi enters the SR and precipitates the free calcium as calcium phosphate i entering the SR may explain why fast-twitch muscles of creatine-kinase-deficient mice  are markedly more fatigue resistant than wild-types In fast-twitch muscles , repeated activation depletes stores of high energy phosphates (ATP and PCr) and there is a graded failure of Ca2+ sensitivity of the contractile proteins, by virtue of the reduction in ionic strength that occurs when water follows creatine osmotically into the muscle fibre 2+] would enhance muscle performance and alleviate some of the force loss resulting from reduced SR Ca2+ release during fatigue.Creatine has also been reported to increase the Caet al.Despite some of the abovementioned benefits of creatine for muscle performance, several studies The aim of our present study was to provide further insight into how creatine may enhance muscle performance, by examining the contractile properties of isolated whole muscles before and after the acute application of creatine, in the absence and presence of fatigue. We used an isolated fast-twitch muscle preparation, using the extensor digitorum longus (EDL) muscle from the mouse . 12 mice (C57BL/10) aged 13 to 14 weeks were used. Animals were sacrificed using an overdose of Halothane  followed by cervical dislocation.In order to assess the water accumulation that resulted from incubation with 10 mM creatine, 7 EDL muscles were dissected out, blotted and weighed, then incubated in oxygenated Krebs solution containing 10 mM creatine for 30 minutes. They were then blotted on filter paper and weighed again. To see whether creatine incubation affects the diameter of muscle fibres, single enzymatically isolated flexor digitorum brevis (FDB) fibres were viewed under a Nikon inverted microscope at ×600, while perfused with oxygenated Krebs solution. A graticule was used to measure the fibre diameter at its widest point. The solution was then switched to one containing 10 mM creatine for 30 minutes, after which the fibres were observed during a washout period for 30 minutes. The isolated fibres were firmly attached to the glass coverslip and the graticule position was monitored throughout the procedure to ensure that its position relative to the fibre did not change during the procedure.2PO4, 1.18 MgSO4, 24.8 NaHCO3, 2.5 CaCl2, 11 glucose, bubbled with 95% O2 5% CO2 to maintain the pH at 7.4. The optimal length was termed L0 and all subsequent measures were made at this muscle length. After determining the set length of the muscle it was allowed to equilibrate for 5 minutes in the bath.Details of the muscle set-up procedure have been reported elsewhere Following this a force-frequency curve was generated using frequencies of 2, 5, 10, 15, 20, 25, 30, 50, 60, 75 and 100 Hz. This served as the “pre-fatigue” force-frequency curve for the muscle. A fatigue run was then carried out using our published fatigue protocol et al.−1Following determination of the force-frequency characteristics of untreated muscle (the control condition), the muscle was incubated in a 9.9 mM creatine solution, prepared by dissolving creatine (Creatine Anhydrous from SIGMA) in the Krebs solution described above. The incubation period was 30 minutes and the muscle was stimulated at 100 Hz for 200 msec once every five minutes. The creatine-specific transporter protein CreaT −3). The wet muscle mass was measured at the end of each experiment and hence represents the muscle mass following creatine incubation. In the water accumulation experiments described above, it was found that creatine incubation resulted in an average 7.3% increase in muscle weight. To allow for this, cross-sectional areas have been reduced by this amount in calculating specific forces before creatine incubation.In the presentation of results, all forces are expressed as specific force . The mean cross-sectional area of each muscle was calculated by dividing the muscle's wet mass by the product of its optimum length and the density of mammalian skeletal muscle (1.06 g cmt-test (GraphPad Prism) was used to test the significance of treatments. The significance level was P<0.05. However, when comparing control force versus creatine-treated force at each stimulation frequency of the force-frequency curve, a different test was used to reduce the possibility of Type I error arising from the large number of comparisons. Here we used Bonferroni multiple comparison post-tests following two-way repeated measures ANOVA, with an overall significance level of 5% (GraphPad Prism).A two-tailed paired P to the stimulation frequency f according to the following equation Pmax is the maximum force, Pmin is the minimum force, fK is the half-frequency  and h is the Hill coefficient (an indication of the steepness of the curve).One further analysis performed on the force-frequency data was to fit a sigmoidal curve to the forces measured in every force-frequency activation sequence. This curve relates the muscle force Creatine enters skeletal muscle by a saturable process, with an accompanying increase in intracellular water that is evidenced by an increase in muscle weight −2 following creatine incubation compared with 14.9±2.13 N cm−2 before creatine treatment  and the Hill coefficient (a measure of the steepness of the curve). The values of these parameters are shown in The rate of relaxation during a muscle twitch, calculated as the fall in force divided by the change in time during the first 50% of relaxation, is shown in The twitch-to-tetanus ratio was unchanged for the creatine-treated condition with respect to the control condition both before fatigue and after recovery. In creatine-treated muscles, however, there was a difference between the ratio in fatigue and the ratios for the pre-fatigue and recovered states. Notably this difference did not occur before creatine treatment. P<0.05) longer than the control for the force to fall to this point (P<0.05). Creatine significantly increased the EDL muscle's resistance to fatigue. The parameter measured during the fatigue runs was the time taken for the maximum force produced by the muscle to fall to 50% of the pre-fatigue maximum. Creatine-treated muscle took 55.1±9.5%  where it precipitates the free Ca2+ resulting in a failure of Ca2+ release i levels lower by using the free inorganic phosphate to regenerate ATP from ADP. In addition the open probability of the phosphate transporter in the SR will be reduced as ATP levels are higher. Supporting this, Gallo et al.Why does creatine have this inhibitory effect on fatigue? One mechanism that can be deduced from earlier studies is that creatine helps to maintain Ca2+ sensitivity of the contractile proteins arising from the reduction in ionic strength that accompanies Cr loading 2+ release from the SR was impaired during fatigue, creatine incubation may attenuate the resulting loss of force by making the contractile proteins more sensitive to Ca2+.However, another mechanism has been proposed where force is enhanced due to the increased Ca2+] falls rapidly 2+ levels relatively constant 2+ handling in dystrophic muscle et al.in vivo displayed an impaired peak tetanic force, a reduced ability to sustain maximal contractions and an increased time to half relaxation. Further, in myotube cultures from the same strain of mouse, both the release and sequestration of Ca2+ were negatively affected as measured by a reduced peak and an extended duration of the Ca2+ released in a twitch.The slowing of contractile kinetics during fatigue is a well established finding in the literature. The