In 2001, the Rehabilitation of the Mentally Disabled Law was implemented, defining a basket of rehabilitation services to which people with mental disabilities are entitled.To describe change over time in the characteristics of applicants to rehabilitation committees, types of referral agencies, and the proportion of those referred who were admitted. To identify factors affecting implementation of decisions to admit people with mental disabilities into different rehabilitation services and predictors of the length of time they remain in the services.The study population consisted of all applicants to rehabilitation committees during 2001-2008. Rehabilitation services included hostels, supported housing, and vocational services. Data were extracted from Ministry of Health rehabilitation and psychiatric hospitalization case registers. Findings were analyzed using descriptive statistics, Kaplan-Meier survival analyses, and Cox regressions.There was a trend over time for more patients with shorter or no psychiatric hospitalization histories to be referred to rehabilitation services. Moreover, there was an increase in the proportion of referrals from the community, although the majority of referrals still came from psychiatric hospitals. Less than half of those recommended for a rehabilitation program were admitted and remained in a rehabilitation facility for one year or more. One factor predicting participants' longer stays in rehabilitation services after hospitalization was the proximity of the committees' decisions to the hospitalization. Another factor was the patient receiving vocational services while in residential care.Although over time the new law has resulted in a broader spectrum of people with mental disabilities receiving rehabilitation services, additional efforts are needed to enable them to remain in the system for a sufficient amount of time. Programs addressing specific needs should be developed accordingly. During the last two decades a change has occurred in treatment of the mentally ill, putting increased emphasis on the development of rehabilitation services in the community -3. AccorIn Israel also, as a consequence of collaborative efforts between social activists, mental health agents, and legislators, new legislation, the Rehabilitation of the Mentally Disabled in the Community Law of 2001 , was pasThe "basket" provides a range of accommodations that reflect a continuum of support and staffing levels, from relatively low levels of support, such as supported housing with one to three participants in a rental apartment with staff visiting them on a regular basis, to high levels of support and staff, such as 24-hour-staffed facilities (hostels with about 20-30 tenants). Currently, about 60% of those in residential care are in supported housing vs. 40% in hostels.Similarly, graded stepwise vocational services were developed in the community, from supported employment in the free market, through sheltered workshops where individuals work under protective conditions, to vocational clubs designed to teach vocational skills. Currently about 20% of those in vocational rehabilitation services are in supported employment in the free market, 30% are in sheltered workshops, and 50% in vocational clubs -13. ThosA number of articles e.g., -15) desc desc15])The objectives of the present study were the following: 1. To describe the changes over the years in the characteristics of the applicants to the rehabilitation committees, the types of referral agencies, and the proportion of referred disabled persons admitted to the different rehabilitation services; and 2. To identify some of the factors affecting implementation of the decisions to admit people with mental disabilities into the different rehabilitation services and the predictors of the time they remain in these services.The study population consisted of all applications during 2001-2008 (excluding those for extensions) to the rehabilitation committees for at least one of the rehabilitation services . In 2007 the main part of the rehabilitation budget (80%) was allocated to residential care and vocational services, while home care services, social and leisure activity services, and supported education services drew only 20% of the budgetary resources . This isData (without any identifying information of the disabled persons) were extracted and merged from two registers of the Ministry of Health: the Rehabilitation Register and the Psychiatric Case Register.The following independent variables were constructed:Age at time of decision of the committee - three categories: young adults (age 18-21 years), adults (age 22-55 years), older adults (age 56+ years). A category of young adults was defined because some hostels are specifically designated for that age group.• Rehabilitation history - six categories: being in a rehabilitation facility at the time of referral to a committee for a different facility (two categories), having been in one of the rehabilitation services in the past (three categories), no rehabilitation history.• Proximity of committee decision to a psychiatric hospitalization - three categories: no hospitalization history, more than two months since release from previous psychiatric hospitalization, two months or less , or within psychiatric hospitalization.• Two outcome variables were examined:Time from the committee decision until its implementation (admission to a rehabilitation unit);• Time in the rehabilitation program after admission. A temporary interruption in the stay in a rehabilitation program of up to three months  was not considered as a termination of the participant's stay in rehabilitation, in keeping with the regulations of the Ministry of Health.• Descriptive statistics, Kaplan-Meier survival analyses, and Cox regressions were used to analyze the findings.Approximately 19,000 applications met the criteria of our study, about half of which were for applicants new to the rehabilitation system. The other applications were for disabled persons who were temporarily released from the rehabilitation system because of a hospitalization of more than three months, for those who dropped out from the system because of other reasons, or who were recommended to change facility  or to add an additional service . The applications were for hostels only (17%), for hostel and vocational rehabilitation (11%), for supporting housing only (16%), for supporting housing and vocational rehabilitation (14%), or for vocational rehabilitation only (42%). Most of the applications to the rehabilitation committees were approved (80-90%), but only 50-70% of the positive decisions were implemented within a year. The probability of remaining at least one year in a hostel was 0.75, in supported housing 0.85, and in vocational rehabilitation services 0.55  Figure  and 4.The rates of application for both residential care (supportive housing and hostels) and vocational rehabilitation have tended to decrease since 2004. In addition, from that year, the rate of applications for supportive housing exceeded that of applications for hostels. Interestingly, this was true even among the applicants without a rehabilitation history, among whom the proportion of referrals to supportive housing increased over time, from 44% to 59%. This shift had already started during 2003-2004. The application for vocational rehabilitation services contributed the bulk of the applications during all the study years , while the percentage of those admitted within one year to supported housing increased considerably, from 60% to 79%, and the percentage for those admitted to vocational rehabilitation increased slightly but still significantly, from 61% to 67%  is significantly correlated with a shorter time until admission to supported housing, but with a shorter stay in the supported housing.Vocational rehabilitation Table :• The young adults take less time to be admitted, and adults aged 22-55 tend to leave more quickly than the younger or older adults.• Those who are referred to vocational rehabilitation and have a vocational rehabilitation history are admitted significantly more quickly than the others. Those who are staying in residential care have a better chance of remaining in the vocational rehabilitation program.• The more distant the referral to the committee is from a psychiatric hospitalization, the sooner the applicant will be admitted to a vocational rehabilitation program, but the probability of remaining in the program is not affected by the proximity of the committees' decisions for psychiatric hospitalization.A limitation of the study is that, although the rehabilitation register contains data on a nation-wide basis about the range and duration of the services provided to each individual, it lacks important personal information, mainly about the severity of the disease and of the disability, the degree of motivation for rehabilitation, the level of functioning, and the quality of life of the participants, as well the amount of available support from the family, Thus only a limited list of variables extracted from the case register could be examined.The strength of the study is its nationwide extent and its longitudinal nature. Trends over the years since the law was implemented in 2001 could thus be analyzed. Before the law was implemented, only 4,600 persons participated in any rehabilitation program. After implementation of the law the number gradually increased, reaching about 16,000 in 2008.This is consistent with the fact that the annual number of hospitalized psychiatric patients increased only from 13,000 to 14,000, although the number of people with mental disabilities eligible for rehabilitation services increased from 44,000 to 59,000 between 2001 and 2007.During the first years, the main objective was to reduce the number of beds of the chronic mentally ill patients with a long psychiatric hospitalization by releasing them from the hospitals to the community . This inInterestingly, although the total number of residents in both hostels and supported housing increased over the years , the perWhen discussing the length of stay in rehabilitation, we assumed that in general the longer the support of the rehabilitation services, the better the outcome. As the majority of the clients in the present study have a history of past hospitalization , most of them need supportive residential care for a long period, otherwise they will have a greater probability of being rehospitalized . LeavingIn the present study, half of the applications were for disabled persons who had already been in the rehabilitation system and were referred again, apparently because they had been hospitalized or had dropped out of the system. They were readmitted to the rehabilitation system more rapidly but, on the other hand, they remained in the system for less time than the new referrals, probably because candidates who dropped out once from the system are less suitable for the existing services. They possibly need more intensive support.An important finding is that the probability of not remaining in any of the rehabilitation services is relatively high in the first year. A similar trend has been found by Struch et al. in Israel , as wellThe young adult age group took more time to be admitted to residential care than the other age groups and remained for less time after being admitted. It seems that separate and more specifically tailored units for youngsters should be established, both for residential care and vocational rehabilitation, where special programs suitable for them should be planned.The closer the proximity of the committee's decision to the release of the patient from psychiatric hospitalization, the sooner the patients are admitted to hostels or to supportive housing, This could be explained by the necessity of the hospital authorities to release patients  and thusThere is a tendency to wait at least two months before admitting patients released from psychiatric hospitalization to a vocational program, although there is no evidence that participants remain for a longer time in vocational rehabilitation when admission is delayed. On the other hand, placing the clients concomitantly in a residential facility does allow them to stay longer in vocational programs, probably because such residential facilities serve as a support for these participants.Another issue that is of interest is why only 60-70% of the positive recommendations by the rehabilitation committees for a rehabilitation program are followed. Possible factors such as discrepancies between regions in the availability of rehabilitation services, lack of case managers, and lack of programs designed for specific groups should be examined.The study was not a full analysis of the extent to which the rehabilitation program has succeeded. A study based on case registers cannot replace a follow up based on a direct examination of the progress of the participants. Such a direct examination is extremely important and should utilize face-to-face interviews with a focus on changes in functioning, quality of life, and realization of personal goals. In addition, case register data contain critical information for an individual follow-up of the sequential transitions between the different levels of residential care and vocational programs. These data enable us to identify points of no further progress and whether they are client-related or service-related, as also suggested by Aviram . This wiOver the years the rehabilitation services have been reaching clients with a shorter hospitalization history, and an increasing proportion of candidates are referred from the community, but effort is still needed to help them remain in the system for sufficient time. Possible conditions to achieve this purpose should be explored and programs for specific needs should be developed.The authors declare that they have no competing interests.All three authors were involved in all aspects of the study. All authors read and approved the final manuscript.Tzipi Hornik-Lurie is a Ph.D. candidate at Ben-Gurion University of the Negev. She specializes in quantitative analysis in the field of the sociology of health. She is presently a researcher at the Falk Institute for Mental Health Research, Jerusalem, Israel.Nelly Zilber, D. ès Sc., is a former member of the French National Center for Scientific Research. She is a neuro-epidemiology and psychiatric epidemiology specialist. She is presently a senior researcher at the Falk Institute for Mental Health Research, Jerusalem, Israel.Yaacov Lerner MD, psychiatrist, is the former director of the Jerusalem Mental Health Center. He is a specialist in mental health service research. He is presently the director of the Falk Institute for Mental Health Research, Jerusalem, Israel.


