RET gene) have led to the development of multitarget inhibitors in different histological subgroups of patients. The correct molecular characterization of patients with thyroid cancer is thought to be a key aspect for the future clinical management of these patients.Despite that thyroid cancer accounts for over 90% of tumors that arise from the endocrine system, these tumors barely represent 2% of solid tumors in adults. Many entities are grouped under the general term of thyroid cancer, and they differ in histological features as well as molecular and clinical behavior. Thus, the prognosis for patients with thyroid cancer ranges from a survival rate of >97% at 5 years, in the case of differentiated thyroid tumors sensitive to radioactive iodine, to a 4-month median survival for anaplastic tumors. The high vascularity in these tumors and the important role that oncogenic mutations may have in the RAS/RAF/MEK pathway and oncogenicity (as suggested by activating mutations and rearrangements of the  Three different types of thyroid cancer have been defined according to their histological features: differentiated thyroid cancer (DTC) deriving from epithelial cells from the thyroid follicles; medullary thyroid cancer (MTC); anaplastic thyroid cancer (ATC). Approximately 90% of diagnosed thyroid cancers correspond to DTC, with papillary (PTC) histology being the most frequent (75%), followed by follicular (FTC) 10%), Hürthle cells (5%), and poorly differentiated carcinomas (1–6%) 0%, Hürth. OverallThyroid cancer is considered a nonfrequent entity because it represents only 1% of all solid tumors in adults. Women are three-times more likely to suffer this disease, and the incidence of DTC has increased 2.4-fold over the past 30 years ..6].Advances in understanding the molecular pathogenesis of different subtypes of thyroid cancer have, currently, made this field of research one of the busiest in the world of endocrine oncology. The application of discoveries in the laboratory to the patient (the “bench-to-bed” transfer) is one of the biggest challenges in translational research area, since these tumors can become a paradigm of therapy based on molecular features that govern the development and growth of tumors in each individual patient. This paper aims to summarize the key molecular determinants of each histological subtype of thyroid cancer and the data derived in recent years from clinical studies conducted with multitarget agents.DTC represents the vast majority of thyroid tumors. Treatment is based on thyroidectomy with or without lymphadenectomy regarding papillary or follicular histology, followed by radioiodine ablation in those patients at high risk of lymph node metastases and levothyroxine in doses that suppress TSH levels. The semiannual or annual monitoring using cervical ultrasound, measurement of thyroglobulin levels as a tumor marker, and whole-body iodine scan is appropriate for these patients.It is estimated that between 10 and 15% of patients undergoing DTC will have disease relapse. In 3 out of 4 cases, recurrence occurs at the cervical level in the thyroid bed or lymph node level. In these patients salvage surgery, radioiodine therapy and external radiotherapy is recommended in highly selected patients.Distant metastases occur in <10% of DTC patients, and only half of them are apparent at the time of diagnosis. The lungs are the main receptor organs of these tumor metastases (50%), followed by bone (25%). Initial treatment of patients with disseminated disease includes TSH-suppressive doses of levothyroxine plus radioiodine administered in conditions of high serum TSH levels in those patients with positive total body scan uptake, albeit complete remission is achieved in only 30% of subjects .A patient is considered resistant to the use of radioactive iodine when at least one tumor lesion is observed that does not show uptake of radioiodine, or when the lesion radiologically progresses in the first 12 months post-radioiodine administration, or when the patient has persistent disease following the administration of an accumulated dose of radioactive iodine of >600 mCi . The medRET gene expression and the abnormal activation of signaling pathways, RAS/RAF/MAPK. There is an increasing body of evidence showing that the role of PI3K/Akt/mTOR pathway may be crucial for tumor development and may become an attractive target in the future [In recent years, several different genetic events have been identified as being related to the genesis of the DTC. Further, a correlation has been reported between the manifestation of histological alterations of the disease and the presence of changes in the regulation of intracellular molecular pathways that confer a distinct clinical behavior. Two major molecular determinants that involve the same molecular pathway are responsible for the appearance of a PTC: the alteration of gene regulation of the e future .RET (rearranged during transfection) with the tyrosine kinase domain of other genes located in the same or other chromosomes results in the fusion oncogene RET/PTC that plays a key role in the pathogenesis of up to 30% of patients with PTC [RET proto-oncogene is activated by fusion of the RET TK domain with the 5′-terminal sequence of one of different heterologous genes via rearrangements that generate a series of chimeric-transforming oncogenes collectively described as RET/PTCs. The results of RET/PTC reorganization are abnormal proteins constitutively activated from RET kinase, independently of the binding to extra-cellular ligands, that activate multiple intracellular signaling pathways leading to clonal expansion and neoplastic transformation of the thyroid follicle cells. It is widely known that RET oncogene rearrangements plays a crucial role in radiation-associated papillary thyroid carcinogenesis. Other proto-oncogene rearrangements have been identified affecting to the NTRK1 gene in PTCs. The frequency of these rearrangements could be seen in up to 25% of patients with PTC. NTRK1 proto-oncogene rearrangements result in the formation of chimeric genes composed of the tyrosine kinase domain of NTRK1 fused to 5′ sequences of different genes . It is estimated that NTRK1 proto-oncogene is activated by rearrangement with a similar frequency in “spontaneous” and radiation-associated thyroid tumours. Moreover, NTRK1 proto-oncogene activating rearrangements play a role in the development of a minority of radiation-associated PTC but not in adenomas [The abnormal chromosome rearrangement of proto-oncogene with PTC . Their iadenomas .(PAX8) and peroxisome proliferator-activated receptor-γγ) sodium iodine and, consequently, a lower uptake of radioiodine by the metastatic lesions of tumors that harbor the mutation. This decreased expression of symport is mediated via the TGFβ autocrine pathway.Genetic alterations in the signaling pathway of RAS/RAF/MAPK constitute the most frequent genetic/molecular anomaly in patients with DTC. BRAF is a serine-threonine kinase that is key in the intracellular regulation of this cellular pathway. Activating mutations have been observed in the V600E codon of the RAS oncoprotein occur to a lesser extent (15%) in PTC, but their frequencies are increased in patients with FTC and less differentiated histologies. Mutations in any gene encoding for RAS  involve abnormal activation of mitogen-activated protein kinase (MAPK) and the PI3K/Akt/mTOR alternative parallel route [Point mutations in the gene encoding the el route , 33.BRAF, RAS and RET rearrangements are involved in >70% of PTC. However, these molecular alterations are mutually exclusive in most cases and are rarely expressed simultaneously within the same tumor. As such, they are independently able to promote PTC.Mutations in PI3CA, encoding for the phosphatidyl-inositol-tri-phosphate kinase (PI3K), play a role as initial triggers that activate the intracellular signaling pathway  receptor. The MET membrane receptor cooperates with VEGF receptor to promote angiogenesis, cell mobility, and survival of thyroid cells. Amplification occurs in 80% of papillary thyroid tumors, while its silencing in animal models induces tumor regression, reduction in tumor size, and the appearance of new distant metastases [Another molecular alteration frequently found in DTC is amplification of the gene encoding for the hepatocyte growth factor (tastases .α and -β) have been shown to be associated with an increased risk of distant metastases, an increased risk of disease recurrence, less protracted PFS, and increased presence of mutations in BRAF. In in vitro experimental models, blockade of the VEGF pathway has succeeded in delaying DTC growth [Angiogenesis is another potential area for the design of new molecular targets. Known receptors for VEGF  and the FGF (FGFR) and PDGF (PDGFR-C growth .