decreased speed of relaxation In the present study it is possible that the increased rates of contraction and relaxation seen in fatigued muscle pre-incubated in creatine  is a con2+ sensitivity of the contractile proteins due to the reduction in ionic strength that accompanies creatine incubation. The improved resistance to loss of force during fatiguing stimulation is also likely to be explained by an increased Ca2+ sensitivity. We have also shown that in fatigued muscle, creatine incubation increases the rate of twitch contraction and relaxation. In fatigued muscle, even small changes in force production and contractile kinetics could be very important in terms of human performance in a race situation. Thus the effects of creatine incubation on isolated muscle demonstrated in our study provide insight into the improved muscle performance observed following creatine supplementation in humans. Providing there are no as yet undiscovered health risks associated with creatine supplementation, creatine may be of great benefit both to athletes and as a therapeutic tool for the treatment of neuromuscular disorders.We have shown that acute incubation with creatine enhances force production in fast-twitch EDL muscle, especially when the muscle is fatigued. Such an effect is likely to occur through an increased Ca


We focused on cystathionine β-synthase (CBS) because of its clinical relevance in homocysteine metabolism and because some variants of the enzyme are clinically responsive to increased levels of its B6 cofactor. Single amino-acid substitutions throughout the CBS protein caused reduced function in vivo, and a subset of these altered sensitivity to limiting B6-cofactor. Some of these B6-sensitive substitutions also had altered sensitivity to limiting heme, another CBS cofactor. Limiting heme resulted in reduced incorporation of heme into these variants, and subsequently increased protease sensitivity of the enzyme in vitro. We hypothesize that these alleles caused a modest, yet significant, destabilization of the native state of the protein, and that the functional impact of the amino acid substitutions caused by these alleles can be influenced by cofactor(s) even when the affected amino acid is distant from the cofactor binding site.Any two individuals differ from each other by an average of 3 million single-nucleotide polymorphisms. Some polymorphisms have a functional impact on cofactor-using enzymes and therefore represent points of possible therapeutic intervention through elevated-cofactor remediation. Because most known disease-causing mutations affect protein stability, we evaluated how the  The interpretation of human genome variation is a defining challenge in human biology for the foreseeable future. The unprecedented increase in both total sequence information and in the number of new gene variants has created a critical bottleneck in the ability to interpret the impact of the genetic variants in an individual. Clearly, advances in interpretation are crucial to realize the potential of personalized medicine. As one measure of the magnitude of the challenge, any two unrelated humans differ by an average of 3 million single-nucleotide polymorphisms (SNPs). Among the SNPs in an individual, a current estimate is that 10,000 cause a change in the sequence of a protein .In the present study we turned to human cystathionine β-synthase (CBS), a vitamin-dependent enzyme whose structure is known, to explore the concept of cofactor remediation more deeply to determine its prevalence, and whether there are structural principles that can be illuminated with such alleles. In addition, well-characterized sets of alleles affecting a protein can serve as a test-bed for efforts such as the Critical Assessment of Genome Information . Homocystinuria due to CBS deficiency (OMIM 236200) is a recessive inborn-error of sulfur-amino acid metabolism, resulting in increased levels of homocysteine in the urine. More than 140 different disease-associated mutations have been identified in the CBS gene  an s of CBS .Protein expression and purification below) as well as a yeast expression vector containing the TEF1 promoter and CYC1 terminator , and served as the parent for all yeast strains used in this study (MATα cys4::KanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0). A hem1Δ strain, deficient in δ-aminolevulinate synthase, was created by the direct transformation of the cys4Δ parent with a hem1::LEU2 construct. Expression plasmids carrying the human CBS major allele, or variants, were transformed into this strain by selecting for uracil prototrophy  or GSH. Although these strains had wild-type versions of all MET genes, growth in minimal media also required the addition of methionine, presumably to augment a limiting intracellular concentration of homocysteine for the human enzyme. All vitamins except B6 (pyridoxine) were added back individually to levels present in standard medium. Pyridoxine was then added back at a range of concentrations, the “high” dose was 400 ng/mL, the concentration of pyridoxine present in standard medium. Intermediate and low doses of pyridoxine were titrated in starting at 4 ng/mL, a concentration slightly less than that required to achieve maximal growth. Note that even though S. cerevisiae requires PLP for growth and makes its own PLP, the level of PLP in wild-type yeast is not sufficient to satisfy the PLP requirement for human CBS when complementing the yeast cys4 mutation.We adopted the strategy of Kruger BS assay , 1995 an595 = 0.01 in a volume of 150 μL). Cell number was measured every 30 min for 72−96 hr in a Tecan GENios plate reader at 27−28° without shaking. For growth-rate calculations, raw OD595 values were normalized to an endpoint read after the cells were resuspended. The normalized OD595 values were then log10 transformed, and the growth rate determined from the slopes calculated between two fixed cell densities . A plate normalization factor was applied to account for plate-to-plate variations, and the variant growth rates expressed as a ratio relative to the growth rate resulting from the major allele.For growth-rate measurements, strains containing CBS mutations were pregrown in minimal medium containing GSH to early stationary phase at 30°. Cells were diluted 1:200 in fresh medium lacking GSH, but with varying amounts of pyridoxine . Substitutions within the CBS coding region were then constructed using site-directed mutagenesis and confirmed by DNA sequencing, as was done with the yeast CBS expression constructs. MBP-CBS fusions were expressed in Rosetta 2 (DE3) cells . Cells were grown in Luria broth with 200 μg/mL ampicillin at 37° to an OD600 between 0.6 and 0.8, and then the temperature was shifted to 20° and the cultures were allowed to grow for an additional hour. Protein expression was then induced by the addition of 1 mM IPTG  and either 0.3 or 0.6 mM δ-ALA  and the cells grown at 20° for an additional 15−20 hr.A bacterial expression clone containing a 6His-MBP-CBS fusion was obtained from Macrolab at UC Berkeley  with a cocktail of protease inhibitors . The cells were then lysed by sonication, and the supernatant fraction collected by centrifugation. Proteins were purified from the lysis supernatant using a Ni-NTA affinity resin , eluted from the column in lysis buffer with 250 mM imidazole, and then dialyzed into lysis buffer to remove the imidazole and concentrate the purified protein. Protein concentrations were determined by measuring the absorbance of the aromatic side chains .Absorbance spectra were taken on