This is an in-depth review of the history of quinacrine as well as its pharmacokinetic properties and established record of safety as an FDA-approved drug. The potential uses of quinacrine as an anti-cancer agent are discussed with particular attention to its actions on nuclear proteins, the arachidonic acid pathway, and multi-drug resistance, as well as its actions on signaling proteins in the cytoplasm. In particular, quinacrine's role on the NF-κB, p53, and AKT pathways are summarized. Quinacrine (IUPAC name 4-N-(6-chloro-2-methoxyacridin-9-yl)-1-N,1-N-diethylpentane-1,4-diamine) is a heterocyclic three-ring compound Figure , and an ®, atebrine, atebrin, mepacrine, quinacrine dihydrochloride, quinacrine dihydrochloride dihydrate, quinacrine dihyrochloride (R)-isomer, quinacrine dihyrochloride (S)-isomer, quinacrine dimesylate, quinacrine hydrochloride, quinacrine monoacetate, quinacrine monohydrochloride, quinacrine monomesylate, quinacrine (R)-isomer, quinacrine (S)-isomer, and 6-chloro-9-[[4-(diethylamino)-1 methylbutyl]amino]-2-methoxyacridine. The most commonly used designations for quinacrine are mepacrine, quinacrine hydrochloride, quinacrine dihydrochloride, and the registered name Atabrine®. Quinacrine is one of several known aminoacridines which include, for example, acridine orange, acriflavine, aminacrin, amsacrine, ethacridine, nitracrine, proflavine and tacrine and which have a range of biological and therapeutic applications. Table Quinacrine formulations and isomers are known by numerous designations some of which are: acrichine, Atabrine® (quinacrine) being one of the acridine derivatives successfully used to combat the disease. Atabrine® was discovered as part of an intensive antimicrobial research program broadly based on biologically active dyes carried out in 1930's in the German laboratories of I.G. Farbenindustrie. The program covered the preparation and trial of over 12,000 compounds leading to the identification of pamaquine and quinacrine as potential therapeutic agents salicyl)-N'-β- amlnoethylvindesine ([125I]NASV) [125I]NASV specifically binds to P-gp and the inhibition of its binding was used as a read out for the affinity of quinacrine for P-gp. In this study the binding affinity of quinacrine to P-gp was correlated to its ability to increase vinblastine sensitivity. It is, however, noteworthy that, although in an earlier investigation published by the same authors quinacrine increased the toxicity of vinblastine (12-fold) and vincristine (15-fold) at 5 μM and had an IC50 of 14 μM when administered alone [125I]NASV labeled P-gp NASV) . [125I]Ned alone , in thisled P-gp .in vitro and in vivo [50 values of 3.1 μM for PC-3, of 4.7 μM for PC-3M (a MDR sub-clone of the cell line) and of 3.5 μM for DU145 cells. Combination therapy of quinacrine and paclitaxel were determined to be synergistic in both, in vitro and in vivo (mouse xenografts) experiments. The exact mechanism of this synergistic effect was not studied however, the authors attributed to quinacrine's effect on phospholipase A2 (PLA2)[The study of quinacrine's role in MDR has not been limited to leukemia but it has also been analyzed in MDR cells from the ovary and prostate cancer. Quinacrine was reported to affect MDR Chinese hamster ovary (CHO) cells at 6 μM in studies measuring the uptake of labeled palmitoyl carnitine and palmitoyl lysophosphatidyicholine. They were more rapidly taken up by the MDR cells and this uptake was reversed after quinacrine treatment back to the rates observed with the parental cell line, hence implicating quinacrine in reversing the MDR . It also in vivo . QuinacrA2 (PLA2).2, hydrolyzes the sn-2-acyl bond of membrane phospholipids to produce arachidonic acid  production in a dose-dependent manner. Prostaglandin, PGE2 and the COX2-PGE2 pathway/arachidonic acid pathway play an important role in the induction of the pro-inflammatory response and ultimately tumorigenesis [2 levels have been implicated in angiogenesis, tumor growth and invasion, apoptosis resistance and suppression of anti-tumor immunity via suppression of T and NK cells, and amplifying Treg [2 have been demonstrated in head and neck cancer [In platelets, the conversion of arachidonic acid to thromboxane is suppressed by quinacrine ,210,223.igenesis -229. PGEing Treg ,230-232.k cancer ,233-237.The deleterious roles of p53 inactivation ,239 and Besides the development of AKT inhibitors, there has been no concerted effort to rationally design drugs that can simultaneously activate p53 and inhibit NF-κB. The opposing nature of these pathways suggests that a drug which activates p53 and simultaneously inhibits NF-κB would have significant clinical potential due to the fact that it is concomitantly modulating two critical cancer targets. In addition, a drug capable of affecting both of these pathways would also be a useful tool to study the interactions between the opposing p53 and NF-κB pathways. The literature  and furtet al. have reported the ability of quinacrine and other acridine derivatives to activate wild-type p53 transcription in ovarian cancer, non-small cell-lung carcinoma and colon adenocarcinoma cell lines, independent of DNA damage and MDM2 [et al. when they found that quinacrine activates p53 in renal cell carcinomas, non-small cell lung carcinoma, colon and breast carcinomas, prostate adenocarcinomas, and fibrosarcomas [Several recent investigations support the notion that quinacrine-induced cell death is caused by a mechanism that is independent of DNA damage -252. Quiand MDM2 . This DNsarcomas . Friedmasarcomas . The celsarcomas , but alssarcomas , therebyet al. [et al. [The unique mechanism of p53 upregulation which differs from the genotoxic upregulation of p53 was not investigated by Wang et al. , but Gur [et al. , found t [et al. . In this [et al. .2- and CoCl2-induced cellular activation of ICAM-1 was inhibited by quinacrine [2 because the enzyme causes the generation of platelet activation factor and eicosanoids [NF-κB is also a key regulator of cytokine-induced expression of endothelial cellular adhesion molecules (CAMs) ,255. Theinacrine . The effosanoids , which aosanoids .2 [NF-κB has been recently shown to also depend on arachidonic acid metabolites  and upst2 . In a st2 .2, based on reports showing that UVB can induce PLA2 in keratinocytes [2 inhibitory activity of quinacrine [Another interesting line of investigation further revealed that reactive oxygen intermediates (ROI) are implicated in UVB-induced expression of TNFα in keratinocytes and that COX products and, more importantly, LOX products, also known as eicosanoids, which are themselves products of an oxidative metabolism, are the main ROI implicated in this induction . The invinocytes -264. Anoinacrine .2 inhibition by quinacrine as the mechanism of NF-κB inhibition and in light of more recent investigations challenging the notion that quinacrine acts primarily as an inhibitor of PLA2 [2 inhibition. Pupe et al. [However, because many studies do not directly implicate PLA of PLA2 ,253,266,e et al.  present et al. [Unlike other NF-κB inhibitors, quinacrine does not inhibit the NF-κB pathway via cytoplasmic sequestration of p65. Instead, published experiments indicate a mechanism involving confinement of the p65 complex in the nucleus in an inactive state . The incet al. .et al. when hydrogen peroxide-induced phosphorylation of the p65 subunit of NF-κB was partially inhibited by quinacrine [Down-regulation of p65 Ser536 phosphorylation by IKKα has been suggested as the primary mechanism of action for quinacrine . The phyinacrine . The effinacrine . As discinacrine .As shown in Figure et al. demonstrated that 9AA inhibits AKT activity and its phosphorylation at Ser473 [Guo t Ser473 . They wet Ser473 . The inht Ser473 . Furthert Ser473 .Since its discovery as a potent antimalarial compound, quinacrine has been effective not only in the treatment, but also as a prophylaxis for malaria as well as a medication for a wide range of other disorders. Due to its anti-inflammatory activity in patients with autoimmune disorders quinacrine has been used to treat lupus erythematosus, rheumatoid arthritis, bronchial asthma and other inflammatory diseases. The safety of and bioavailability of quinacrine has been demonstrated as patients with these diseases used quinacrine for months at a time to control their symptoms. The pharmacokinetics and safety of quinacrine has been extensively studied as it was administered as a protective measure to millions of US soldiers in the Pacific region during World War II.Some of the more serious side effects of quinacrine are mild in comparison to other anti-cancer chemotherapeutics and most of the conditions can be easily reversed after treatment cessation or dose reduction. Many of quinacrine's side effects develop gradually, starting from minor lesions in the case dermatitis or a slight decrease in blood counts in the development of anemia, and have been found to be completely and easily reversible, if quinacrine use is discontinued at this early stage ,64,276. Furthermore, the polypharmacology of quinacrine make it an attractive drug in the use of different cancer types. In addition, as inflammation is now being considered the seventh hallmark of cancer , quinacrThe authors declare that they have no competing interests.All authors contributed to the writing of this manuscript and approved the final version.