β (n = 16) achieved a median PFS of 84 weeks versus 54 weeks for those who did not have this mutation [Sorafenib is a multitarget agent for several major molecular pathways involved in the development and progression of PTC, including the serine-threonine kinase BRAF and the tyrosine kinases from the membrane receptors VEGFR-2 and PDGFR-β . From a β n =  achievedmutation . In the mutation . In a momutation . Most admutation . Taken tRET/PTC and on RET mutations. Vandetanib has been the subject of report of the largest randomized clinical trial conducted to date in patients with DTC refractory to radioiodine. A total of 145 patients received vandetanib at doses of 300 mg/day or placebo. PFS, which was the primary endpoint, was significantly higher from the clinical as well as statistical point of view, in patients receiving the study drug (11 months) as compared to those receiving placebo (5.8 months) with a relative risk (hazard ratio) of 0.63 (95% CI: 0.43–0.92). However, the objective response rate was less than 5% in the vandetanib arm, and is difficult to explain. The most frequent adverse events found in the vandetanib arm were diarrhea (74%), asthenia (26%), fatigue (23%), nausea (25%), hypertension (34%), hyporexia (26%), and skin rash (25%). Updates from this study are expected in the near future [Vandetanib (ZD6474) is a low molecular weight inhibitor of tyrosine kinases, which acts mainly against VEGFR-2, VEGFR-3, and RET and, in higher concentrations, on the epidermal growth factor receptor (EGFR) . Vandetar future .β, and c-KIT, in the subnanomolar range  (Lenvatinib (E7080) is a potent inhibitor in the nanomolar range of VEGFR-1 and -2 as well as PDGFR-(FGFR-1) . In an iMotesanib (AMG706) is an oral inhibitor of multiple kinases, including VEGFR-1, -2, and -3 as well as the wild and mutant forms of the membrane receptor RET. In one of the initial phase I studies, focusing on recommended doses and major toxicities, motesanib was shown to achieve objective radiographic partial response in 3 of the 5 patients with thyroid cancer. This activity was not expected in these patients and led on to a prospective phase II study in 93 patients with DTC being carried out. One in every 3 patients remained on treatment for 48 weeks after commencement. The most common adverse events found at any grade were diarrhea (59%), hypertension (56%), fatigue (46%), and weight loss (40%). The radiological response rate assessed by independent reviewers was 14%, and up to 35% of patients benefited from a stabilization of their disease beyond 6 months. The median PFS was 9.3 months and, although the drug does not inhibit BRAF, the investigators observed that patients carrying a mutation in this pathway were less likely to progress if they received motesanib than patients without the mutation This finding suggested a greater involvement of angiogenesis in patients with the BRAF mutation .RET/PTC and BRAF that is, its activity in these tumors is due mainly to its antiangiogenic effect rather than any intrinsic antitumor effect  and the European Medicine Agency (EMA). Following the failure of initial treatment by thyroidectomy and cervical lymphadenectomy, patients have received only symptomatic care measures. Therefore, this tumor has represented, and currently represents, a niche for the development of new active drugs, especially considering that between 35 and 50% of patients diagnosed with metastatic disease are likely to be diagnosed as having lympho-ganglionar metastases, and up to 15% of patients will develop distant metastases after diagnosis. Half of the patients diagnosed with MTC will ultimately present distant metastases during the clinical evolution of the disease.MTC is considered a neuroendocrine tumor caused by the uncontrolled proliferation of para-follicular C cells producing calcitonin derived from the neural crest. It accounts for between 4 and 10% of thyroid cancers. In up to 30% of cases, the CMT is associated with an inherited syndrome and multiple endocrine neoplasia type 2 (MEN 2A or 2B) or with familial MTC (FMTC). Most hereditary MTC occur within MEN 2A that is often associated with pheochromocytoma or hyperparathyroidism .Sporadic MTC is diagnosed in the 5th or 6th decade of life, while the hereditary versions of the disease can be diagnosed in 5-year-old children. Conventional therapy with chemotherapy has not proven to be particularly useful in patients with metastatic MTC. The most commonly used chemotherapy regimen has traditionally been that of doxorubicin monotherapy. This treatment achieves a clinical benefit, including the partial responses plus stable disease of 6 months, in about 21% of patients, with 79% of patients progressing within 5 months of treatment . RegimenRET gene. These mutations are considered “gain of function” type. The presence of this mutation is also associated with a high probability of lympho-ganglionary metastases, recurrent/persistent disease, and decreased survival. By contrast, germinal mutations at different loci of the RET gene are present in virtually all hereditary syndromes associated with MTC. There is a strong genotype-phenotype correlation between the location of the mutation in RET and the associated MEN2 type. This enables the patients to be classified into different risk groups according to the mutation they carry and as such, to be referred (or not) for prophylactic thyroidectomy [Approximately 50% of patients with sporadic MTC have a somatic mutation at codon 918 of the idectomy , 37.RET by any of the several mutations affecting the coding part of the gene can trigger activation of multiple intracellular signaling pathways such as RAS/RAF/MAPK, PI3K/Akt/mTOR, or Rac/JNK.The abnormal activation of P < 0.0001) after 24 months of followup. The objective radiological response rate was also higher in the vandetanib treatment arm . Patients receiving vandetanib showed a statistically significant delay in time-to-pain worsening compared with patients receiving placebo . Patients receiving this agent also showed significant decreases in serum concentrations of calcitonin and carcinoembryonic antigen (CEA). Adverse effects found were asthenia, weight loss, diarrhea, mucositis, hand-foot syndrome, and hypertension, most of them of grades 1 and 2 severity. Very rarely, some grade 3/4 events were observed. Interestingly, those patients harboring M918T mutation in RET had higher objective response rate (54.5%) than those with RET mutation unknown (34.1%) or negative (0%) [Vandetanib is the first drug approved in the US by the FDA and in Europe by the EMA for treatment of MTC in the last 30 years. The approval was as a consequence of the data reported in the ZETA study which was a phase III, double-blind trial with 2 : 1 randomization of patients to receive vandetanib at doses of 300 mg/day versus placebo. Between December 2006 and November 2007, a total of 331 patients with sporadic or inherited MTC were enrolled. The median PFS in the placebo arm was 19.3 months while the vandetanib treatment arm has not yet been concluded  .RET/PTC rearrangements (RET (in both sporadic and hereditary cases) and tumor response to cabozantinib. A phase III study comparing cabozantinib and placebo in patients with metastatic MTC is currently recruiting patients (NCT00215605).Cabozantinib is an oral inhibitor that, in addition to acting against VEGFR-1 and -2, and c-KIT as are most of the drugs discussed above, is able to inhibit c-MET which, as also discussed above, is amplified in a large proportion of patients with DTC and MTC. It also acts against RET and against ngements . In the in vitro activity of sorafenib in MTC cell lines has stimulated investigators to design a phase II study in patients with hereditary (n = 5) and sporadic (n = 16) MTC. Analysis of the M918T mutation of the RET gene was performed in 12 of these patients, with 8 showing positive for the mutation. Preliminary results of the activity of sorafenib as a single agent in patients with MTC show that one (6%) patient achieved a partial positive response and 10 (62%) of 16 patients with sporadic MTC obtained a stabilization of their disease for >6 months. Serum levels of calcitonin and CEA decreased by over 62% and 44%, respectively [The observed ectively .RET mutation was present. There were no significant differences between the effectiveness of motesanib in patients in relation to the presence or absence of the RET mutation [In a multicentered, international, open-label phase II trial, motesanib demonstrated that it was able to induce a clinical benefit in 50% of 91 patients with locally advanced or metastatic MTC. The PFS was 12 months. Most study patients (84%) corresponded to sporadic MTC in 72% of whom the mutation .RET mutation and treated with sunitinib, the probability of remaining progression-free at one year was 88% (95% CI: 43%–98%).In the study by Carr et al.  that meaATC contributes less than 2% of all thyroid-derived tumors but is responsible for 14 to 39% of deaths from thyroid cancer. With a frequency 5-fold higher in men than in women, the median survival range depends on status between 3 and 9 months after diagnosis. Approximately 50% of patients diagnosed with ATC already have metastases at the time of diagnosis, and another 25% will eventually develop metastases. Treatment options are surgery, palliative radiotherapy, and chemotherapy. ATC tumors are considered chemo resistant. Doxorubicin in monotherapy achieves positive radiological response rates in <20% of patients, and this response is rarely protracted. Other platinum-based combinations such as taxanes, gemcitabine or vinorelbine can achieve up to 53% radiological response but does not have much impact on the overall survival of patients . p53 gene in up to 70% of patients. Genomic aberrations of ATC also include RAS and β-catenin mutations in more than 50% of cases, and mutations in the oncogene BRAF have only been observed in around 20% of patients. Different genomic profiles between DTC or MTC and