purified proteins ranging in concentration from 20 to 100 mM and then normalized to protein concentration for comparison. The ratio of the heme 430 nm absorbance peak and the aromatic 280 nm absorbance peak was one-to-one, indicating nearly 100% heme incorporation in the major allele protein  was loaded with the protein sample by use of a pressure bomb and washed extensively with buffer A. The column was then directly coupled to an electrospray ionization source mounted on a Thermo-Fisher LTQ XL linear ion trap mass spectrometer. An Agilent 1200 HPLC equipped with a split line, so as to deliver a flow rate of 30 nL/min, was used for chromatography. Peptides were eluted with a 90-min gradient from 100% buffer A to 60% buffer B. Buffer A was 5% acetonitrile/0.02% heptaflurobutyric acid; buffer B was 80% acetonitrile/ 0.02% heptaflurobutyric acid. The programs SEQUEST and DTASELECT were used to identify peptides and proteins from a database consisting of common contaminants and the engineered sequences of the proteins  expression of the human clone can be driven by a heterologous promoter and terminator so as to minimize transcriptional effects on protein levels; (2) the expression level of the complementing human clone can be modulated so as to restore growth but remain limiting, thereby providing a sensitive assay for both partial loss-of-function alleles or enhanced-function alleles; (3) defined growth media allow for the titration of exogenous pyridoxine or bypass of CBS function entirely by the addition of cysteine in the form of GSH to the growth medium; and (4) the yeast genome can be engineered so as to alter other intermediates of the metabolic pathway. This assay was used to rank order variant activities as well as vitamin, or heme, responsiveness. We recently used this assay to catalog the functional impact of the majority of CBS polymorphisms identified clinically , and (2) distal from the PLP cofactor-binding site. Also, catalytic residues were excluded because mutations of catalytic sites would have clear deleterious effects independent of their effect on the free energy of folding. We used Rosetta-based predictions of the thermodynamic impact of single amino acid substitutions as an approximate guide to be able to test a presumed range of destabilizing mutations. The resulting set of 31 single amino-acid substitutions was predicted to have a range of destabilizing impacts, from mild to severe. The second strategy was random mutagenesis designed to be unbiased with respect to protein residue and substitution and resulted in another set of 27 single-amino-acid substitutions. In total, 58 single amino acid substitutions were constructed in the human CBS gene by these two strategies, and functionally assayed in vivo. The complete list of substitutions and their predicted impact on the thermodynamic stability of the protein based on the Rosetta profile is shown in The focus of this study was to examine the effect of single amino acid substitutions on CBS function and the associated impact of cofactor availability in a more direct fashion. Two strategies were used to construct a new collection of CBS variants, specifically trying to enhance moderate functional impacts. The first was a directed strategy aimed at protein residues not directly involved in either the binding of cofactor or the catalytic pocket, but dispersed throughout the protein, that might result in a range of impacts on protein function. The primary criteria were that the residue be (1) buried within the hydrophobic core . Although the majority of these substitutions exhibited slower growth rates relative to wild type, particularly at low pyridoxine concentrations, three substitutions  conferred better growth than wild type. We refer to this category of 14 substitutions as being cofactor sensitive.The observed growth phenotypes were assigned to one of three categories. The first comprised those changes that failed to confer growth even at the highest concentration of exogenous pyridoxine supplementation (400 ng/mL). These 19 substitutions created a nonfunctional enzyme as defined by the entation . Twenty-Some alleles led to proteins with decreased yet remediable function. For these mutants, their loss or reduction in function could be compensated by the binding of a cofactor. If the effect of these mutations on the enzyme were local to the altered site, remediable alleles would be expected to be close in structure to the cofactor-binding site. If, however, the effect were more global, such as a general destabilization of the protein, then remediable alleles may occur at a considerable distance from the cofactor-binding site.We used the structure of a truncated version of the major allele protein as a framework for exploring the relative distribution of different classes of variants. We mapped the positions of the 44 mutated residues with positional information  to examine if there were any “hot spots” for the phenotypic categories . There wTo explore possible associations between physical characteristics of substitutions and observed growth phenotype in a more quantitative fashion, alleles were grouped based their growth phenotype: nonfunctional, sensitive, or benign; each of these three groups were further divided based on the method of molecular construction, directed or random . For each category, five physical characteristics were determined , and category averages were calculated. For comparison, an equal number of alleles were randomly assigned to each category 10,000 times and category averages for the same five measurements performed allowing a rigorous statistical test for the probability that the test category is higher or lower than random.As expected, the directed variants had significantly less solvent accessibility than the randomized controls because this was a criterion in their selection. This correlation provided a positive control that the analysis could detect correlations if they existed among the data. Among the randomly generated variants, those with nonfunctional or sensitive phenotypes were also less solvent accessible than either the controls or the benign random substitutions. However, no other physical parameter examined was characteristic of any of the phenotypic categories. The remediable alleles were no closer to either of the cofactor binding sites, nor did they have altered occurrence in regular secondary structure, or correlate with high or low crystallographic B-factor values.Although the Rosetta algorithm was used to select mutations with a range of predicted effects on stability, the correlation between the predicted effects on stability and observed effects on growth was poor. The only reliable correlations were for those variants predicted to be severely destabilizing . Nine of the 11 substitutions in this category resulted in a no-growth, no-remediation phenotype . The twoIn contrast, the majority of mutations were predicted to cause a more modest ΔΔG effect and were selected precisely because of their predicted intermediate effects on stability. These variants fell into all three growth-phenotype categories . There whem1Δ strain in which we could control the relative level of intracellular heme, the other cofactor for CBS, by supplementing with either high or low concentrations of δ-amino levulinic acid (δ-ALA). δ-ALA is the product of Hem1p and a precursor in heme biosynthesis . Of these, two variants exhibited heightened sensitivities to levels of both cofactors, pyridoxine and heme (K267E and L345P), an additional three variants showed sensitivity