Fifty-eight patients (51 families) were included. Age at first evaluation, weight and height at birth, consanguinity, familial recurrence, severity of AG, penile length, LH, FSH, T, dihydrotestosterone (DHT), Δ4-androstenedione (Δ4), and T/DHT and T/Δ4 ratios were evaluated. The AR and SRD5A2 genes were sequenced in all cases. There were 9 cases (7 families) of 5α-RD2, 10 cases (5 families) of PAIS, and 39 patients had normal molecular analysis of SRD5A2 and AR genes. Age at first evaluation, birth weight and height, and T/DHT ratio were lower in the undetermined group, while penile length was higher in this group. Consanguinity was more frequent and severity of AG was higher in 5α-RD2 patients. Familial recurrence was more frequent in PAIS patients. Birth weight and height, consanguinity, familial recurrence, severity of AG, penile length, and T/DHT ratio may help the investigation of 46,XY patients with AG and normal T synthesis.The aim of this study was to search for clinical and laboratorial data in 46,XY patients with ambiguous genitalia (AG) and normal testosterone (T) synthesis that could help to distinguish partial androgen insensitivity syndrome (PAIS) from 5 Before puberty, the phenotypes of 46,XY DSD due to androgen insensitivity syndromes or 5α-reductase type 2 deficiency are, in general, indistinguishable, particularly when there is no parental consanguinity  or family history consistent with X-linked inheritance (androgen insensitivity syndromes) [The main diagnosis for patients with ambiguous genitalia and 46,XY karyotype  with normal testosterone secretion and normal Müllerian duct regression is PAIS or 5Despite the multiple genetic causes of 46,XY DSD, around 30–40% of cases remain without diagnosis , 8.α-reductase type 2 deficiency and from cases without molecular defects in the AR and SRD5A2 genes.For that reason, the aim of this study was to search for clinical and laboratorial features of 46,XY patients with ambiguous genitalia and normal testosterone synthesis that could help to distinguish PAIS from 5In the last 10 years (from January 2001 to December 2010), the Interdisciplinary Group for the Study of Sex Determination and Differentiation (GIEDDS) from the Clinical Hospital of the Faculty of Medical Sciences of State University of Campinas (UNICAMP), Brazil evaluated 58 patients (51 families) with ambiguous genitalia having a 46,XY karyotype and normal testosterone secretion after hCG stimulation. This study was performed according to the Helsinki declaration and was approved by the Ethical Research Committee of Faculty of Medical Sciences of UNICAMP. Informed consent was obtained from all participants and from parents of participants under 18 years of age.All patients included in this study were born at term. They underwent laparoscopy during genitoplasty, orchidopexy, and/or gonadectomy, and no Müllerian structures were found.Data were obtained regarding age at first evaluation, weight and length at birth, history of parental consanguinity, family history of ambiguous genitalia or infertility, severity of the ambiguous genitalia  followed by sequencing the fragments with Big Dye Terminator Cycle Sequencing Kit V3.1 Ready Reaction (ABI PRISM/PE Biosystems). The sequences were compared with the normal sequence of each gene (ENSEMBL: ENSG00000049319 and ENSG00000169083) using CLC Sequence Viewer v.6.2 (free software).Molecular analyses of α-reductase type 2 deficiency, (2) PAIS, and (3) undetermined (without molecular defects in either AR or SRD5A2 gene).According to clinical and molecular data, the cases were classified in three groups of diagnosis: (1) 5P < 0.05 was adopted.Data were processed in the SPSS program for Windows, version 16.0, and descriptive analyses for continuous variables were made by calculating range, means and standard deviation. The data were compared among the three groups using Chi-square test or Fisher's exact test for categorical variables and Kruskal-Wallis test for continuous variables. For all analyses, a significance level of SRD5A2 gene  = 19.86, P = 0.00005), whereas familial recurrence was significantly more frequent in the groups of PAIS (χ2(2) = 8.14, P = 0.02). The severity of ambiguous genitalia according to Sinnecker et al. [χ2(2) = 15.49, P = 0.0004) and according to Ahmed et al. [χ2(2) = 20.89, P = 0.00003) was significantly higher in the group of 5α-reductase type 2 deficiency and PAIS in relation to undetermined cases. Analyzing only the patients with 5α-reductase type 2 deficiency and PAIS, the Sinnecker et al. [χ2(2) = 6.41, P = 0.04), while the Ahmed et al. [r = −0.675, P = 0.0001).d et al.  score . The age at first evaluation was significantly lower in idiopathic cases (P = 0.02). Weight (P = 0.002) and length (P = 0.02) at birth and T/DHT ratio (P = 0.0001) were significantly lower in undetermined cases, and penile length was significantly higher in this group (P = 0.0001). The T (P = 0.07) and Δ4-androstendione (P = 0.12) levels and T/Δ4 ratio (P = 0.32) did not differ among the three groups. All patients showed normal Δ4-androstendione levels for age and pubertal stage and T/Δ4 ratio above 0.8. α-reductase type 2 deficiency, PAIS, and disorders in testosterone synthesis may be indistinguishable in newborns [α-reductase type 2 deficiency should be established as soon as possible because individuals with PAIS are usually recommended to be raised as females, whereas those with 5α-reductase type 2 deficiency as males, when the diagnosis is made early in childhood [Clinical and laboratorial investigation must include careful and precise anatomical and hormonal studies  prior to gender assignment in 46,XY patients with undermasculinization , which can be difficult in most cases , 8, 12. hildhood .α-reductase type 2 deficiency and 46,XY karyotype raised as girls develop a male gender identity and change the gender behavior in adolescence or early adulthood [A correct and early diagnosis is very important because as a result of pre- and/or postnatal brain exposure to androgens, almost 70% of individuals with 5dulthood –18. The α-reductase type 2 deficiency can be easier due to the presence of gynecomastia, little genital virilization, and body hair in patients with PAIS, whereas in patients with 5α-reductase type 2 deficiency, there is genital virilization, although not always with adequate penile growth, absence of gynecomastia, and absent or hypoplastic prostate [α-reductase type 2 deficiency [α-reductase type 2 deficiency, but it was important to differentiate these two groups from undetermined cases. Probably, this result was due to the sensitivity of laboratory methods. Unfortunately, we could not confirmed these results using more specific methods like liquid chromatography linked with tanden mass spectrometry (LC-MS/MS) or immunoassays after organic solvent extraction [β-hydroxysteroid dehydrogenase III deficiency in infants younger than 6 months can be excluded with a basal T/Δ4 ratio above 0.8, with a sensitivity of 100% and in prepubertal children with the same value of T/Δ4 ratio after hCG stimulation test, with a sensitivity of 90%. At puberty, the differential diagnosis of PAIS and 5prostate . Howeverprostate . In addificiency . Surpristraction . Δ4-andrtraction , the diaSRD5A2 gene, confirming the diagnosis of 5α-reductase type 2 deficiency, 5 (9.8%) showed hemizygous mutation in AR gene, confirming the diagnosis of PAIS, and the remaining 39 (76.4%) cases showed normal molecular analysis of SRD5A2 and AR genes. Based on the literature available, we expected more cases of PAIS [AR gene if the AR promoter region and 3′UTR were evaluated, and we must also remember that the AR gene in target tissues from patients with hypospadias is more methylated than in control children, resulting in a decreased expression of the AR. This epigenetic alteration of the AR gene might be involved in the pathogenesis of hypospadias [In the present study, among 51 families evaluated, 7 (13.7%) showed homozygous or compound heterozygous mutations in  of PAIS , 22 and ning 39 7.4% casesncy, 5 9.% showed ospadias .α-reductase type 2 deficiency, which is a male-limited autosomal recessive disorder (OMIM 264600), whereas the familial recurrence was higher in PAIS, an X-linked disorder (OMIM 300068).As expected, the frequency of parental consanguinity was higher in patients with 5α-reductase type 2 deficiency in comparison with PAIS and undetermined cases. The 5α-reductase type 2 deficiency has frequently a classical syndrome of pseudovaginal perineoscrotal hypospadias, characterized by a predominantly female phenotype at birth and significant virilization at puberty [Weight and length at birth were lower in undetermined cases, suggesting that metabolic and endocrine disorders as fetal malnutrition could play a role in poor external genitalia development. Morel et al. showed that, in comparison with PAIS, undetermined 46,XY DSD was characterized by a high incidence of prematurity and/or intrauterine growth retardation (30%) . In the  puberty . Recent  puberty .α-reductase type 2 deficiency and PAIS groups have more cases with Sinnecker et al. [The age at first evaluation was lower in undetermined cases, and this data can be associated with severity of ambiguous genitalia: 5r et al. , classifr et al.  score beIn conclusion, birth weight and length, parental consanguinity, familial recurrence, severity of ambiguous genitalia, penile length, and T/DHT ratio may help the investigation of 46,XY patients with ambiguous genitalia and normal testosterone synthesis.