ATC probably are related with the higher aggressiveness of these tumors. Similarly, histological findings indicate high vascularity with overexpression of VEGF, the levels of which appear to correlate with stage and tumor size, lymph node, and distant metastases. Finally, EGFR is overexpressed in most of the patients with ATC. The implication of overexpression of EGFR on the clinical and biological behavior of these tumors is not known [Most frequent mutations observed in ATC affectot known .In a phase II study conducted with single-agent axitinib in patients with thyroid cancer of all histology subtypes, there was an objective radiological response in approximately 50% patients with ATC who entered the study .In the only study performed specifically in patients with ATC, 16 patients were treated with sorafenib alone. The rate of control of the disease was 40%, although time-to-progression barely reached 1.5 months. Of the 15 evaluable patients, 2 (13%) showed an objective radiological response that was maintained for a median of 5.3 months. The most frequently observed grade 3 and 4 adverse events were lymphopenia (31%), skin rash (12%), weight loss (12%), and chest pain (12%) .CA4P is a vascular-disrupting agent, also called fosbretabulin tromethamine or combretastatin A4 phosphate. Unlike other antiangiogenesis agents that block tumor blood vessel formation, CA4P prevents blood flow in existing vessels. This drug has shown some activity as a single agent in patients with metastatic ATC who have not responded to at least one prior systemic therapy but in whom, on rare occasions, positive responses were observed and objective radiological PFS of 3 months was achieved. The final results in overall survival of the largest randomized trial conducted in patients with metastatic ATC were presented at American Society of Clinical Oncology (ASCO) annual meeting in 2011. A total of 80 of the planned 180 patients with ATC were recruited and randomized to receive carboplatin + paclitaxel with or without associated CA4P in a 2 : 1 randomization. The median overall survival was higher in the CA4P arm but did not reach statistical significance ; 27% of patients were alive at 1 year in the arm that received the experimental therapy compared to 9% in the standard treatment arm. The planned total patient sample could not be reached due to low recruitment .Thyroid cancers are one of the greatest challenges for those treating advanced tumors. Thyroid cancer offers extensive opportunities to work in interdisciplinary teams that include not only oncologists and endocrinologists but also specialists in pathology, nuclear medicine, surgery, otolaryngology, radiology, clinical laboratory, and radiotherapy. Coordinated administration  of the different therapeutic options for these patients make collaborative and efficient functioning of surgical and medical professionals more important than ever. The opportunity that presents itself is to apply the various treatments becoming available based on the genotypic characteristics of patients. Moreover, we have to consider the possibility of administering tyrosine kinase inhibitors for a long time with caution. We assume that these drugs are quite specific cytostatic and not cytotoxic agents, therefore, there is a need to give these agents until progression of the disease or toxicity. The concept of long-term treatment management of these new agents to patients in a childbearing age, with known cardiovascular toxicity, and perhaps many different side effects that could arise in the long-time followup, makes the monitoring of patients receiving inhibitors tyrosine kinase a must to be done very closely.Maximizing clinical outcomes of tyrosine kinase therapy requires clear, effective communication, anticipation of side effects, and early intervention to avoid treatment delays and dose-limiting toxicities. There is a direct correlation between the plasmatic levels of the tyrosine kinase inhibitors with the activity of the drug, therefore, we would need to maintain as far as we can the highest dose of the targeted agent we consider is the most appropriate to our patients. In this sense, the experience in the management of tyrosine kinase inhibitors is crucial and probably the development of therapy management guidelines would be necessary in the next years.To summarize, there is a need for a greater understanding of the molecular basis of the disease by the various health professionals charged with the care of these patient that could lead to a better optimization of treatment options in the advanced setting.


The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine. Approximately 2 weeks later, antibodies are formed against the immunodominant gp120 region and these are also non-neutralizing. Neutralizing antibodies appear for the first time around 3 months post-seroconversion. These antibodies are mostly strain-specific and cannot neutralize heterologous viruses. Autologous neutralizing antibodies can rapidly select for escape variants of HIV-1 and do not seem to have a protective effect against progression to AIDS . MAb b12 was the first broadly neutralizing antibody and obtained from the bone marrow of an asymptomatic HIV-infected man  had been isolated that could potently neutralize a majority of primary HIV-1 strains -based assay that requires primary virus isolates and a single-round infection assay using a reporter cell line, such as TZM-bl, that requires pseudoviruses produced by 293T cells. The neutralization sensitivity of viruses does vary between these two assays , explaining at least in part the absent effect of potent CrNA on the clinical course of infection  infection in macaque models .The screening for patients with CrNA in serum has intensified and so have the methods to isolate the broadly neutralizing antibodies from these patients. Several high-throughput techniques have been implemented  in which all areas except the CD4-binding site of the virus were altered or masked, making those unfavorable for recognition . With thIn another large scale study, the approach of non-specific high-throughput memory B cell culture was taken , 2011. IThe non-specific high-throughput memory B cell culture system was performed with the B cells of four elite neutralizers, which resulted in the isolation of a new class of broadly neutralizing antibodies, termed the PGT series. These antibodies are directed against mannose structures and appear to be 10- to 100-fold more potent than other antibodies . CrystalIn order to find even more potentially conserved regions within the virus envelope, the undertaking of extensive epitope mapping of broadly neutralizing activity in sera of patients with CrNA has revealed various antibody specificities. There appears to be a difference in the number of antibody specificities between individuals with CrNA that is not so potent, and the so-called elite neutralizers . PreviouA better understanding of how CrNA develops during the natural course of infection in some individuals will give valuable clues for vaccine design. CrNA is positively correlated with viral load, especially during the primary stage of infection . This moAs intact CD4 helper cells are necessary for class switching and multiple rounds of somatic hypermutation, this seems counterintuitive. Interestingly, in a lymphocytic choriomeningitis virus (LCMV) mouse model, lower CD4 T cell counts resulted in less HIV-1-induced polyclonal B cell activation, which in turn boosted virus-specific antibodies . MoreoveAlthough CrNA was associated with overall lower CD4 T cell levels prior to infection and during acute infection, there was a positive association with the fraction of CD4 T cells expressing high levels of PD-1 . PD-1 isIn a recent genome wide association study (GWAS) on the development of CrNA, we have found several single nucleotide polymorphisms (SNPs) in the gene region of MHC class I chain-related protein A (MICA) on chromosome 6 that were associated with the development of CrNA at 3 years post-serconversion . The association of these SNPs with CrNA was strong but did not reach genome wide significance after correction for multiple testing and confirmation of this finding is required. Interestingly, previous studies have shown strong associations between SNPs on chromosome 6 and viral load set point  which asOther covariates such as HLA class II alleles, risk group, history of using ARVs, race, ethnicity, gender, and age were not associated with the development of CrNA (Obviously, characteristics of the HIV-1 variant that is transmitted can also have an impact on the induction of CrNA. In order to find viral genetic signatures associated with CrNA, The known broadly neutralizing antibodies have unique characteristics, which also complicates the induction of these antibodies. A majority of broadly neutralizing antibodies have a long CDR3 in their immunoglobulin heavy chains. Two such antibodies, PG9 and PG16, have an exceptionally long CDR3, which forms a type of “hammerhead” with which it interacts with HIV-1 Env. Interestingly, it appears that B cells with long CDR3 regions undergo negative selection at the naïve B cells stage . PolyreaHowever, polyreactivity is no prerequisite for antibodies to neutralize HIV, as b12, 2G12, and VRC01 have very little polyreactivity . Also thIn vitro reverted unmutated ancestor antibodies from HIV-1-specific broadly neutralizing