to reduced pyridoxine levels but not heme levels , and the final variant selected was benign , which facilitated the solubility of bacterially expressed CBS variants. To aid in the intracellular production of heme and its subsequent incorporation into the CBS protein during expression, the bacterial growth medium was supplemented with δ-ALA , but displayed differing phenotypes with respect to heme-level sensitivity. Three variants,  exhibited sensitivity to reduced pyridoxine levels relative to the major allele, but no heightened sensitivity to reduced heme levels based upon growth-rate measurements. However, there was a range of heme incorporation among these alleles in vitro. The L181A variant appeared to incorporate a level of heme similar to the protein encoded by the major allele, although the other two CBS variants incorporated less heme. These variants established that heightened B6-level sensitivity was not necessarily coupled with discernable effects on heme loading or conformational stability, and suggested that there may be a threshold with respect to heme loading and protein function in vivo.Five of the six single amino acid substitutions of CBS selected for physical analysis exhibited altered sensitivity to the B6 cofactor in vivo sensitivity to reduced levels of either pyridoxine or heme. Upon purification both variant proteins bound far less heme than wild-type CBS, and exhibited the most enhanced thermolysin sensitivity of the seven CBS versions tested. Heme incorporation into CBS protein appears to be obligatorily co-translational, and reduced heme binding in turn affects PLP binding (in vivo. It would appear that variants with heightened sensitivity to the levels of two cofactors commonly have heightened conformational flexibility, as measured by protease sensitivity, relative to variants with more specific sensitivities.Perhaps the most interesting alleles were those that encode K267E and L345P, which exhibited the most extreme Taken to a logical conclusion, these striking examples raise the possibility that a small molecule ligand that binds to one position of a protein can suppress the impact of a destabilizing mutation at a distant site. This notion is consistent with the observation that the physical impacts of a number of clinical CBS alleles result in subtle alterations of protein structure .Clearly, experimental evaluation of all amino acid substitutions in the human proteome would be technically daunting. Hence robust computational prediction of the impact of variation would be desirable if personalized medicine is to reach its full potential. However, the results described here demonstrate that single amino acid substitutions that are calculated to have a modest impact on protein stability can nevertheless impact protein function, ranging from nonfunctional to benign. The needed improvements in predictive methods rely on datasets that interrogate the impact of many alleles of a protein. The 58 alleles of CBS described here, combined with previous studies, offer an increasingly useful resource for such developments.


The expression profiles found in the analyses of breast cancer tissue must be evaluated with caution. Different profiles may simply be the result of differences in the surgical trauma and timing of when samples are taken and not necessarily associated with tumor biology.Gene expression studies on breast cancer have generally been performed on tissue obtained at the time of surgery. In this study, we have compared the gene expression profiles in preoperative tissue (core needle biopsies) while tumor is still in its normal milieu to postoperative tissue from the same tumor obtained during surgery. Thirteen patients were included of which eleven had undergone sentinel node diagnosis procedure before operation. Microarray gene expression analysis was performed using total RNA from all the samples. Paired significance analysis of microarrays revealed 228 differently expressed genes, including several early response stress-related genes such as members of the  Breast cancer is detected either by clinical signs such as palpable tumour or in mammographic screening. In both cases biopsies are taken from the tumour to determine whether the tumour is benign or malign. If malignancy is detected, the patient will be scheduled for surgery within a few weeks. Before the surgery, sentinel node (SN) diagnostics is generally performed to examine the spread of cancer cells to axillary lymph nodes. The SN can be identified using a blue dye, a radioactive colloid, or a combination of the two , 2.Microarray technology enables scientists to study thousands of genes simultaneously. The resulting molecular profile can be used to study complex multifactorial diseases such as breast cancer , 4. GeneThe time of procurement, which refers to the point of when the biopsies are taken  as well This study includes 13 patients from which both a pre- and postoperative samples were available. Histopathological characteristics are listed in Preoperative needle biopsies were obtained by an experienced radiologist using a 16 Gauge core needle device through a small skin incision in a sterile field. Three samples were processed for routine histological diagnosis while one sample for molecular analysis was put directly into RNAlater . The postoperative samples were taken by the breast cancer surgeon upon removal of the breast and were preserved in RNAlater. The RNAlater-stabilised tissue samples were stored at −80°C. The time delay between the sampling of the pre- and postoperative specimens were 2–8 weeks.RNA was prepared using the method of Wei and Khan  but modiμg cRNA was fragmented and applied to Agilent Whole Human Genome 4 × 44 k microarrays (G4112F). After hybridisation for 17 h at 65°C the microarray slides were washed and scanned with the Agilent Microarray Scanner. Microarray data were extracted using Agilent Feature Extraction (v. 10.7.1.1) and further quantile normalized and analyzed using J-Express 2009 [http://www.ebi.ac.uk/microarray-as/ae/) accession number E-MTAB-470.mRNA amplification, labelling, and hybridization were done following the manufacturer's instructions . Briefly, 500 ng RNA was amplified and labelled with Cy3 using the Quick Amp labelling kit and the labelled cRNA purified using the Qiagen RNeasy Mini Kit. Amplification and labelling efficiency were controlled on the NanoDrop before 1.65 ess 2009 . For expGene functional classification of the significant genes was performed using DAVID , 17 and GAPDH, 18S, and ACTB were used.To confirm the results of the microarray experiment, qRT-PCR using TaqMan low density arrays  were performed using primer-probe pairs for 13 of the significant genes . The gen FOSB, response to oxidative stress such as DUSP1,9 as well as genes earlier identified as differentially expressed in cancer   .FOSB, ERK, MAPK3, CYR61, and the RAS-genes  while the downregulated genes mainly correspond to transmembrane proteins . Gene fuAS-genes ; DUSP1, nd RASD1 ; CYR61 and NFRκB  amongst ACTB, CYR61, DUSP1, EVI2b, FOSB, GAPDH, and RASD1.To confirm the results of the microarray experiment, qRT-PCR was performed using primer-probe pairs for the top significant genes. The genes were selected to contain both up- and downregulated genes. The microarray and the qRT-PCR results were in agreement with the following genes : ACTB, CImmunohistochemistry was performed on the pre- and the postoperative samples. Overall the tumour content in the two samples were comparable and there was no systematic bias , indicatMicroarray studies have