Specifically, β1-AR and β2-AR coupled to Gαs exert a proapoptotic action, while β2-AR coupled to Gi exerts an antiapoptotic action. β1 integrin signaling protects cardiac myocytes against β-AR-stimulated apoptosis in vitro and in vivo. Interaction of matrix metalloproteinase-2 (MMP-2) with β1 integrins interferes with the survival signals initiated by β1 integrins. This paper will discuss background information on β-AR and integrin signaling and summarize the role of β1 integrins in β-AR-stimulated cardiac myocyte apoptosis.Increased sympathetic nerve activity to the myocardium is a central feature in patients with heart failure. Accumulation of catecholamines plays an important role in the pathogenesis of heart disease. Acting via  Heart failure is a leading cause of morbidity and mortality in the western world. Cardiac myocytes are generally viewed as terminally differentiated and incapable of reentering the cell cycle. Myocyte apoptosis is shown to occur in human myocardium and animal models of cardiovascular disease under a variety of pathological states of the heart –4. The aβ-AR antagonist in improving the clinical outcome as well as benefit in long-term morbidity and mortality of patients with chronic heart failure [α- and β-adrenergic receptors (ARs), a family of G protein-coupled receptors (GPCRs). Specific stimulation of β1-AR using NE or isoproterenol (β-AR agonist) induces apoptosis in cardiac myocytes in vitro and in vivo [Increased sympathetic nerve activity is a central feature in patients with heart failure . Initial failure  has conf in vivo –9. Increα and β subunits. Integrins play a significant role in cell-matrix interactions. They are involved in a variety of functions like gene expression and regulation, organogenesis, cell proliferation, differentiation, migration, and death. In the heart, integrins are shown to regulate cellular phenotype in the developing and postnatal myocardium. They also serve as mechanotransmitters during normal development and in response to physiological and pathophysiological signals [β1 integrins. β1 integrins play an important role in β-AR-stimulated LV remodeling with effect on myocyte apoptosis [Integrins are a large family of heterodimeric transmembrane receptors composed of  signals –12. Cardpoptosis . β-AR-stimulated myocyte apoptosis, and (c) role of β1 integrins in the regulation of β-AR stimulated cardiac myocyte apoptosis.This paper will discuss (a) the expression of AR subtypes in the heart and their role in cardiac myocyte apoptosis, (b) molecular signals involved in β-AR  and three subtypes of the α1-AR  [α2-AR in most species [β1-AR generally predominates, although the ratio of β1 to β2-AR varies among species and under various pathophysiological states [Nine subtypes of AR have been identified . Cardiacnd α1C) , 15. Car species . Based ol states . β1- and β2-AR couple to the stimulatory G protein (Gαs). This coupling normally leads to the activation of adenylyl cyclase, production of cAMP, and activation of protein kinase A (PKA). β2-AR also couples to Gi, a different heterodimeric G-protein whose Gα subunit can inhibit adenylyl cyclase, thereby inhibiting synthesis of cAMP and activation of PKA. This coupling may require phosphorylation of β2-AR by cAMP-dependent protein kinase A [β2-AR may induce compartmentalization of Gαs-stimulated cAMP signal affecting only plasma membrane effectors such as L-type calcium channel, without affecting cytoplasmic target proteins such as phospholamban and myofilament contractile proteins [β2-ARs are localized exclusively to the deep transverse tubules, whereas β1-ARs are distributed across the whole cell surface. Redistribution of β2-AR from transverse tubules to the cell crest occurred in myocytes isolated from a rat model of heart failure. This redistribution led to cAMP signals in both cell crest and T-tubule, a pattern similar to that observed for β1-AR stimulation [β2-AR-Gi pathway, redistribution of β2-AR during heart failure, and changes in the compartmentalization of cAMP may have implications during the development of heart failure. Both kinase A . The duaproteins . In normmulation . Thus, tβ3-AR and a putative β4-AR are present in rodent heart [β3-AR pathway by catecholamines may contribute to the myocardial dysfunction during sepsis [β4-AR may represent propranolol-insensitive state of β1-AR [Pharmacological approaches have suggested that nt heart , 21. Theg sepsis , and putf β1-AR , 24. β-AR antagonist propranolol, but not by α-AR antagonist, and was mimicked by β-AR agonist isoproterenol. Communal et al. provided evidence that stimulation of β-AR increases apoptosis in adult rat ventricular myocytes (ARVM) [β-AR blockers such as metoprolol and carvedilol reduced myocyte apoptosis and improved cardiac systolic function in animal models of chronic heart failure [β-AR-stimulated apoptosis of cardiac myocytes.Chronic exposure to catecholamines is known to be toxic to cardiac myocytes . Tonic es (ARVM) . Similars (ARVM) , 28, 29. failure , 31. Theβ1-AR induces proapoptotic signals, while specific linkage of β2-AR to Gi plays an antiapoptotic role in ARVM [β1-AR exhibited increase in basal contractile function at young age. This difference in contractility was lost at 16 weeks (age), and contractility continued to decline thereafter, going to less than 50% of wild-type value at 35 weeks [β2-AR (60–100-fold over the endogenous level) in the myocardium show enhanced cardiac function without deterioration into heart failure [β2-AR overexpression could be attributed to the enhanced coupling of β2-AR to Gαs. It is interesting to note that β2-AR overexpression (200-fold over the endogenous level) preserved LV contractility in a mouse model of myocardial infarction while exhibiting similar cardiac hypertrophy and chamber size as wild-type mice [αs also had deleterious effects on the heart with chamber dilation, reduced ejection fraction, and increased myocardial fibrosis and myocyte apoptosis [Pharmacologic manipulations indicated that specific stimulation of  in ARVM , 33. Tra35 weeks . This de failure –39. In cype mice . Overexppoptosis , 42. In poptosis .β-AR-stimulated apoptosis is influenced by the intracellular and extracellular signals. With respect to the intracellular signals, the apoptotic pathway (β1-AR-Gαs) involved activation of PKA [β2-AR-Gi) involved activation of phosphatidylinositol 3-kinase (PI3-kinase) and Akt [β-AR-stimulated apoptosis. This is based on the findings that prolonged β-AR stimulation may decrease the levels of β1-AR and Gαs may become uncoupled from adenylyl cyclase, leading to activation of calcium/calmodulin kinase II (CaMKII) [β1-AR systems (adult cardiac myocytes isolated from β2-AR knockout mice and adenovirus-mediated transfer of the mouse β1-AR in myocytes isolated from β1-AR and β2-AR double knockout mice), it was demonstrated that sustained β1-AR stimulation delivers a powerful cardiac apoptotic signal via a CaMKII-dependent, rather than a PKA-dependent, mechanism [β1-AR knockout mice demonstrated that β1-AR stimulates CaMKII and enhances cardiac dysfunction after myocardial infarction [c release. The increase in cardiac myocyte apoptosis and cytochrome c release was attenuated by coexpression of anti-apoptotic protein Bcl-X(L). Furthermore, expression of a dominant negative mutant of CaMKII(deltaC) not only prevented CaMKII(deltaC)-mediated apoptosis but also protected cells from multiple death-inducing stimuli [n of PKA , 33, whi and Akt . However(CaMKII) . Using techanism . Recent farction . Cardiac stimuli . β-AR stimulation has been shown to activate these three subgroups of MAPKs [β-AR-stimulated apoptosis. Inhibition of mitochondrial permeability transition pore opening or caspase activation also decreased β-AR-stimulated apoptosis [β-AR-stimulated apoptosis in ARVM involves reactive oxygen species/JNK-dependent activation of the mitochondrial death pathway. In this pathway, small GTPase Rac 1 may act upstream in the activation of JNKs [β-AR stimulation activates glycogen synthase kinase-3β (GSK-3β), and activation of GSK-3β plays a pro-apoptotic role in β-AR-stimulated apoptosis via the involvement of mitochondrial death pathway [β increased JNK phosphorylation, suggesting that GSK-3β may also act upstream in the activation of JNKs [Mitogen-activated protein kinases (MAPKs), a large family of serine-threonine kinases, have important functions as mediators of intracellular signal transduction. Three subgroups of MAPKs have clearly been identified: c-jun N-terminal kinases (JNKs), p38 kinase, and ERK1/2. of MAPKs . Activatof MAPKs . Superoxpoptosis . These s of JNKs . Studies pathway . Adenovi of JNKs . Furtherβ-AR-stimulated apoptosis. On the other hand, SB-202190, an inhibitor of p38 kinase, potentiated β-AR-stimulated apoptosis in ARVM, suggesting a protective role for p38 kinase [β1- and β2-AR to the activation of p38 kinase, although β-AR-stimulated activation of p38 kinase could be inhibited by inactivation of Gi using pertussis toxin. In adult mouse myocytes, β2-AR activated p38 kinase independent of Gi [α. Inhibition of p38 kinase(α), by mating mice expressing dominant negative p38 kinase(α) with mice overexpressing β2-AR, reversed depressed LV function and reduced apoptosis in mice overexpressing β2-AR. Inhibition of p38 kinase(α) had no effect on β1-AR overexpressing mice [α) plays a pro-apoptotic role during the development of cardiomyopathy following chronic β2-AR stimulation. These differential effects of p38 kinase may reflect nonspecific effects of pharmacological inhibitor and/or overexpression of β2-AR. It may also reflect acute versus chronic stimulation of β-AR. The transgenic mice studies were carried out in 11–14-months old mice versus acute stimulation of β-AR in vitro.Inhibition of ERK1/2 using PD98059 had no effect on 8 kinase . Pharmacnt of Gi . Transgeing mice . These tβ-AR-stimulated cardiac myocyte apoptosis. Matrix metalloproteinases (MMPs), a large family of endopeptidases, have the ability to degrade extracellular matrix (ECM) proteins, and therefore, play a fundamental role in tissue remodeling, including the heart [β-AR stimulation  increases expression of MMP-2 and TIMP-1, and decreases expression of TIMP-2 in ARVM [β-AR stimulation had no effect on the expression or activity of MMP-9. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), or purified TIMP-2 (tissue inhibitor of MMP-2) inhibited β-AR-stimulated apoptosis in ARVM. This decrease in apoptosis associated with inhibition of JNK activity decreased cytosolic cytochrome c levels and maintenance of mitochondrial membrane potential. On the other hand, treatment with active MMP-2 alone increased cytosolic cytochrome c levels and the number of apoptotic cardiac myocytes [β-AR-stimulated cardiac myocyte apoptosis and provide evidence that MMP-2 is capable of modulating JNK-dependent mitochondrial death pathway.Extracellular signals also modulate he heart –57. Tisshe heart . Cardiache heart . Targetehe heart . Our lab in ARVM . β-AR stmyocytes , 62. Theβ-AR increased levels of extracellular ubiquitin in the media. Treatment of ARVM using purified ubiquitin inhibited β-AR-stimulated apoptosis. This inhibition of apoptosis associated with inactivation of GSK-3β/JNK and mitochondrial death pathways [Recently, our laboratory has identified ubiquitin in the conditioned media of ARVM. Stimulation of pathways . Growingpathways –65. Usinpathways . α and β subunits, with α subunits ranging from 120 to 180 kDa, while β subunits range from 90 to 110 kDa [Integrins link the ECM proteins and the intracellular cytoskeleton. Integrins consist of  110 kDa , 67. Int 110 kDa . Integri 110 kDa –71. β modulate the expression of several integrins as well as specific ECM components such as interstitial collagens, osteopontin, fibronectin and laminin. The myocyte integrin-ECM interactions may play a fundamental role in the pumping function of heart. The ECM surrounding the individual myocytes coordinates the transduction of force to the whole ventricular chamber, so that the heart can function as a single pump [β1 integrin may subject the cell to altered mechanical force that can be detrimental to the long-term cell survival. Shedding of β1 integrins is described in the heart during the transition from cardiac hypertrophy to heart failure [β-AR induces β1 integrin shedding in the mouse heart [A significant role of integrins in the heart is their ability to serve as mechanotransducers during normal development and in response to physiological and pathophysiological signals . Mechanigle pump . These mgle pump  and in pgle pump . This digle pump . It was gle pump . The mecgle pump –79. The  failure . Chronicse heart . β1 integrins. However they express β1D, a differentially spliced variant of β1 integrin. β1D has a unique cytoplasmic domain of 50 amino acids with the last 24 amino acids encoded by an additional exon. However, both integrin isoforms, β1A and β1D, were found to be functionally similar with regard to integrin signaling [β1 integrins can heterodimerize with integrin α subunits  [β1 integrins in myocytes, the paper summarizes the role of β1 integrins in β-AR-stimulated apoptosis.Cardiac myocytes predominantly express ignaling , 82. In and α7b) . Due to β1 integrins alter AR signaling and influence myocyte phenotype with respect to hypertrophy and apoptosis. β1 integrins participate in α1-AR-induced hypertrophy of neonatal rat cardiac myocytes [β2-AR agonist shown to induce cardiac hypertrophy, demonstrated upregulation of genes associated with integrin-mediated cell adhesion and signaling [β1 integrin gene exclusively in cardiac myocytes, Shai et al. [β1 integrins play an important role in myocardial fibrosis and cardiac failure [In vitro, stimulation of β1 integrin signaling using laminin or adenoviral-mediated overexpression of β1A integrin protected ARVM from β-AR-stimulated apoptosis [c levels. On the other hand, expression of a cytoplasmic domain of β1 integrin, present as a result of integrin shedding, induced apoptosis in ARVM [c suggesting involvement of caspase-8 and mitochondrial death pathway. Deficiency of β1 integrins as demonstrated using β1 integrin heterozygous knockout mice associated with increased cardiac myocyte apoptosis in the heart after myocardial infarction and isoproterenol infusion [β1-integrin-deficient mice exhibited enhanced LV dysfunction and dilation after myocardial infarction when compared to the wild-type mice. Myocyte cross-sectional area (a measure of myocyte hypertrophy) and myocardial fibrosis were significantly lower in β1-integrin-deficient mice after chronic β-AR stimulation [β1 integrins influence β-AR responsiveness and play a crucial role in β-AR-stimulated myocardial remodeling with effects on cardiac myocyte hypertrophy and apoptosis. Increased cardiac myocyte apoptosis and decreased myocardial hypertrophy and fibrosis during β1 integrin deficiency may induce LV dilation due to side-to-side slippage of myocytes during deficiency of β1 integrins. It is interesting to note that a combined deficiency of dystrophin and β1 integrins in cardiac myocytes decreased ventricular function and blunted adrenergic responsiveness [β1 integrin signaling has the potential to negate the apoptotic effects of β-AR stimulation in cardiac myocytes. Integrins can themselves signal through a host of pathways. However, integrins are capable of collaborating with growth factors and their receptors leading to changes in intracellular signals. Many studies now indicate that myocytes , 85. A gignaling . Using Ci et al.  demonstr failure . In vitrpoptosis , 88. Thi in ARVM . This ininfusion , 89. β1-siveness . Thus, ββ [β1A integrin increased FAK phosphorylation at both Tyr397 and Tyr576 residues without affecting expression of FAK. Inhibition of MMP-2 using SB3CT or TIMP-2 significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). On the other hand, active MMP-2 significantly inhibited FAK phosphorylation [In vivo, chronic stimulation of β1-AR impaired FAK signaling during early compensated mitral regurgitation in dogs [β1 integrins inhibited adenylyl cyclase activity via the involvement of FAK/PI3-kinase/Akt pathway [β1A integrin or inhibition of MMP-2 inhibits β-AR-stimulated activation of GSK-3β. On the other hand, active MMP-2 protein increased GSK-3β activity. Inhibition of PI3-kinase using wortmannin reversed the effects of β1 integrins on GSK-3β activity and inhibited the protective effect of β1 integrins [β1-integrin-deficient mice when compared to wild type following chronic β-AR stimulation [β1 integrin engagement, resulting in the activation of a JNK-dependent mitochondrial death pathway. Of note, extracellular ubiquitin also inhibited β-AR-stimulated activation of GSK-3β, and inhibition of PI3-kinase using wortmannin reversed the protective effects of extracellular ubiquitin [β1 integrins. Co-immunoprecipitation studies demonstrated physical association of MMP-2 with β1 integrins in ARVM. β-AR stimulation increased the level of interaction between these two proteins, while inhibition of MMP-2 using SB3CT or stimulation of β1 integrin signaling using laminin inhibited β-AR-stimulated interaction of MMP-2 with β1 integrins [β1 integrins may interfere with the survival signals induced by β1 integrins, leading to apoptosis.Integrin engagement with ligands initiates autophosphorylation of focal adhesion kinase (FAK) at Tyr397. This autophosphorylation site provides a binding site for Src, which phosphorylates FAK at Tyr576 and Tyr577 to further activate FAK . Activatβ . In ARVMrylation . In vivo in dogs . In cat  pathway . Our labntegrins . Activatbiquitin , suggestntegrins . It is lβ1-AR and β2-AR coupled to Gαs exert a pro-apoptotic action via a cAMP-dependent mechanism which appears to involve mitochondria and ROS and is associated with the activation of JNK and GSK-3β. Pro-apoptotic action of β1-AR may involve activation of CaMKII. Conversely, β2-AR coupled to Gi exerts an anti-apoptotic action which is mediated by PI3-kinase/Akt (β-adrenergic stimulation may have important clinical implications. Identification of molecular targets involved in the activation of JNKs and GSK-3β following β-AR stimulation and understanding how these kinases activate mitochondrial death pathway may provide new targets for prevention of heart failure. β1 integrins play an important role in chronic β-AR-stimulated cardiac myocyte apoptosis and myocardial remodeling via the involvement of FAK and PI3-kinase/Akt pathways (β1 integrins. Further studies aimed at determining the molecular mechanism by which interaction of MMP-2 with β1-integrins affects β-AR-stimulated apoptosis in cardiac myocytes may have important implications for the regulation of myocyte survival.Catecholamines play an important role in remodeling of the heart, when the heart is subjected to pathophysiological stressors. nase/Akt . Elucidapathways . The str