antibodies have shown little or no reactivity with the epitopes of their progeny broadly neutralizing antibodies (Another unique characteristic of broadly neutralizing antibodies is the high degree of somatic mutations within the CDR3 region as compared to antibodies in other infections which typically accumulate only 5–15% changes in this region during affinity maturation . The degtibodies . This intibodies . This sutibodies  which mitibodies  as immunin vivo. In one recent study, in order to program the B cell response, the natural evolution within HIV-infected individuals was mimicked in an animal model by sequential immunization with different Env proteins. Compared to animals that received a cocktail of Envs and only one clonal Env, the sequential immunizations resulted in the highest heterologous neutralization titers (Current strategies to unravel the pathway of affinity maturation that leads to the formation of broadly neutralizing antibodies includes 454 pyrosequencing of B cells of HIV-1 individuals to identify their antibody repertoire or antibodyome . In the n titers , suggestRecently, we have shown that HIV-1 has become more resistant to antibody neutralization over the course of the epidemic . This efDespite the increased neutralization resistance, currently circulating HIV-1 strains are sensitive to neutralization by multiple of the newly identified broadly neutralizing antibodies at low concentrations, in particular to Mabs PG9, PG16, VRC01 as well as PGT121 and the HAADS 3BNC117, 3BNC60, and NIH45–46 , contempin vitro. However, in vivo, naturally developed broadly neutralizing antibodies have no effect on disease progression as the virus easily escapes. Therefore, an effectively neutralizing humoral immune response needs to be in place prior to encountering HIV in order to prevent infection. As soon as infection is established, the humoral immune response is not likely to play a role in controlling viral replication.Many challenges remain in the development of an immunogen that is capable of eliciting broadly neutralizing antibodies. Besides the structural constraints on the immunogen, there are immunological barriers that need to be tackled, including the long affinity maturation that B cells seem to require before they can produce broadly neutralizing antibodies, and the risk of eliciting antibodies that have potential self-reactivity. If polyreactivity is a key feature of broadly neutralizing antibodies, then the chances of eliciting them with an envelope-based immunogen that is presented in the absence of a membrane context may be complicated or even impossible. The identification of even more broad and potently neutralizing antibodies against not previously identified epitopes will hopefully bring us ever more closer to the design of an immunogen that can overcome these challenges. Most circulating HIV strains are sensitive to the majority of broadly neutralizing antibodies known to date An HIV vaccine is not available but huge progress has been made in understanding what is required to neutralize HIV-1 and how this type of immunity may be elicited. Understanding the problem may pave the way to its solution.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The results revealed that infants distinguish between emotions during an early perceptual processing stage, whereas adults recognize and evaluate the meaning of emotions during later semantic processing stages. While infants do discriminate between emotions, only in adults was genetic variation associated with neurophysiological differences in how positive and negative emotions are processed in the brain. This suggests that genetic association with neurocognitive functions emerges during development, emphasizing the role that variation in serotonin plays in the maturation of brain systems involved in emotion recognition.The ability to effectively respond to emotional information carried in the human voice plays a pivotal role for social interactions. We examined how genetic factors, especially the serotonin transporter genetic variation  The human voice is the most important sound in our environment. Our ability to analyze and effectively respond to information carried in the voice plays a pivotal role for social functioning. The voice not only carries speech information but it can also be seen as an “auditory face” that enables us to recognize individuals and their emotional states SLC6A4/5-HTTLPR) on the processing of emotional information conveyed by visual stimuli 5-HTTLPR) in the regulatory regions of the serotonin transporter gene has a short (s) and a long (l) allele  that alters promoter activity: the s variant produces significantly less serotonin transporter mRNA and protein than the l variant, resulting in higher concentrations of serotonin in the synaptic cleft. Individuals carrying the s allele appear to have increased anxious temperament, resulting in an elevated risk to develop depression One successful approach to studying variability in emotional sensitivity has been to examine in adults how genetic variation within specific neurotransmitter systems impacts differences in the brain responses to emotional stimuli 5-HTTLPRAnother critical aspect to consider when assessing the association between genetic variation in the serotonergic system and emotional brain processes is the role that genetic variation may play in development 5-HTTLPR on the processing of emotional tone of voice  in adults and infants by using event-related brain potentials (ERPs). We had adults and infants listen to neutral, positive (happy), and negative (angry) voices. We measured ERPs for two reasons, first, because it allows for the precise measurement of the timing of brain processes and second, because ERPs are the method most readily used to study and compare brain processes in infants and adults 5-HTTLPR we therefore tested between three possibilities: genetic variation is associated with differences (i) in early perceptual processing in infants, (ii) in later cognitive processing in adults, and (iii) in both early perceptual processing in infants and later cognitive processing in adults.In the present study, we addressed the question of whether and how genetic variation in the serotonin transporter is associated with differences in processing emotional information carried by the human voice across development. We thus examined the effects of M = 25 years, Range = 19 to 32 years). The adult participants were paid for their participation. The adult participants had no prior history of psychiatric illness. The infant sample consisted of 48 7-month-old infants . An additional five 7-month-olds were tested but not included in the final sample due to fussiness. All infants were born full-term (37–42 weeks gestation) with normal birthweight (>2500 g). All adult participants and parents of the infants gave written informed consent before the study. This study and the written consent procedure were approved by the Ethics committee of Leipzig University Medical School.The adult sample consisted of 57 participants , (b) mean fundamental frequency in Hz , and (c) mean intensity in dB . These three parameters were then used to compare acoustic differences across the three emotions. Means were compared using t- tests, which revealed that angry stimuli were significantly longer in their duration than happy stimuli , and happy stimuli were longer than neutral stimuli . Furthermore, mean fundamental frequency was significantly higher for happy stimuli than for angry  and neutral  stimuli, whereas neutral and angry stimuli did not differ in their fundamental frequency . The three stimuli did not differ with respect to their mean intensity.The stimulus material consisted of previously used and validated material consisting of 74 semantically neutral German verbs The adult participants sat in a dimly-lit, sound-attenuated, and electrically-shielded room facing a computer screen. Adults were instructed to sit still and watch the screen but no task was given in order to ensure that the data could be compared between adults and infants. Infants were seated on their mother’s lap in a dimly lit, sound-attenuated, and electrically-shielded room. Mothers were listening to music via headphones during the experimental session so that they could not hear the acoustic stimuli presented to their infant. The session continued until the infant had attended to the maximum number of trials (222) or got tired of the experiment. All adult participants listened to the maximum number of trials. The experimental session consisted of consecutive presentations of 74 words from each emotional prosody category . Stimuli from the different emotional categories were randomly distributed over the session with no more than two stimuli of the same category occurring consecutively. The inter-stimulus interval varied randomly between 1500 and 2000 ms. During the presentation of the acoustic stimuli, an abstract screensaver without social stimuli was presented to adults and infants on a computer screen placed at a 60 cm distance in order to reduce eye movement artefacts.SD = 8.8) for happy and 63.6 (SD = 9.3) for angry voices. The mean number of artifact-free trials in infants was 36.6 (SD = 15.3) for happy and 36.1 (SD = 15.1) for angry voices. Mean ampltiude ERP effects were analyzed by repeated measures ANOVAs with within-subject factors of emotion , hemisphere , and the