influenced breast cancer research over the last decade revealing breast cancer as a heterogeneous disease opening for individual treatment in a clinical perspective. Therefore, the results from microarray studies need to be validated. Multiple studies have generated different gene list and studied the reproducibility and correlation with prognosis –22. DespCYR61, MALAT1, RASD1, CX3CL1, FOSB, and CYP2D6. Some of these genes have been studied by others in relation to oxidative stress [MAPK3, κBNFR, FOS, and ERK.One important question may be if the differences in gene expressions are related to tumour biology or reflect the surgical trauma of the patient or the manipulation of the tumour tissue during the operative procedure or the time of specimen handoff. If altered gene expression is caused by such exogenous factors, the results may differ considerably between studies depending on the operative procedure and the time spent at the operation before taking the tissue samples. It is therefore important to evaluate if gene expression patterns differ between biopsies taken before and after surgical procedure. This has been done in our study with 13 patients and the gene list of 228 genes was dominated by stress-related genes like e stress –27 and aFOS, FOSB, FOSL1, and FOSL2. These genes encode leucine zipper proteins that can dimerise with proteins of the jun-family, and the Fos-proteins have been implicated as regulators of cell proliferation, differentiation, and transformation. Another gene, CYR61 , most strongly associated to differential expression in pre- and postoperative samples, belongs to the CCN-family [CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αvβ3 and augments growth factor-induced DNA synthesis in the same cell type [CYR61 is claimed to play a critical role in oestrogen, as well as growth factor-dependent breast tumour growth [CYR61 is repeatedly connected in most of the involved pathways. Further studies will be necessary to confirm and explain this association.Upregulation of Fos has been associated with breast cancer in a number of studies –31. The N-family  and mediell type . In thisr growth . In our DUSP1. Expression of this gene may be associated with resistance to further administration of chemotherapy. In our study DUSP1 was one of the significantly upregulated genes in postoperative tissue. DUSP1 is a stress response gene of the mitogen-activated protein (MAP) kinase phosphatase family and is located in the cytoplasm, mitochondria, and the nucleus. The gene has been shown to be overexpressed in human breast cancer [MAPK pathway. Later studies have implicated that DUSP1 is controlled by p53 during cellular response to oxidative stress [DUSP1 was also among the genes suggested to be useful diagnostic and prognostic markers. Our study suggests that such findings have to (1) demonstrate upregulation above the one observed here by us attributable to the pre- and postoperative factors and (2) that deregulation attributable to the pre- and postoperative factors is similar in the compared case/control or treatment arm groups.It is of particular importance to take into consideration knowledge about gene expression differences in pre- and postoperative tissue samples in the case of treatment response studies in the neoadjuvant setting, when the first sample is frequently taken by biopsy and the second during operation. In a study comparing gene expression profiles before and after doxorubicin and cyclophosphamide neoadjuvant chemotherapy  one of tt cancer  through e stress . A simile stress , where DIn the present study, it is not possible to separate the effects of operative manipulation, anaesthesia, or the injection of radioactive substance to examine spread of cancer cells. For the latter, we should have had a biopsy after the application of radioactivity not before operation. However, both ethical and logistical considerations make collection of such a sample infeasible. Wong et al.  studied Another confounding factor in the analysis of gene expression profiles of breast cancers is intratumour heterogeneity , 40. EveGRB7, which is upregulated in the postoperative samples. GRB7 was associated with an increased risk of recurrence in TNBC (tripple negative breast cancer) treated with adjuvant doxorubicin-containing chemotherapy, suggesting that GRB7 or GRB7-dependent pathways may serve as potential biomarkers for therapeutic targets [in vitro [As we have seen there are genes that are differently expressed between the pre- and postoperative samples. We compared our gene lists to some of the publically available gene list to see if there were any overlapping genes. The Oncotype DX consists of 21 genes, 16 cancer related genes, and 5 reference genes . One of  targets , 44. We in vitro –47, it iNDRG1) in common for the 70 gene list in Mammaprint with our gene list separating pre- and postoperative samples. NDRG1 (N-myc downstream-regulated gene 1) is a member of the N-myc downregulated gene family which belongs to the alpha/beta hydrolase superfamily. The protein encoded by this gene is a cytoplasmic protein involved in stress responses, hormone responses, cell growth, and differentiation. The encoded protein is necessary for p53-mediated caspase activation and apoptosis. Expression of this gene may be a prognostic indicator for several types of cancer . The gene is significantly upregulated in the postoperative samples of the present study. It is known to be induced by stress, through hypoxia [We also wanted to compare the 70 genes listed in the Mammaprint which were based on the intrinsic gene list . As with hypoxia , like maNDRG1 is correlated with poor clinical outcome in breast cancer [NDRG1 is downregulated upon estradiol stimulation, and its expression is correlated with favorable prognosis in breast cancer patients [NDRG1 could be used as a biomarker for differentiation of breast cancer for both diagnostic and therapeutic purposes. Still it is very important to be aware of at what material the gene is measured upon.Low expression of t cancer . It has patients . On the patients  concludeOur study shows the expression profiles found in the analyses of breast cancer tissue must be evaluated with caution. Different profiles may simply be result of differences in the surgical trauma and timing of when samples are taken, and not necessarily associated with tumor biology. Supplementary Figure 1: Hierarchal clustering with all probes present on the array of the pre- and postoperative samples together with corresponding adjacent normal tissue shows overall similar expression profiles for the majority of the pre- and postoperative tumor pairs.Supplementary Figure 2: Hierarchical clustering of the samples using the differently expressed genes (FDR < 2.5%) separates the majority of the preoperative tumors from the corresponding postoperative tumors.Supplementary Figure 3: Molecular network showing the interaction between all the 228 differently expressed genes. The gene identifiers and corresponding expression values were uploaded into in the Ingenuity Pathway Analysis. Networks were then algorithmically generated based on their connectivity in Ingenuity's Knowledge Base. Molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. Nodes are displayed using various shapes that represent the functional class of the gene product.Supplementary Table 1: Significantly differently expressed genes between pre- and postoperative samples. All probes with FDR < 2.5% ranked according to SAM. Systematic names shown in brackets.Click here for additional data file.