Androgen receptor (AR) gene has an essential role in the hormone-dependent stage of sexual development. In addition, longer AR polyglutamine repeat lengths encoded by CAG repeats are associated with lower transcriptional activity in vitro. In the present study, we aimed at investigating the role of the CAG repeat length in the AR gene in hypospadias cases as compared to the controls. Our study included 211 hypospadias and 208 controls of Caucasian origin.Hypospadias is a birth defect of the urethra in males, and a milder form of 46,XY disorder of sexual development (DSD). The disease is characterized by a ventrally placed urinary opening due to a premature fetal arrest of the urethra development. Moreover, the We amplified the CAG repeat region with PCR, and calculated the difference in the mean CAG repeat length between the hypospadias and control group using the T-test for independent groups.We detected a significant increase of the CAG repeat length in the hypospadias cases when compared to the controls ; p-value: 0.001). In addition, the odds ratios between the hypospadias and controls revealed that the hypospadias cases are two to 3 times as likely to have longer CAG repeats than a shorter length for each repeat length investigated.AR gene have a clear effect on the risk of hypospadias in Caucasians.We have investigated the largest number of hypospadias cases with regards to the CAG repeat length, and we provide evidence that a higher number of the CAG repeat sequence in the  Androgen receptor (AR) gene is located on chromosome Xq12 and is involved in the generation of the male internal and external genitalia through the actions of testosterone and 5α-dihydrotestosterone (DHT) [The ne (DHT) .AR gene that severely impact the function of the receptor can cause the syndrome of partial or complete androgen insensitivity, and have also been observed in a few cases of isolated hypospadias [Mutations in the ospadias -10. The ospadias .Hypospadias is usually diagnosed during physical examination of the newborns, with subsequent surgery in their first two years of life. Still, the molecular mechanisms behind this disease, particularly in the sporadic form, are largely unknown.AR gene results in longer polyglutamine tracts in the AR, and cause the AR to have a reduced transcriptional activity and to associate with moderate to severe undermasculinized genitalia in XY males [AR gene with the disease of hypospadias in a significantly larger patient material.Since AR mediates important biological effect of testosterone and DHT, it is an obvious candidate in the development of hypospadias. Moreover, greater number of CAG repeats within the XY males . In addiXY males , and anoXY males , assesseAR gene in each case from the hypospadias group with the repeats in individuals from the control group. We first calculated the skewness for each group, and detected the CAG repeats to be normally distributed within each group  was calculated between the hypospadias and the control group for a range of repeat sizes using logistic regression , or Kennedy’s disease [Several pathways have up until now been implicated in male urethra development, and the strong focus of genetic research on the hormone-dependent stage of sexual development has revealed promising candidate genes, among which the function . An even disease , where t disease ,18. It h disease ,19. Thes disease , were no disease ,15.Our analysis suggests a role of expanded CAG repeat length in the development of hypospadias. Based on our results, we propose that the CAG polymorphism modifies the risk of hypospadias in Caucasians.We investigated DNA from 211 cases with hypospadias. The cases composed of 33 patients with penoscrotal, scrotal or perineal phenotype, and 178 with glandular, coronar, mid or distal penil phenotype, as classified by A.N . The control group consisted of 208 healthy voluntary anonymous blood sample donors, all males, from the Karolinska University Hospital. The cases and controls were of Caucasian origin. Genomic DNA was extracted from either tissue harvested at surgery or from peripheral blood according to standard procedures reported elsewhere [AR gene was PCR amplified and mixed with 500-Rox size standard, and electrophoresed on the ABI3730 Sequencer (Applied Biosystems). Primer and PCR amplification conditions have been reported previously [The CAG repeat region located in the eviously . In totaThe authors declare no competing interests.TA and AN conceived and designed the study. TA and AN performed the PCR and statistical analysis. TA and AN interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/13/109/prepub


Nrp1cyto) have normal angiogenesis but impaired developmental and adult arteriogenesis. The arteriogenic defect was traced to the absence of a PDZ-dependent interaction between NRP1 and VEGF receptor 2 (VEGFR2) complex and synectin, which delayed trafficking of endocytosed VEGFR2 from Rab5+ to EAA1+ endosomes. This led to increased PTPN1 (PTP1b)-mediated dephosphorylation of VEGFR2 at Y1175, the site involved in activating ERK signaling. The Nrp1cyto mutation also impaired endothelial tubulogenesis in vitro, which could be rescued by expressing full-length NRP1 or constitutively active ERK. These results demonstrate that the NRP1 cytoplasmic domain promotes VEGFR2 trafficking in a PDZ-dependent manner to regulate arteriogenic ERK signaling and establish a role for NRP1 in VEGF-A signaling during vascular morphogenesis.Neuropilin 1 (NRP1) plays an important but ill-defined role in VEGF-A signaling and vascular morphogenesis. We show that mice with a knockin mutation that ablates the NRP1 cytoplasmic tail (  •The NRP1 cytoplasmic domain promotes VEGF receptor (VEGFR) 2 endocytic trafficking•In its absence, VEGR2 trafficking is delayed in sorting endosomes•1175 site of VEGFR2PTP1b binds to Rab5+ sorting endosomes and dephosphorylates the Y•Loss of the NRP1 cytoplasmic domain impairs developmental and adult arteriogenesis Lanahan et al. find that the VEGF coreceptor NRP1 is required for proper VEGFR2 endocytic trafficking. In knockin mice lacking NRP1’s cytoplasmic domain, VEGFR2 passes through early endocytic compartments more slowly, increasing its exposure to the inhibitory phosphatase PTP1b and attenuating VEGF-stimulated ERK signaling and arteriogenesis. Abnormal ERK signaling in Nrp1cyto endothelial cells could be rescued by full-length NRP1, but not NRP1 lacking the cytoplasmic domain or its PDZ-binding sequence. Furthermore, abnormal endothelial cell (EC) tubulogenesis in vitro could be rescued by expression of full-length NRP1 or constitutively active ERK. These findings establish NRP1 as a specific regulator of VEGF-A-induced VEGFR2 trafficking and ERK signaling that is essential for VEGF-A-dependent arterial morphogenesis.e retina . We showNrp1cyto knockin mice, in which a stop codon in exon 17 prevents translation of the cytoplasmic domain  littermates  or the NRP1-binding isoform of VEGF-A, VEGF-Ar 7 days F and S1Gr 7 days . We founf 7 days H and S1INrp1cyto mice compared to littermate controls in developing renal vasculature at P7  to visualize arterial circulation in the kidneys of neonatal (P7) and in the heart, hindlimbs, and kidneys of young adult (6–8 weeks of age) mice, using contrast injections that fill only arterial, but not capillary or venous beds . The mCTre at P7 A and 1B re at P7 C–1I. Oveull mice . Consistt weight A as wellt weight B compareNrp1cyto mutants and WT littermates immediately after induction of ischemia  led to a rapid upregulation of VEGFR2 phosphorylation at the Y1175 residue that is involved in the activation of PLCγ/ERK signaling in control mice and was accompanied by increased ERK1/2 phosphorylation  or vectors expressing full-length NRP1 (Ad-Nrp1), NRP1 lacking the entire cytoplasmic domain (Ad-Nrp1cyto) or NRP1 lacking only the PDZ binding site (Ad-Nrp1PDZ). As NRP1cyto expression levels are variable in vitro (but not in vivo), we chose cells with NRP1cyto levels that best matched Nrp1 levels following transduction of various mutants noted above  mice that were transduced with a CRE-expressing vector (Ad-Cre) to ablate NRP1 expression and then transduced with Ad-Nrp1, Ad-Nrp1cyto, or Ad-Nrp1PDZ  that mimics many features of arteriogenesis in vivo  ERK (Nrp1cyto EC.To link changes in ERK activation in  in vivo . Knockinogenesis E and 4F.(CA) ERK E and 4F.Previous studies demonstrated that reduced VEGFR2 endocytosis or delayed trafficking reduces its signaling output . Thus, lNrp1cyto and WT EC after exposure to VEGF-A165 showed a comparable extent and rate of NRP1 uptake  or moves faster to downstream compartments (Rab7+ and/or Rab11+ endosomes). To distinguish these possibilities, we compared colocalization of VEGFR2 and NRP1 in peripherally located Rab5+ endosomes 10 min after VEGF-A165 stimulation and in Rab7+ versus Rab11+ endosomes at 30 min after stimulation. At 10 min, the VEGFR2/NRP1 complex was present at significantly higher levels in Rab5+ endosomes in Nrp1cyto compared to WT EC  have previously been described (Mice lacking the cytoplasmic tail of NRP1 (escribed . All ani165 (293-VE R&D Systems), PTP siRNAs FlexiTube ; QIAGEN 1027416), EZ-Link Sulfo-NHS-SS-Biotin (Pierce 21331), and immobilized Neutravidin protein (Pierce 29200). Antibodies include rat monoclonal VEGFR2 , goat polyclonal NRP1 (R&D Systems AF566), goat polyclonal NRP1 (Santa Cruz 7239), goat polyclonal anti-EEA1 (Santa Cruz 6415), rabbit polyclonal anti-EEA1 (Santa Cruz 33585), rabbit monoclonal Rab5 , rabbit monoclonal Rab7 , goat polyclonal anti-VE-cadherin (Santa Cruz SC6458), anti-phospho-VEGFR2 , anti-total VEGFR2 , anti-phospho p44/42 MAP kinase , anti-p44/42 MAP kinase , anti-PTP1b , anti-DEP-1/CD148 (R&D AF1934), anti-PTPmu , and anti-SHP1 . We used appropriate secondary antibodies that were conjugated to horseradish peroxidase (Vector Laboratories) or fluorescently labeled (Life Technologies).The following regents were used: transferrin from human serum, Alexa Fluor 488 conjugate (Invitrogen T-13342), human fibronectin (BD Biosciences 356008), VEGF-ANrp1 EC were grown to 60%–70% confluence on gelatin-coated dishes and treated with Cre adenovirus overnight in media containing 20% FBS at a multiplicity of infection of 100. The washed cells or Nrp1cyto EC were then treated with control CMV, Nrp1cyto, Nrp1PDZ, or full-length NRP1 overnight, washed, grown an additional 2 days, and then starved overnight in 0.5% FBS. Cells were stimulated for 5 min with 50 ng/ml VEGF-A165 and lysed in NP40 buffer.Floxed 165 and lysed in NP40 buffer.EC were grown to 60–70% confluence on gelatin-coated dishes and treated on 2 consecutive days for 4 hr with 200 pmol/dish of siRNA (QIAGEN FlexiTube siRNA or Santa Cruz siRNA) in 2 ml growth media containing 0.5% FBS and