between-subjects factor 5-HTTLPR genotype . In adults and infants time windows were chosen by visual inspection around the peaks of the major ERP components (N100-P200-N300/400 complex) at frontal and central electrodes commonly investigated in auditory processing 5-HTTLPR variation using visual stimuli in adults In adults the EEG was recorded from 67 Ag/AgCl electrodes, referenced to the left mastoid. In infants the EEG was recorded with Ag-AgCl electrodes from 19 scalp locations of the 10–20 system, referenced to Cz. Horizontal and Vertical EOGs were recorded bipolarly. Sampling rate was at 250 Hz. EEG data was re-referenced to the algebraic mean of the left and the right mastoid electrodes, and band-pass filtered with 0.3 to 20 Hz (1501 points). Data were baseline corrected by subtracting the average voltage in the 200 ms baseline period from each post-stimulus data point. For elimination of artifacts caused by eye and body movements, EEG data for the whole trial were rejected off-line whenever the standard deviation within a 200-ms gliding window exceeded 80 µV for the vertical or horizontal electro-oculogram and 50 µV at any electrode. The mean number of artifact-free trials in adults was 63.4 . Swabs were placed in a lysis buffer and DNA was extracted as described previously 5-HTTLPR indel polymorphism was carried out in an MJ Research Thermal Cycler . Each 25 µl PCR reaction consisted of an initial DNA denaturation and Taq activation step at 95° for 15 min followed by 34 repeated cycles of denaturation at 95° for 30 sec, an annealing step for 30 sec min at 66° and extension at 72° for 45 sec. The reactions included 20 ng of template DNA, 1× PCR buffer mix 1 , 500 µM dNTPs , 400 nM of each primer  and 1.25 unit of Extensor Long PCR Enzyme . Primers  were those described previously . Following amplification products were electrophoresed through a 2% SeaKem LE gel  for 1.5 hours at 120V and stained with ethidium bromide, with a 512 bp product corresponding to the long (l) allele and a 469 bp product corresponding to the short (s) allele. One limitation of the current approach is that we did not include in our genotyping protocol two more recently described variants that have been identified in the long version of 5-HTTLPR (LG and LA), with the LG allele showing similar levels of serotonin transporter expression as the short allele . However, individuals carrying the LG variant of the long allele are very rare (approximately 10%) and it is thus unlikely that this impacted the current results. The distribution of genotypes at the 5-HTTLPR polymorphism was as follows: long/long (15), short/long (26), short/short (16) in the adult sample, and long/long (19), short/long (21), short/short (8) in the infant sample. Genotype frequencies did not deviate significantly from Hardy-Weinberg expectations. In our adult sample, in order to examine potential variation in personality traits associated with genotypic variation, we administered the NEO-Five Factor Inventory (NEO-FFI) PCR-amplification for genotyping the rs4795541 Fp<0.001, partial 2η = 0.361) angry voices elicited a more negative-going waveform when compared to happy voices. This difference had its maximum at anterior  sites. There were no ERP differences between emotions during earlier time windows. Our analysis further revealed an interaction between the factors emotion and 5-HTTLPR genotype  during that time window, suggesting that genetic variation at this locus was associated with differences in the ERP responses to emotional tone of voice. As shown in 5-HTTLPR showed a significantly decreased differentiation between happy and angry tone of voice (Fp = 0.006): posthoc tests showed that the difference between the ERP response to happy and angry voices was signigicantly greater in the long/long genotype group than in the short/long genotype group (p = 0.017) and in the short/short genotype group (p = 0.002). More specifically, for happy tone of voice the number of short alleles was associated with a linear decrease in the amplitude of this ERP component . Moreover, for angry tone of voice there was a non-linear (inverted u-shaped) relation between the number of short alleles and the amplitude of this ERP component in response to an angry tone of voice  and individuals homozygous for the long allele  showed a more negative-going ERP response than the heterozygous individuals . However, posthoc comparisons revealed no statistically significant differences between genotype groups with respect to the ERP response to angry voices. Importantly, there were no differences in personality traits such as neuroticism or extraversion as measured by the NEO-FFI questionnaire associated with genetic variation of 5-HTTLPR. This indicates that the genetic association effects with the ERP data oberved in adults are unlikely to be related to general differences in personality.As shown in nent see , statistoice see . IndividFp = 0.039, partial 2η = 0.088), that is, earlier than for adults. This difference had its maximum at anterior electrode sites. For this time window there was no interaction between the factors emotion and 5-HTTLPR genotype  on the processing of emotional voice processing in adults and infants. The results revealed that in adults 5-HTTLPR variation was associated with differences in the brain responses to emotional tone of voice. This is the first study that goes beyond facial expressions and visually presented emotionally valenced material In the current study, we examined the effects of serotonin transporter genetic variation 5-HTTLPR variation was associated with differences in the ERP responses to emotional tone of voice over anterior brain regions  starting around 400 ms after voice onset (N400). This late ERP component is thought to reflect evaluative (cognitive) processes related to the recognition of emotion conveyed through the voice Our adult findings show that in adults 5-HTTLPR in adults. Our results revealed that carriers of the short allele of 5-HTTLPR showed a decreased differentiation in the ERP responses elicited by happy and angry tone of voice, indicating that genetic variation impacts the way in which positive and negative emotional information in the voice is differentiated and evaluated. Specifically, the number of short alleles was associated with a linear decrease (more negative-going) in the amplitude of this ERP component in response to a happy tone of voice, suggesting that the facilitatory effects of processing positive (happy) voices were reduced for individuals carrying the short allele and facilitation was strongest for individuals homozygous for the long allele. Furthermore, there was a non-linear association (inverted u-shape) between the number of short alleles and the amplitude of this ERP component in response to angry voices, with individuals homozygous for either the short or the long allele showing the most negative-going amplitudes, suggesting greatest cognitive effort in the homozygous groups when processing angry voices. Taken together, this pattern of findings is in line with recent behavioral work showing that while affectively positive visual stimuli had the most facilitating effects on selective visual attention, affectively negative stimuli had the most slowing effects on selective attention in individuals homozygous for the long allele 5-HTTLPR is critically associated with the way in which positive and negative emotions differentially bias information processing. Such differential sensitivity to emotional information in individuals is thought to have far-reaching effects on daily emotional experiences and general well-being While angry voices when compared to happy voices had this general effect, there were critical differences in the ERP responses associated with variation in 5-HTTLPR with emotional voice processing across development, we had tested between three hypotheses: genetic variation is associated with differences (i) in early perceptual processing in infants, (ii) in later cognitive processing in adults, and (iii) in both early perceptual processing in infants and later cognitive processing in adults. Our findings showed that 7-month-old infants’ brain responses indicated that they can discriminate between emotions carried by the human voice as reflected in the early perceptual ERP effect. However, no late cognitive N400-like effect as a function of emotional valence could be observed for the infants. This was expected on the basis of prior language work which revealed no semantic N400 effect before the second year of life With regard to the association of genetic variation in 5-HTTLPR was associated with differences in infants’ brain responses to facial expressions 5-HTTLPR while this is not the case for emotional voice processing. This might have to do with the fact that emotional facial expressions and other nonverbal means of emotional communication provide more powerful and effective means of communicating emotions from an evolutionary perspective than emotional tone of voice that needs to be extracted from speech 5-HTTLPR is associated with differences in the frequency of producing emotional expressions in daily life, in particular positive expressions such as smiling 5-HTTLPR contributes to differences in emotional voice processing.For our interpretion of the differences between infants and adults in the genetic association oberved in emotional voice processing, it is important to note that prior work with the same group of infants at the same age showed that genetic variation in In line with our predictions based on prior work 5-HTTLPR variation and differences in emotional voice processing emerges during development and is not present in infancy raises interesting hypotheses about how genetic variation may bias specific brain processes during development and thereby give rise to individual differences that ultimately contribute to complex phenotypes such as temperament and personality.In conclusion, to our knowledge this is the first study that systematically compared how genetic variation is associated with infants’ and adults’ brain responses to emotional information carried in the voice. Taking such a genetic imaging approach has been shown to be of great value for the understanding of individual differences in adults, and studying the association of genetic variation with brain responses as intermediate phenotypes, or so-called endophenotypes, has been argued to be a more powerful approach than studying gene effects on behavior  . Using this approach to compare adults and infants in the current study has revealed novel insights into our understanding of emotional voice processing by adding a developmental dimension to the complex picture of how genetic variation may affect human emotional sensitivity. The finding that the association between