Current Portuguese HIV treatment guidelines recommend initiating antiretroviral therapy with a regimen composed of two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor (2NRTI+NNRTI) or two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor (2NRTI+PI/r). Given the lower daily cost of NNRTI as the third agent when compared to the average daily costs of PI/r, it is relevant to estimate the long term impact of each treatment option in the Portuguese context.We developed a microsimulation discrete events model for cost-effectiveness analysis of HIV treatment, simulating individual paths from ART initiation to death. Four driving forces determine the course of events: CD4+ cell count, viral load, resistance and adherence. Distributions of time to event are conditional to individuals’ characteristics and past history. Time to event was modeled using parametric survival analysis using Stata 11®. Disease progression was structured according to therapy lines and the model was parameterized with cohort Portuguese observational data. All resources were valued at 2009 prices. The National Health Service’s perspective was assumed considering a lifetime horizon and a 5% annual discount rate.In this analysis, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor reduces the average number of switches by 17%, saves 19.573€ per individual and increases life expectancy by 1.7 months showing to be a dominant strategy in 57% of the simulations when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor.This study suggests that, when clinically valid, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor is a cost-saving strategy and equally effective when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor as the first regimen. Human immunodeficiency virus (HIV) infection remains a major public health concern in Europe, with evidence of increasing transmission in several countries. From 2000 to 2009, the rate of newly HIV diagnosed cases reported has almost doubled in the European Region, from 57 to 92 cases per million. On the other hand, the number of acquired immune deficiency syndrome (AIDS) cases has continued to decline, with the exception of eastern countries, where it has increased Since 1983, a total of 39,347 cases of HIV infection have been notified in Portugal of whom 23% have died. Within alive, 29% have been diagnosed with AIDS, 11% have developed symptoms and the remaining 60% are in an asymptomatic stage of the infection Ministério da Saúde, Administração Central do Sistema de Saúde - Inpatient care episodes in the Portuguese National Health Care Service Database).In 2009, antiretroviral drug expenditure in Portugal was estimated in 193.23 million Euros ) Between 2008 and 2009, pharmaceutical expenditure grew 7% in the NHS hospital market and antiretroviral drugs were a major driver Current Portuguese HIV treatment guidelines Aragão F. Budget Impact, in terms of antiretroviral costs, of switching patients on a regimen containing boosted protease inhibitors or the components of the single tablet regimen to the single tablet regimen for treatment of HIV-1 Infection. 13rd Conference of the European AIDS Clinical Society, Belgrade, Out 2011. P7.5/5). Consequently, in a search for a more efficient use of resources, especially given the increasing cost containment pressure in Portugal, it is relevant to compare the effectiveness of each treatment option in routine care setting and to estimate the corresponding long term impact within a cost-effectiveness framework.NNRTI and PI/r are considered clinically equivalent, in the sense that elements of both classes are considered as first choices in clinical recommendations, being differently prescribed according to clinical criteria . The average daily cost of PI/r is significantly higher than that of NNRTIs (In line with models such as the Cost-Effectiveness of Preventing AIDS Complications (CEPAC) model Our model differs from other microsimulation models in that we take a discrete event approach. In discrete-event simulation, event occurrences determine time advances and outcomes are updated at the time the event occurs and not at the end of a predetermined time period. Moreover, discrete-event simulation is particularly useful when interaction between individuals is of relevance as is the case with infectious diseases 10 of HIV RNA copies per mL (log10VL) <50 copies/mL), regimen switch without virological failure (any change in current regimen), line switch , resistance development, hospitalization, AIDS-defining event, and death. At the occurrence of the event, evolving state variables , costs and benefits are updated.Regarding line switch, we assumed that the individual switches from first-line to second-line (and subsequently from second-line to third-line) if viral suppression is not reached within 12 months of line initiation or if virological failure (HIV-1 RNA level ≥50 copies/mL) is confirmed after viral suppression has been achieved. While a 6-month period is currently recommended, 12 months was the threshold used in clinical practice in the first half of the 2000 decade. Moreover, it should be noted that the 12 months period to reach viral suppression reflects 12 months to reach viral suppression and being tested, which depends on testing frequency. When entering in the third-line, the model assumes that the individual will stay on this same line as long as resistance is not in the highest class and regardless of the number of virological failures. Once the highest resistance class is reached, the individual starts non-suppressive therapy. This process is repeated until death occurs and the individual simulation is stopped.Cost-effectiveness results are obtained by simulating the paths of one million individuals. The statistical analyses were performed in Stata 11®.The model was parameterized with Portuguese observational data and when such was not available estimates available in the international literature were used. Portuguese observational data sources include three main databases with data at the patient level  database, the Egas Moniz Hospital (LVHEM) laboratory database and the Cascais Hospital (CHC) database) along with aggregate national data regarding deaths due to HIV/AIDS and inpatient cost per episode. Written consent for access to the databases was approved by the Ethics Committees of Hospital de Cascais, Centro Hospitalar de Lisboa Ocidental and Instituto Nacional de Saúde Dr. Ricardo Jorge, as required. The Ethics committees waived the need for informed consent from participants as the data were analyzed anonymously and retrospectively.The CVEDT laboratory database is the national registry of HIV infected individuals . The LVHEM database provides data on approximately 80% of HIV infected individuals tested for resistance in Portugal but it has a selection bias since the database tends to include those individuals with suspected resistance  along with pre-ART resistance test results. This database was used to parameterize the initial resistance level (using pre-ART resistance tests) and lines 3 and 4 of the model.The CHC database provides data on 1,306 HIV-1 infected individuals followed at one hospital unit in the vicinity of Lisbon, between 2001 and 2008. The data resulted directly from the electronic software used, on a daily basis, for patients follow-up. While not nationally representative, this was the most complete database available in the country providing socio-demographic information, clinical markers and resource consumption data. For the most part, CHC data was used to parameterize lines 1 and 2 of the model.The model simulates individual paths from the moment of ART initiation to death. At ART initiation the individual is characterized with respect to age, gender, employment status, hepatitis C virus (HCV) co-infection, AIDS diagnosis, mode of transmission, CD4+ cell count, viral load, adherence, resistance, and regimen characteristics .10VL (p = 0.056), and median adherence measured by pharmacy refills (p = 0.327). Quantitative data was