TransPass R2 transfection reagent-siRNA suspension (New England BioLabs). EC were then starved overnight in growth media containing 0.5% FBS, stimulated for 5 min with 50 ng/mlVEGF-ACells were starved overnight with 0.5% FBS, placed on ice for 20 min, incubated with Alexa Fluor488 conjugated transferrin on ice for 30 min, rinsed three times with cold PBS and then stimulated with serum at 37°C for various times. After rinsing twice with ice cold PBS pH 2.5, cells were fixed at room temperature in 4% formaldehyde in PBS for 10 min and permeabilized with 2.5% formaldehyde containing 0.1% Triton X-100 and 0.1% NP-40 for 10 min at room temperature. Cells were then labeled with rabbit anti-EEA1 followed by chicken anti-rabbit Alexa 568 antibody. Samples were mounted with ProLong Gold DAPI (Life Technologies) and imaged using an UltraVIEW VoX spinning disc confocal microscope with a 60× oil immersion lens (Perkin Elmer) and equipped with a Ti-E microscope stand (Nikon). Image analysis was performed using the colocalization module of Volocity software.165, incubated at 37°C for various times, washed twice with ice cold PBS pH 2.5, rinsed three times with cold PBS and then fixed for 10 min at room temperature in 4% formaldehyde in PBS and permeabilized with 2.5% formaldehyde containing 0.1% Triton X-100 and 0.1% NP-40 for 10 min at room temperature. Cells were labeled with rabbit anti-EEA1 followed by chicken anti-rabbit Alexa647 secondary antibody. For detection of VEGFR2 and NRP1, cells were labeled with chick anti-rat alexa488 and donkey anti-goat alexa568 secondary antibodies. For Rab5, Rab7, and PTP1b costaining, NRP1 primary antibody was labeled with chick anti-goat alexa647, whereas primary antibodies for Rab5 or Rab7 were labeled with anti-rabbit alexa568, and PTP1b with anti-mouse alexa568. For Rab11 staining, a Rab11-RFP adenovirus was used to transduce cells prior to labeling. Samples were mounted with ProLong Gold DAPI and imaged with a Perkin Elmer UltraVIEW VoX spinning disc confocal microscope equipped with a 60× oil immersion lens and a 14 bit electron-multiplied, charge coupled device camera . Thresholds for z stack images were acquired using the MaxEntropy automatic threshold method provided by the ImageJ MultiThresholder plugin of (NIH Bethesda) . The overlap coefficient according to Manders was calculated using the ImageJ colocalization analysis plugin JACoP (Bolte & Cordelieres).Cells were plated on gelatin-coated glass bottom dishes (MatTek), starved overnight with 0.5% FBS, and then placed on ice. Cells were incubated with 10 μg/ml rat anti-mouse VEGFR2 and goat anti-rat NRP1 for 15 min and rinsed three times with cold PBS. Cells were stimulated with 50 ng/mlVEGF-A2 CCD cameras with a pixel size of 0.080 μm  on the OMX version 3 system (Applied Precision) equipped with 488, 561, and 642 nm solid-state lasers (Coherent and MPB communications). Samples were illuminated by a coherent scrambled laser light source that had passed through a diffraction grating to generate the structured illumination by interference of light orders in the image plane to create a 3D sinusoidal pattern, with lateral stripes approximately 0.270 nm apart. The pattern was shifted laterally through five phases and through three angular rotations of 60° for each z section, separated by 0.125 nm. Exposure times were typically between 200 and 500 ms, and the power of each laser was adjusted to achieve optimal intensities of between 2,000 and 4,000 counts in a raw image of 16-bit dynamic range, at the lowest possible laser power to minimize photo bleaching. Raw images were processed and reconstructed to reveal structures with 100–125 nm resolution  and CoolSNAP HQsolution . The cha6/ml to assess their ability to form lumens and tubes  at 2 × 10nd tubes . EC werend FGF-2 . The culescribed . After 3escribed .As previously described , dissect165 fusion protein was incubated with freshly dissected E12.5 hindbrain tissue, washed three times for 20 min each with PBS, fixed with 4% formaldehyde in PBS for 1 hr, and washed again. Endogenous AP was heat-inactivated at 65°C for 3 hr. Tissue-bound, heat-stable, recombinant AP activity was detected as an insoluble reaction product after incubation with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) in a buffer containing 100 mM Tris pH 9.5, 100 mM NaCl, and 5 mM MgCl2. Images were recorded using an MZ16 microscope (Leica) equipped with a Micropublisher 3.3 camera (Q Imaging).As previously described , alkalinAll data are shown as mean ± SEM or SD as bars in the histograms. Differences were considered statistically significant if p ≤ 0.05 by Student’s t test.


Yes, biophysics is the study of biological systems and biological processes using physics-based methods or based on physical principles.Physics provides the fundamental theories for understanding biomolecules. For example, statistical mechanics, a cornerstone of modern physics, is also the foundation for understanding the behaviors of biomolecular systems. Electron transfer within protein matrices, which drives respiration and photosynthesis, can only be understood with the help of quantum mechanics. In essence, an electron can hop from one position to another within a protein matrix only when the energy levels before and after the hop are equal.Importantly, many of the powerful tools for investigating biomolecules were initiated by physicists. X-ray crystallography provides a telling example. X-rays were discovered by Wilhelm Röntgen (1901 Nobel Prize in Physics) and their diffraction by crystals was first demonstrated by Max von Laue (1914 Nobel Prize in Physics). The subsequent mathematical formulation of the diffraction pattern by the Braggs, father and son (1915 Nobel Prize in Physics), ushered in the new field of X-ray crystallography. This made possible the determination of the first protein structures by Max Perutz and John Kendrew (1962 Nobel Prize in Chemistry), the structure of DNA by Francis Crick, James Watson, and Maurice Wilkins (1962 Nobel Prize in Physiology or Medicine), and the structures of the photosynthetic reaction center (1988 Nobel Prize in Chemistry), ion channels (2003 Nobel Prize in Chemistry), RNA polymerase II (2006 Nobel Prize in Chemistry), and the ribosome (2009 Nobel Prize in Chemistry). Similar paths can be traced for nuclear magnetic resonance spectroscopy , atomic force microscopy (1986 Nobel Prize in Physics), electron microscopy (1986 Nobel Prize in Physics), and single-molecule techniques such as optical tweezers (1997 Nobel Prize in Physics).Many computational techniques - for example, molecular dynamics simulation - that are now widely used for modeling biomolecular systems also have their origins in physics.You are in good company: some of the giants in modern biology, including Max Delbrück, Francis Crick, and Seymour Benzer, made the transition from physics. As Crick learned, you have to adjust from the "'elegance and deep simplicity"' of physics to the "'elaborate chemical mechanisms that natural selection has evolved over billions of years."' As Crick put it, the adjustment is "'almost as if one had to be born again."' You have to take the time to learn the biology. The transition can be eased by collaborating with another biophysicist or a biologist.However, despite the significant barrier to the transition from physics to biology, intellectually it is probably still far easier than the transition in the opposite direction!An important contribution of biophysicists to modern biology is the perspective that biological processes can be understood from the interactions between and within the constituent molecules. Therefore, the behaviors of biological systems can be predicted from physical principles.A biological problem that has been mostly tackled by biophysicists is protein folding, by which a nascent polypeptide chain coming off the ribosome finds its unique structure in its native environment. The broad outlines of how the protein avoids the vast number of alternative conformations and quickly finds its native structure are now clear. Some may go as far as claiming the problem is solved. Biophysicists are now using very similar approaches to study the binding of proteins and other biomolecules as well as more complex biological processes.Biophysicists are largely responsible for dramatic increases in the spatial resolution of structural characterization and the temporal resolution of dynamical characterization, and for bringing the study of biological processes to the single-molecule level.Biophysicists have demonstrated that many essential features of complex biological systems can be emulated by relatively simple computational models. In particular, artificial neural networks are shown to produce associative memory, an essential function of the brain.How the modeling work is labeled is less important than the fact that it is able to demonstrate that many essential biological features seem generic and robust. That is, they emerge from relatively simple models and are insensitive to details of the models. There are now similar efforts dealing with signaling and gene regulatory networks. Still, a fundamental understanding of these processes will require considering the physical interactions between the molecules involved.Yes. One example is the theory of complex systems, in which a key concept is emergent properties. These are properties that are not intrinsic to the individual components of a system but are only produced when the components work together as a whole system. For instance, a neural network can produce memory only through the interactions of all the neurons in the network.In addition, biological problems have stimulated renewed interest in areas like stochastic processes and open, driven systems.Indeed. Many biophysical concepts, theories, and tools were originally developed in physical chemistry. Binding affinity, key to characterizing