PRKCI gene showed an association with risk of prostate cancer . We confirmed this in a meta-analysis using as replication set the data from the second phase of our study jointly with the first phase of the Cancer Genetic Markers of Susceptibility (CGEMS) project. In conclusion, we found an association with prostate cancer risk for two SNPs belonging to PRKCI, a gene which is frequently overexpressed in various neoplasms, including prostate cancer.The mTOR  signal transduction pathway integrates various signals, regulating ribosome biogenesis and protein synthesis as a function of available energy and amino acids, and assuring an appropriate coupling of cellular proliferation with increases in cell size. In addition, recent evidence has pointed to an interplay between the mTOR and p53 pathways. We investigated the genetic variability of 67 key genes in the mTOR pathway and in genes of the p53 pathway which interact with mTOR. We tested the association of 1,084 tagging SNPs with prostate cancer risk in a study of 815 prostate cancer cases and 1,266 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC). We chose the SNPs (n = 11) with the strongest association with risk (p<0.01) and sought to replicate their association in an additional series of 838 prostate cancer cases and 943 controls from EPIC. In the joint analysis of first and second phase two SNPs of the  Within the prostate tissue, tumor-promoting effects of endogenous hormones and growth factors are thought to be associated with the stimulation of cellular growth and mitosis, and inhibition of apoptosis. In addition to signaling by IGF-I , the growth and proliferation of cells are co-determined by amounts of energy and essential amino acids available to the cell TSC1) and 2 (TSC2) genes), ras-homologue enriched in brain (Rheb), regulatory associated protein with mTOR (raptor), and mammalian target of rapamycin (mTOR). mTOR activation in turn leads to phosphorylation of downstream elements that directly control ribosome biogenesis and ribosomal mRNA translation for protein synthesisRecent studies have showed that the mTOR  signal transduction pathway integrates these various signals, regulating ribosome biogenesis and protein synthesis as a function of available energy and amino acids, and assuring an appropriate coupling of cellular proliferation with increases in cell size PI3K, AKT1) and tumor suppressor genes . These genes are often mutated or aberrantly expressed in human malignancies, including prostate tumorsThis pathway includes several established proto-oncogenes  We hypothesized that genes in the mTOR pathway and genes of the p53 pathway that directly relate to mTOR may be centrally implicated in prostate carcinogenesis, and that polymorphic alleles in these genes could modify their expression or activity, thus conferring altered prostate cancer susceptibility. SNPs in genes belonging to the mTOR pathway have already been studied in relation to cancer risk, with some promising results In this report we investigated the genetic variability of 67 key genes in the above mentioned pathways. We tested the association of 1,084 tagging SNPs with prostate cancer risk in a case-control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC). To our knowledge this is the first report on polymorphisms of these genes and prostate cancer risk.Summary characteristics of the study populations are shown in −5) and were thus not analyzed further. Four SNPs were monomorphic in this population. This left a total of 1,084 SNPs  in the 67 candidate genes to be analyzed.We had 1,163 SNPs on our GoldenGate array, of which 30 were included as quality controls and 1,133 were in the candidate gene regions of interest in this study. Thirty-four SNPs were dropped because they had a call rate lower than 75%, which is usually indicative of poor genotyping quality. Eleven SNPs  showed strong departure from Hardy-Weinberg Equilibrium .Thirty SNPs were included, which had previously been genotyped on the same samples in the context of a different study. The concordance of the new genotypes with the old genotypes was 100%.We initially included 2,099 samples, and after removing subjects samples with a call rate lower than 75% (n = 39), we had a dataset including 815 prostate cancer cases and 1,239 controls. The incidence density sampling led to duplicate selection of 27 controls so that 1,266 controls were included in the conditional analyses.Random duplicate samples (∼5%) were also included and concordance of their genotypes was 100.0%.trend<0.01 or p2df<0.01) . Supplementary Eleven SNPs were significantly associated with prostate cancer risk, at a threshold of p<0.01 .We genotyped the eleven SNPs from the first phase in an additional set of 838 prostate cancer cases and 943 matched controls. In the second phase SNP rs546950 in the PRKCI SNPs showed an association with risk . Results for the first phase, the replication set and for the joint analysis are shown in When we analyzed jointly the results from the two sample sets, both eff values for each candidate gene separately and for the whole study  were observed between any of the polymorphisms genotyped and overall prostate cancer risk.We calculated MPRKCI were also genotyped in the context of the Cancer Genetic Markers of Susceptibility (CGEMS) project (http://cgems.cancer.gov/), one of the first genome-wide association studies on prostate cancer susceptibility. The associations observed in the first phase of CGEMS  were similar to those observed in the present report. In a meta-analysis using the unconditional OR-estimate from the data of the second phase of our study jointly with results from CGEMS, the two SNPs showed very similar results as those obtained with the EPIC data alone . A meta-analysis performed considering the joint data of the first and second phase of our study with results from CGEMS showed essentially the same results .The two SNPs in We analyzed associations of SNPs with prostate cancer risk by grouping cases according to disease aggressiveness, but we did not observe statistically significant (p<0.05) heterogeneity between strata. Results for the eleven SNPs that were genotyped on the complete dataset are shown in supplementary The mTOR pathway is implicated in tumor development, and analogues of rapamycin – a natural antibiotic that specifically interferes with mTOR action  – are showing great promise as potential therapeutic agents for treating certain types of solid tumors PRKCI gene, rs546950 and rs4955720, with a decreased risk of prostate cancer. The first SNP showed an association at the first screening, in a replication set and in a meta-analysis of our second phase with data from CGEMS, while rs4955720 showed an association only in the screening set and in the meta-analysis. Since the two SNPs were selected as tagging SNPs they are not in strong LD, however we cannot exclude that they might reflect the same signal due to a moderate underlying LD (r2 between the two SNPs is 0.53).In this study, we thoroughly captured common genetic variation across 67 genes in the mTOR pathway and to our knowledge, this is the most comprehensive evaluation of common and coding variation in the mTOR pathway genes in relation with prostate cancer risk. We found an association of two SNPs in the PRKCI gene in prostate cancer aetiology is plausible given that atypical protein kinase C lambda/iota (aPKCλ/ι), encoded by the PRKCI gene, is a protein kinase C isozyme, which plays multifunctional roles in cellular maintenance and growth of epithelial cellsA role of genetic variation in the in vivo and in vitroPRCKI gene, which has been reported in lung and ovarian cancer PRCKI gene which has been reported in prostate cancer cell lines PRCKI promoter PKC isozymes are also involved in cell proliferation, survival, differentiation and apoptosis. Studies on lung, ovary, colon, and breast cancers have demonstrated a relationship between aPKCλ/ι expression and cancer progression and suggest that aPKCλ/ι expression might