compared between groups using t-test for independent data, or Mann-Whitney if normality assumption was not accepted. For qualitative data, Chi-square test was used. A statistically significant difference was found regarding year of ART initiation with the 2NRTI+PI/r, on average, initiating 2 years later . Reflecting the study period and clinical practice at the time, in that cohort, the most frequently used pair of NRTI was zidovudine/lamivudine, and lopinavir/ritonavir was the PI/r used in 73% of patients on PI-based regimen. Initial patient characteristics of the first ART regimen were mainly obtained from the subsample of ART naïve patients in the CHC database, who initiated treatment with 2NRTI+NNRTI (n = 158) or 2NRTI+IP/r (n = 159) during the follow-up period. Patients were not randomized to each group, nonetheless, no statistically significant difference was found with regard to gender (p = 0.751), median age (p = 0.649), co-infection with hepatitis C (p = 0.971), initial resistance to at least one drug (p = 0.792), median CD4 cell count (p = 0.500), median logThe model assumes that most of the initial characteristics will evolve over the lifetime of the individual being updated upon event occurrence and thereby influencing (together with cumulative history of events) the distribution of time to next event. Employment status, HCV co-infection, mode of transmission and gender are assumed constant over time due to either being immutable or due to lack of information in the data sources used.10VL was estimated by weighted non-linear least squares regression conditional on therapy line. In the estimation, we used a logarithmic function with line specific parameters to reflect the fact that CD4+ and log10VL evolution may not be linear  and may differ according to the accumulated number of virological failures. For modeling purposes, if the individual does not respond to the new regimen, log10VL will continue the estimated path until virological failure is declared due to lack of response. If viral load suppression is achieved, log10VL will decrease instantaneously and remain constant until a rebound occurs. The log10VL rebound value is sampled from a Uniform distribution obtained from the CHC database.Based on individual-level data from CHC database, the average monthly variation of CD4+ and logIn this analysis, pharmacy refills were used to quantify adherence. Our estimate of adherence is the percentage of days, in each regimen, the individual could not have had medication in his/her possession - given the quantities dispensed, the dates of refill and accounting for the fact that often individuals will refill before the stock has gone down to zero. In order to model changes in adherence level over time, and given the fact that adherence is likely to depend upon individual and ART regimen characteristics, a generalized linear model with a binomial family and a Logit link Resistance level was defined as the sum of the inverted Genotypic Sensitivity Score obtained from the REGA 8.0 algorithm, after inverting this score (which measures sensitivity to the drugs) to reflect rather the resistance to the drugs. The resistance score was grouped in four classes  and it was assumed that individuals could move only to an adjacent resistance class. When the next event occurring to the individual corresponds to a resistance development, the individual moves to the following resistance class and a new resistance level is assigned sampled from a Uniform distribution within that class.3, the individual is permanently classified as having AIDS. Subsequent AIDS-defining events may occur with impact on both costs and effectiveness indicators.The simulated individuals may have been diagnosed with AIDS at ART initiation. If not, upon the occurrence of the first AIDS-defining event or a CD4+ <200 cells/mmRegimen characteristics  are updated on occurrence of line switch or regimen switch without virological fail. Upon event occurrence, a random draw from a Table distribution  will determine the new value for each of the three variables.et al.et al.In simulation survival analysis was used to link time to event to individual characteristics, accumulated history and regimen characteristics depending on data availability. Weibull distributions were considered in the parameterization of survival curves for each clinical event . Virological failure may occur for several reasons but since those were not available, a unique event was considered regardless of the cause. A Weibull distribution was fitted for time to line switch for line 1 and line 2 using the CHC sample whereas LVHEM sample was used to model time to event regarding line 3.The information to parameterize the model with respect to time to first resistance development was obtained from the CHC sample. Time to resistance class switch once the first resistance has developed was estimated on the LVHEM database since it provides a sample of individuals who have some positive level of resistance (and 76% are resistant to at least two drugs). LVHEM database does not however contain information on adherence and consequently the relationship between adherence and resistance could not be estimated. Predicted median time to resistance was much longer for the first resistance than for when some level of resistance had already been developed (28 years versus 7 years) likely reflecting cross-resistance and cumulative exposure.When the individual reaches non-suppressive therapy, he will be in the highest class of resistance. In order to account for the fact that resistance will eventually increase within class 4, we have used the model in Data in CHC database was considered for modeling time to hospitalization. The discharge date was the only information available and therefore was used as a proxy for the date of the hospitalization. Hospitalizations occurring immediately before ART were excluded since those were considered to be related with the absence of ART.Due to lack of information on national databases, time to AIDS-defining event was assumed to follow the Weibull proportional hazards model stratified on CD4+ and transmission risk published by May et al. Time to death among HIV infected individuals was adjusted to a Weibull distribution stratified by disease stage (AIDS/non-AIDS) using data from CVEDT database. In order to account for the fact that mortality due to age may not yet be correctly reflected in the CVEDT database (due to 30 years of the epidemics and the young age of those infected.), the model assumes that the estimates provided by the CVEDT refer to HIV related deaths whereas deaths due to other causes were included as a competing risk. Parameterization of the model, regarding death due to other causes was based on Portuguese general population mortality rates in the 2006–2008 period published by the National Institute of Statistics database While, in the model a parametric survival analysis approach was used, it is worth nothing that in the CHC naive sample, a statistical difference was found in Kaplan-Meier curves of time to first regimen switch without virological failure with medians  of 6.7 (3.5–7.1) and 2.7 (2.3–3.7) years for 2NRTI+NNRTI and 2NRTI+PI/r , respectively. No statistically significant difference was found between groups in terms of time to viral suppression (p = 0.072), virological failure (p = 0.579), first resistance development (p = 0.561).Unitary drug costs were extracted from the Catalogue of Health Procurement First regimen ART and non-ART costs were obtained from the subsample of ART naive patients in the CHC database. A statistically significant difference was obtained for the median [interquartile range] of first regimen monthly cost  and these estimates were used in the model.In line with the approach followed to estimate effectiveness, the CHC sample was the main source for ART resource consumption in first and second line and LVHEM sample for third line and non-suppressive therapy.A monthly ART cost was estimated based on individual characteristics through a generalized