specificity and selectivity in molecular interactions, derives from equilibrium constants of chemical reactions. Rate theories and the stopped-flow technique for measuring rate constants are other examples. However, in adapting these physical chemistry concepts and theories to biology, it is important to recognize the much higher complexities of biomolecules and their native environments Figure .Many biophysicists have focused on biology at the molecular level, but more and more of them are now studying processes at the cellular level. For example, the National Cancer Institute has funded 12 Physical Sciences-Oncology Centers, where physicists and cancer biologists are teamed up to uncover the physical principles that govern the emergence of cancer and its behavior at different scales.Tackling the challenging biological problems of the future will require ever closer integration of biology and physics in advancing new concepts and new experimental techniques. A life scientist with a solid training in physics will have unique strengths in this integration.Research at the intersection of the physical and life sciences is full of opportunities. These have been targeted by the National Institutes of Health and the National Science Foundation as well as by the Burroughs Wellcome Fund.The blurring of the disciplinary boundary is a good sign! That said, at present most people doing biological research have been trained in traditional departments. As a result, there are still cultural differences. For example, a biochemist may be interested in reducing a complex biological process such as protein synthesis into a sequence of binding events and chemical reactions, whereas a biophysicist may be interested in the rate constants of these events. So the biochemist identifies the constituent molecules and frames the biologically interesting questions, and the biophysicist then asks how do I explain the biochemical observations based on the structures and the interactions of the constituent molecules? Both are needed to discover how the biological process actually works.One clear trend is that biology is becoming more and more quantitative. This trend is well justified, since, for example, even a six-fold decrease in DNA-binding affinity of a mutant protein may be responsible for a change in phenotype Figure . After sAs knowledge at the molecular level expands, studies at the network and cellular levels will come into focus. Here again biophysics will make unique, important contributions, in terms of new concepts, new theories, and new experimental and computational tools.st century, dramatic progress can be anticipated in early diagnoses and treatments of cancer, Alzheimer's, and other diseases, in the development of biologically inspired materials, devices, and energy sources, and in understanding how the human brain works. In each of these endeavors, biophysics will be a central player.In the 21th century, one cannot help but wonder: does another revolution await biophysics?These directions paint a tantalizing future for biophysics. Still, given that modern physics came out of revolutionary (not evolutionary!) developments in the early 20What Mad Pursuit: A Personal View of Scientific Discovery. New York: Basic Books; 1988.Crick F: Research at the Intersection of the Physical and Life Sciences. Washington, DC: National Academies Press; 2010.National Research Council (US) Committee on Forefronts of Science at the Interface of the Physical and Life Sciences: Bridging the cultural gap. Nature 2002, 419:244-246.Knight J: The biological frontier of physics. Phys Today 2006, 59:38-43.Phillips R, Quake SR. http://physics.cancer.gov/Physical Sciences-Oncology Centers: http://grants.nih.gov/grants/guide/pa-files/PAR-10-142.htmlNew Biomedical Frontiers at the Interface of the Life and Physical Sciences: http://www.bwfund.org/pages/129/Career-Awards-at-the-Scientific-Interface/Career Awards at the Scientific Interface:


Drosophila melanogaster and Caenorhabditis elegans each carry a single representative of the Forkhead box O (FoxO) family of transcription factors, dFOXO and DAF-16, respectively. Both are required for lifespan extension by reduced insulin/Igf signaling, and their activation in key tissues can extend lifespan. Aging of these tissues may limit lifespan. Alternatively, FoxOs may promote longevity cell nonautonomously by signaling to themselves (FoxO to FoxO) or other factors (FoxO to other) in distal tissues. Here, we show that activation of dFOXO and DAF-16 in the gut/fat body does not require dfoxo/daf-16 elsewhere to extend lifespan. Rather, in Drosophila, activation of dFOXO in the gut/fat body or in neuroendocrine cells acts on other organs to promote healthy aging by signaling to other, as-yet-unidentified factors. Whereas FoxO-to-FoxO signaling appears to be required for metabolic homeostasis, our results pinpoint FoxO-to-other signaling as an important mechanism through which localized FoxO activity ameliorates aging.  •dfoxo/daf-16 is not required beyond gut/adipose tissue to counteract aging•dfoxo in other cell typesNeuroendcrine dFOXO extends lifespan independently of •dfoxo in other tissues to alter metabolismIn contrast, gut/adipose dFOXO requires  Drosophila foxo may signal to itself in distal tissues to alter fly metabolism.Activation of Forkhead box O (FoxO) transcription factors in key tissues can extend lifespan. FoxOs may promote longevity cell nonautonomously by signaling to themselves (FoxO-to-FoxO) or other factors (FoxO-to-other). The findings of Partridge and colleagues emphasize the importance of FoxO-to-other signaling in the antiaging effects of tissue-restricted induction of FoxO in the fly and worm. At the same time, the authors reveal that  Foxo3A are robustly associated with human longevity (Forkhead box O (FoxO) transcription factors (TFs) are involved in a plethora of cellular processes to regulate whole-organism physiology and are major determinants of animal lifespan . Activatongevity . Indeed,ongevity .Drosophila melanogaster, tissue-restricted activation of Drosophila foxo (dfoxo) is sufficient to extend lifespan  activates the transcription of Drosophila insulin-like peptide (dilp) 6 in the fat body, whereas in muscle dfoxo represses the activin ligand dowdle, and these endocrine signals have a distal effect on the median neurosecretory cells (mNSCs) in the brain, resulting in lowered DILP2 peptide in circulation . We used females, where the effects of dfoxo activation on aging are clearly observed  model (−4) and the presence of genomic dfoxo (p < 10−15) had a significant effect on lifespan, but their interaction did not (p = 0.95). The absence of a significant interaction confirms that the effect of RU486 did not differ between the lines and hence that the presence of dfoxo elsewhere in the body does not affect the extension of lifespan by induction of dfoxo in the gut and fat body. Thus, tissue entrainment through dfoxo-to-dfoxo signaling is not required for longevity.Flies lacking ifespans C, possibifespans , complicH) model . Both RUdfoxo acts cell autonomously to extend lifespan or that dfoxo in one tissue activates dfoxo-independent longevity-assurance mechanisms in other tissues. The latter would occur through dFOXO-to-other signaling, as has been observed for DAF-16  and its induction in dilp2GAL4>dfoxo flies  on lifespan but no evidence for a significant interaction between them , including dilp6  and dfoxo (p = 5.4 × 10−15) were both significant but that their interaction was not (p = 0.12). Hence, local action of dfoxo in the gut and fat body has a beneficial effect on the performance of a distal organ system. This could occur through systemic effects of healthy gut and fat body or through specific signaling events. In the latter case, its independence from dfoxo in the distal cells is again consistent with dfoxo-to-other signaling.Induction of climbers E. This edfoxo-to-other signaling, the gut/fat body dfoxo may regulate the expression of a secreted factor other than dilp6. To identify such a factor, we determined the whole-fly, genome-wide, transcriptional changes induced by RU486 in the S1106>dfoxo flies . The mature product of this gene is a YSY peptide of previously unknown function  but significant reduction in total protein content of S1106>dfoxo females and that this effect was blocked by deletion of dfoxo   .daf-16 from the intestinal-specific ges-1 promoter in otherwise wild-type worms and asked whether this effect required the presence of daf-16 in other tissues (daf-16-deficient (daf-16(mu86)) worms fed HT115 bacteria  and revealed a significant interaction of the two main effects . For both lifespan and protein content, the alteration of phenotype between OP50 and HTT15 is reminiscent of the lifespan effects of certain sensory mutants in C. elegans  on whole-organism aging  and has been kept in population cages maintaining its lifespan and fecundity at levels similar to freshly caught stocks. The lines were maintained, and all experiments performed, at 25°C with 60% humidity and 12 hr:12 hr light:dark cycle on sugar-yeast-agar (1SYA) food (All transgenes and the YA) food . ExperimYA) food . For furWorms were maintained at 20°C unless otherwise indicated. Prior to experiments, animals were maintained at the permissive temperature and grown for at least one generation in the presence of food to assure full viability. Lifespan assays were performed on HT115 bacteria carrying empty pL4440 vector, or OP50 bacteria, in the presence of 10 μM FUdR. Worms were placed on these plates at the L4 stage and scored as dead or alive every 2–3 days. For further details, see dfoxoΔ/Δ S1106>dfoxo as the dependent variable and the response in S1106>dfoxo (continuous) and gene set  as explanatory variables, testing for the significance of the interaction term.Analyses were performed in JMP (SAS) or R. Further details are given in