predict poor survivalPRKCI gene were associated at a study-wise significant level with a decreased risk of prostate cancer. rs546950 and rs4955720 are not situated in the coding region of the gene and it is not immediately evident how to relate the genetic variability to the gene function. We searched public databases for any reported functions of the two SNPs but we did not find evidence pointing to a differential gene expression or mRNA stability. No report to date has been published on either SNP in relation to disease suceptibility. We also ran in silico analysis on the possible changes on trasciption binding sites. These analyses predict a differential binding to the alleles of the two SNPs of various transcription factors. In particular MYOD1 is predicted to bind only to the minor allele (A) of rs546950 and POU3F3 to the minor allele (A) of rs4955720. Both transcription factors have pro-differentiation, anti-proliferation effect PRKCI, very close to the beginning of the gene, therefore this is consistent with a possible involvement in the regulation of transcription of the gene. Since the variant allele exerts a protective effect on prostate cancer risk we can hypothesize that it decreases or increases the ability of transcription factors to bind and in such way that results in a decrease of the gene expression, and consequently decreasing the risk of prostate cancer.In this report we found that two allelic variants of the PRKCI. However, all known common PRKCI SNPs in high LD with rs4955720 are located in introns of the gene, and do not seem functionally relevant. In conclusion it is difficult to understand the biological mechanism that could explain the association of the two polymorphism with prostate cancer risk and further functional studies are warranted.It is more difficult to understand the association between rs4955720 and decreased risk of prostate cancer from a biological point of view. However, in the joint analysis the p value of this SNP was the more significant of the two. This SNP is located at the 3′ of the gene, after the end of the last exon, and a possible function is not immediately evident. Also the possible differential binding with transcription factors seems to be less relevant in this case. rs4955720 could be in high linkage disequilibrium (LD) with another SNP that directly affects the transcription and/or function of PIK3C2B, was shown to be associated with increased prostate cancer risk. In our study we genotyped rs4951384, which tags rs7556371 (r2 = 1), but we did not observe any evidence of association  (Koutros 2010). In that study, rs7556371, a SNP of −5 (study-wide significance p-threshold) for a SNP with a minor allele frequency of 0.30.The intensive SNP tagging approach used provided a close to exhaustive analysis of possible mono-allelic (main effect) associations of prostate cancer risk with common polymorphic variants known for each of the loci studied. We had sufficient power  to detect OR = 1.40 (or OR = 0.71 for associations with a reduced risk) at alpha = 5.9×10Although over 97% of the EPIC subjects are estimated to be of Caucasian origin, differences in allelic frequencies across Europe could in theory cause confounding by population stratification. However, we did not observe major variations in allele frequencies across countries for the SNP studied here (data not shown).A possible drawback of this work is the large number of tests performed, although several hints suggest a real association: 1) we observed associations (p<0.05) in both stages of the study, 2) after correcting for the number of tests (n = 11) performed in the second phase the association between rs546950 and prostate cancer risk remains significant, 3) the association is biologically plausible and 4) the meta-analysis CGEMS/EPIC  showed very similar results as those obtained with the EPIC data alone  of subjects recruited in the EPIC cohort are of European (‘Caucasian’) origin. All EPIC study subjects provided anthropometric measurements  and extensive, standardized questionnaire information about medical history, diet, physical activity, smoking, and other lifestyle factors. About 260,000 women and 140,000 men provided a blood sample.Cases of cancer occurring after recruitment into the cohort and blood donation are identified through local and national cancer registries in 7 of the 10 countries, and in France, Germany, and Greece by a combination of contacts with national health insurances and/or active follow-up through the study subjects or their next of kin. Follow-up on vital status is achieved through record linkage with mortality registries.Case subjects were selected among men who developed prostate cancer after blood collection. Control subjects (1–2 controls per case) were selected randomly by incidence density sampling, matching the cases for centre of recruitment, age at blood donation and duration of follow-up. A total of 815 invasive prostate cancer cases and 1,266 controls, for a total of 2,081 subjects, were included in the first phase of the present study. Each control should have been free of cancer up to the duration of follow-up of the index case.2 = 0.8 between each pair of tagged and tagging SNPs, pairwise tagging (we observed an average mean r2 between tagging SNPs and the SNPs they tag of 0.95). SNPs that were predicted to perform poorly with Illumina GoldenGate genotyping technology were either replaced by SNPs in high LD , or dropped from the list if no proxy was available. This gave us a list of 1,133 SNPs. Finally, for quality control purpose, we added 30 SNPs that had been genotyped on the same samples in an unrelated project.For each of the 67 candidate genes we selected a genomic region between 5 kb 5′ of the beginning of the first known exon and 5 kb 3′ of the end of the last known exon. A list of SNPs in all 67 gene regions was compiled using data from HapMap , and tagging SNPs were selected by use of the Tagger algorithmDNA was extracted from blood samples on an Autopure instrument  with Puregene chemistry . The order of DNAs from cases and controls was randomized on PCR plates in order to ensure that an equal number of cases and controls could be analyzed simultaneously.Genotyping was carried out using the Illumina GoldenGate technology , according to the protocol specified by the manufacturer.−5) deviations from Hardy-Weinberg equilibrium (HWE) among controls.Any sample where greater than 25% of the SNPs failed had all of the SNPs set to missing and these subjects were dropped from analysis. We then filtered data to remove poorly performing SNPs: all SNPs that failed on 25% of samples or more were set to missing, as were all SNPs that showed statistically significant  or as two indicator variables, one for heterozygotes and one for minor-allele homozygotes (two degrees of freedom test).We performed also analyses in subgroups of disease aggressiveness (aggressive disease was defined as extraprostatic extension (stage C/D) or high histologic grade (Gleason score ≥8).All statistical analyses were performed using SAS 9.11.eff, by use of the SNP Spectral Decomposition approach eff value for each gene and also a study-wide Meff value, by adding up the gene Meff's.In order to take into account the large number of tests performed in this project, we calculated for each gene the number of effective independent variables, Mtrend<0.01 or p2df<0.01; n = 11) on an additional set of 838 cases and 943 controls selected within the EPIC cohort. All the additional genotyping was carried out using the Taqman assay. The MGB Taqman probes and primers were purchased from Applied Biosystems  as pre-designed assays. The reaction mix included 10 ng genomic DNA, 10 pmol each primer, 2 pmol each probe and 2.5 µl of 2x master mix (Applied Biosystems) in a final volume of 5 µl. The thermocycling included 40 cycles with 30 s at 95°C followed by 60 s at 60°C. PCR plates were read on an ABI PRISM 7900HT instrument (Applied Biosystems).We replicated the SNPs showing the strongest associations with prostate cancer risk  showed a concordance of 100.0%.http://genomatix.de/cgi-bin/matinspector_prof/mat_fam.pl) Potential binding sites of transcription factors within the sequence encompassing the two study-wise significantly associated SNPs were performed with MatInspector Professional Click here for additional data file.Table S1Candidate genes and their SNPs.(DOC)Click here for additional data file.Table S2Main effects of 1,084 SNPs genotyped in the first phase. Columns in this table show: gene name, NCBI dbSNP rs number, numbers of cases and controls for each of the three genotypes, odds ratios for heterozygotes, homozygotes for the rare allele  and per allele, with 95% confidence interval, p-value of the test with two indicator variables (2 d.f. test), p-value of the trend test.(XLS)Click here for additional data file.Table S3eff for each gene in the study.M(XLS)Click here for additional data file.Table S42 statistic, p-value of the test for heterogeneity, p-value of the trend test for each stratum, name of the stratum.Analyses in subgroups of disease aggressiveness. Columns in this table show: gene name, NCBI dbSNP rs number, possible genotypes, numbers of cases and controls for each of the three genotypes, odds ratios for heterozygotes and for homozygotes for the rare allele , with 95% confidence interval, Cochran's Q parameter, I(XLS)Click here for additional data file.