linear model . For the remaining therapy lines, a monthly non-ART cost conditional on individual characteristics was estimated using a generalized linear model with a Gamma distribution and a log link function   and the cost of a hospitalization to be the average cost of the remaining HIV related code episodes .The present cost-effectiveness analysis considers HIV-1 infected treatment naïve adult individuals. In accordance with the Portuguese guidelines, both costs and effectiveness were discounted at 5% Life-years were converted in quality-adjusted life years (QALYs) applying published utility estimates Cost-effectiveness analysis was performed considering the total costs of each treatment option along with two effectiveness indicators were used: life-years and QALYs. Incremental cost-effectiveness ratios included the incremental cost per life-year gained and per QALY gained.Microsimulation outputs were obtained by simulating 1 million individuals. Clinical outcomes predicted by the model are described in −15.753€) and non-ART costs (−3.821€). The most significant cost differences are seen in first line since treatment in subsequent lines is only slightly conditional on first line ART in each group (through the number of PIs and the number of accumulated different regimens). In both regimens, ART is the cost-driver representing 79% of the total cost. This cost distribution reflects the fact that progress in ART has improved overall health and thus decreased the frequency and severity of opportunistic infections/AIDS-defining episodes.Annual non-ART costs increases along the therapy lines indicating a more intense consumption of health resources as the disease progresses. Except for non-suppressive therapy, annual ART-costs are also increasing in therapy line reflecting the need for more complex and expensive drugs as resistance develops. Annual ART costs in non-suppressive therapy are estimated to be lower than those for third line which is an unrealistic result given the currently available option for highly resistant patients (data not shown). This result is a consequence of the 2001–2008 period of analysis in the datasets.As presented in According to this analysis, initiating ART with 2NRTI+NNRTI results in lower total health care costs over the life span of the individual and such gain is obtained with a marginal increase in effectiveness as measured by the estimated number of QALYs. Selecting NNRTI as the third agent class is thus a dominant strategy when compared to initiating ART with 2NRTI+PI/r.In the context of microsimulation models it is relevant to differentiate two categories of uncertainty: the first-order which is associated with the variability among individuals and the second-order related with parameter uncertainty. The first is reflected in the microsimulation outputs whereas the second was incorporated in the current probabilistic sensitivity analysis, which is performed by allowing all input parameters to vary simultaneously. The acceptability curve was obtained by simulating 50.000 individuals for 133 different set of combinations of parameter values sampled from appropriate probability distributions via Monte Carlo simulation. The ranges and distributions used in the uncertainty analyses were derived from the estimated standard errors (when related to parameters obtained from databases) or published sources .Given the estimated small difference regarding the total number of QALYs in the two strategies, it is of special interest to evaluate the impact of the assumed parameter values on the base-case results. In the probabilistic analysis 57% of the simulations yielded 2NRTI+NNRTI as a dominant strategy. The acceptability curve  shows thIn the current cost containment environment, there exists an urgent need to identify strategies that will reduce costs without compromising the health benefits ART has generated. Economic evaluation, by providing information on the cost per QALY gained, provides policy makers and health care professionals an efficiency oriented tool to support decisions.Two cost-effectiveness analyses have been performed comparing NNRTI to PI/r as a third agent in the initial treatment of HIV-1 infected individuals Although a model represents a simplification of reality, it should be able to reproduce the main aspects under consideration. In our simulation, the percentage of individuals attaining viral suppression in the first line (68%) was lower than in clinical trials like in Study 934 The percentage of individuals who reaches viral suppression decreases with the number of previous therapy failures and is in line with clinical trial results of naïve and experience patients. Model predictions for the percentage of experienced individuals reaching viral suppression were similar to those observed in clinical trials CD4+ yearly increase was estimated to be 28, 24 and 21 cells/mm3 in first, second and third line, respectively, which are similar to those estimated by Phillips et al. According to the model, at ART failure, the viral load increases about 83% of the level at ART initiation (not shown). This value is close to the estimates reported in the literature where rebound viral load levels are about 10% less than the original levels The total number of regimen switches without virological failure (4.2 in the NNRTI group and 5.0 in PI/r group) is the variable where the highest percent difference between groups was found, what is in alignment with CHC sample  and clinical trials results The ART Cohort Collaboration Of the 24 predicted years, the model estimates that an average of 9 years will be spent in first line. This value is identical to the predicted value obtained using a Markov model parameterized with the same CHC database We predicted a 5 months difference in the average time spent in first line in the two groups, favoring the 2NRTI+NNRTI option. Once again our results contrast with those of Beck et al. The overall cost of treating an HIV infected individual was 375,470€ in the 2NRTI+PI/r group (7.5% less in 2NRT+NNRTI group) with 79% being related with ART acquisition. This proportion seems reasonable given the increasing weight of ART cost in the overall treatment cost of HIV, from 50% in 1996/97 (at the time HAART was introduced) to 70% in 2000/2001 (at the time PIs became available) The predicted average annual cost of ART in first line was 9,010€ in 2NRTI+NNRTI and 23% higher in 2NRTI+PI/r suggesting that the annual payment of 11,040€ per infected individual established by the Portuguese Government in the HIV financing program for ART naïve individuals covers first line ART costs, although not first line total costs.Our analysis has limitations that should be considered when interpreting the results.First, it is of relevance to highlight the fact that the present analysis is based on cohort observational data rather than clinical trials. Observational cohort data highlights reality beyond clinical trials and is of relevance to correct factors that are overestimated in clinical trial settings, such as adherence. Nonetheless, utilization of observational cohort data has implications on results which should be taken into consideration. Our results indicate that, on average, a patient found  more suitable to initiate ART with a NNRTI based regime, will have a lower total lifetime cost than a patient found  more suitable to initiate ART with a PI/r based regime. This result does not imply that, on average, 19,573€ could be saved per patient initiated with a NNRTI based regimen instead of a PI/r based regimen.Second, while all efforts have been made to parameterize the model so as to reflect Portuguese clinical practice, given the unavailability of a national database complete enough for estimation of all parameters of interest, several databases were used. Moreover, due to sample size restrictions, the complete observation period had to be considered for estimation reflecting clinical practice from 2001 to 2008. One implication is that our analysis  and its consequences quantified.