Physical activity promotes health and longevity. Further elaboration of the role of physical activity for human health in epidemiological studies on large samples requires accurate methods that are easy to use, cheap, and possible to repeat. The use of telecommunication technologies such as cell phones is highly interesting in this respect. In an earlier report, we showed that physical activity level (PAL) assessed using a cell phone procedure agreed well with corresponding estimates obtained using the doubly labeled water method. However, our earlier study indicated high within-subject variation in relation to between-subject variations in PAL using cell phones, but we could not assess if this was a true variation of PAL or an artifact of the cell phone technique.Our objective was to compare within- and between-subject variations in PAL by means of cell phones with corresponding estimates using an accelerometer. In addition, we compared the agreement of daily PAL values obtained using the cell phone questionnaire with corresponding data obtained using an accelerometer.2 to 33.6 kg/m2). The results were evaluated by fitting linear mixed effect models and descriptive statistics and graphs.PAL was measured both with the cell phone questionnaire and with a triaxial accelerometer daily during a 2-week study period in 21 healthy Swedish women  of the variation was within subjects, while with the cell phone, within-subject variation was 76% (95% CI 59%-83%). The day-to-day variations in PAL observed using the cell phone questions agreed well with the corresponding accelerometer results.Both the cell phone questionnaire and the accelerometer showed high within-subject variations. Furthermore, day-to-day variations in PAL within subjects assessed using the cell phone agreed well with corresponding accelerometer values. Consequently, our cell phone questionnaire is a promising tool for assessing levels of physical activity. The tool may be useful for large-scale prospective studies. Large epidemiological studies on physical activity and health require accurate methods that are easy to use, cheap, and possible to repeat. Recently, the potential of using cell phones, either through short message service (SMS) or Web-like applications, in behavior change intervention studies have been explored . HoweWhen assessing physical activity, one important aspect is the total amount of energy expended due to physical activity. This variable can be obtained as total energy expenditure divided by basal metabolic rate, that is, the so-called physical activity level (PAL) . We have2 . PAL was measured daily during a 14-day study period using the cell phone questions as well as a tri-axial accelerometer, the RT3 . The first day of the study period was excluded since the women did not wear the accelerometer until the afternoon that day; thus, results are reported for 13 days. One woman accidently broke her accelerometer; thus, results are reported for only 21 women. The study was approved by the central ethics board in Stockholm, Sweden.In all, 22 healthy nonsmoking Swedish women were recruited during August 2007 through May 2008 as previously described [Our JAVA-based questionnaire for assessing PAL using cell phones has previously been described in detail . BrieflyEach woman was instructed to wear the accelerometer during all her waking hours except when in water. She recorded in a notebook when she took off the device and the activities performed without it . Recorded movements were converted to total energy expenditure according to the manufacturer. All women wore the RT3 all 14 days and the recordings obtained during these days covered 97% ± 2% of time awake. For activities reported in the notebook, energy expenditure was estimated based on published energy costs of specific activities . PAL wasIntraclass correlation coefficients (ICC) were calculated as the between-subject variation divided by the total variation.The coefficients were estimated by means ofa linear mixed effect model with the two components of variance and a fixed effect for days 1 to 13. The ICC was used to compare the between-subject variation in relation to total variation for PAL values at days 1 to 13. The ICC was calculated from models fitted separately for the cell phone data and the accelerometer data. To test the robustness against the influence of single gross outliers, we repeated the ICC calculations for both methods when we had replaced the 10 as well as 20 most deviating daily PAL values out of 273 values with the mean PAL for these women. Furthermore, the agreement of daily PAL values obtained using the cell phone questionnaire and corresponding accelerometer data was evaluated using descriptive statistics. All statistical analyses were carried out using the SAS software proc mixed, version 9.2 .For PAL obtained using the cell phone, the variance between and within subjects was estimated to be 0.009 and 0.029, respectively. Thus, 24% of the total variation was between subjects, and 76%, within subjects. The corresponding estimates for the accelerometer were 0.017 and 0.023, showing that 43% of the variation was between subjects and 57%, within subjects. The 95% CI for the within-subject variation was 59% to 83 % for the cell phone while it was 40% to 66% for the accelerometer. The variance components for PAL obtained using the cell phone and the accelerometer remained similar when we replaced the 10 and 20 most deviating values with the mean PAL of these women.                Both the cell phone questionnaire and the accelerometer showed high within-subject variations, indicating that there is considerable true day-to-day variation in PAL. First, this makes it more likely that the relative low between-subject variations we reported in our earlier paper are not artifacts of the cell phone technique. Second, this is an interesting finding since few reports have described the specific sources of variance in daily physical activity using objective measures in adults -13.PAL obtained using the accelerometer was generally on a lower level than the cell phone value. The RT3 as well as several other accelerometers have shown a tendency to underestimate energy expenditure during free-living conditions compared with the doubly labeled water method -15 also With the accelerometer, 43% of the variation in PAL was between subjects, while between-subject variation was 24% with the cell phone. For comparison, Matthews et al  reportedThe major limitation of this study is the small sample size. First, it makes our analysis sensitive to single gross outliers since these may largely increase the within-subject variations. However, we found no evidence for any important effect of such outliers in our analysis since the variance components were stable when we replaced the 10 or 20 most deviating PAL values  with the average PAL for those women. Second, the small sample size limits our ability to make firm conclusions about the between-subject variations. We cannot exclude that the low between-subject variation obtained by both methods to some extent is due to the fact that our participants were a relatively small group of women. Other limitations are that we estimated energy expenditure for activities when the women were not wearing the RT3, but this amount of time was small, and that this study was conducted in healthy, moderately active women. The results should be repeated in other populations including men as well as elderly and obese subjects.In conclusion, (1) Both the cell phone questionnaire and the accelerometer showed high within-subject variations in PAL and (2) changes in daily cell phone PAL values agreed well with corresponding accelerometer values. This study adds further evidence to our earlier findings  that the


To examine whether prenatal exposure to parental type 1 diabetes, type 2 diabetes, or gestational diabetes is associated with an increased risk of malignant neoplasm or diseases of the circulatory system in the offspring.We conducted a population-based cohort study of 1,781,576 singletons born in Denmark from 1977 to 2008. Children were followed for up to 30 years from the day of birth until the onset of the outcomes under study, death, emigration, or December 31, 2009, whichever came first. We used Cox proportional hazards model to estimate hazard ratios (HR) with 95% confidence intervals (95% CI) for the outcomes under study while adjusting for potential confounders. An increased risk of malignant neoplasm was found in children prenatally exposed to maternal type 2 diabetes . An increased risk of diseases of the circulatory system was found in children exposed to maternal type 1 diabetes , type 2 diabetes , and gestational diabetes , but results were attenuated after excluding children with congenital malformations. An increased risk of diseases of the circulatory system was also found in children exposed to paternal type 2 diabetes  and the elevated risk remained after excluding children with congenital malformations.This study suggests that susceptibility to malignant neoplasm is modified partly by fetal programming. Diseases of the circulatory system may be modified by genetic factors, other time-stable family factors, or fetal programming. The number of pregnant women with diabetes is increasing as a result of an increasing prevalence of diabetes, a higher age at the time of birth, and earlier onset of diabetes. We therefore conducted a population-based cohort study of 1.7 million children who were followed for up to 30 years to examine whether children born to mothers or fathers with diabetes are more susceptible to malignant neoplasms or diseases of the circulatory system. If parental diabetes affects the outcomes in the offspring mainly through changes in the intrauterine environment, one would expect only maternal diabetes but not paternal diabetes to be associated with the outcomes. If mainly time stable family conditions including genetic factors play a role, one would expect to see both maternal diabetes and paternal diabetes to be associated with the outcomes.According to Danish laws, register-based studies do not need to obtain consents from individuals, when personal identifiers have been encrypted and stored by a trusted third part (Statistic Denmark). The data recruitment was approved by the Danish Data Protection Agency (J.nr.2008-41-2680).We identified 1,927,343 singletons born in Denmark from 1977 to 2008 in the Danish Medical Birth Register which includes all births in Denmark since 1973. th revision of the International Classification of Diseases (ICD-8) from 1977 to 1993, and the 10th revision (ICD-10) from 1994 onwards. The Danish National Hospital Register holds nationwide data on all admissions to any Danish hospital since 1977 and on all outpatient visits since 1995. The information on diabetes was based on the Danish version of the 8We also extracted information on diabetes from the Danish National Diabetes Register that aims at including persons with diabetes who were only treated outside the hospital. The Danish National Diabetes Register holds information on diabetes in the Danish population from 1 January 1995 to 1 January 2007 and it is based on existing registers including the Danish National Hospital Register, the National Health Insurance Service Registry, and the Register of Medicinal Product Statistics. Children were classified as prenatally exposed to maternal  diabetes if they were born after the time when their mothers (or fathers) were recorded with diabetes. Firstly, exposed children born from 1987 to 1993 were categorized as exposed to type 1 or type 2 diabetes at the time of birth. Secondly, Exposed children born from 1994 to 2008 were categorized as exposed to type 1, type 2, or gestational diabetes at the time of birth. Finally, any types of diabetes were grouped in a single category and all exposed children born from 1977 to 2008 and were categorized as exposed to maternal  diabetes regardless of types of diabetes.Information on the outcomes of interest was obtained from the Danish National Hospital Register. Malignant neoplasm were identified as hospitalizations with ICD-8 (140–209) and ICD-10 (C00–C97), diseases of the circulatory system were identified as hospitalizations with ICD-8 (390–458) and ICD-10 (I00–I99), and congenital malformations were identified as hospitalizations with ICD-8 (740-759) and ICD-10 (Q00–Q99).Information on gestational age, birth weight, maternal age at birth, and parity was obtained from the Danish Medical Birth Registry. The children were followed from the day of birth until the first hospitalization or first outpatient visit for the outcomes under study, death, emigration, or December 31, 2009, whichever came first.Since some children were born to the same mothers, we used robust inference for the Cox proportional hazards model to estimate hazard ratios (HRs) with 95% confidence interval (95% CI) for hospitalization due to malignant neoplasm, diseases of the circulatory system, or congenital malformation for children exposed to maternal or paternal diabetes compared to the cohort of unexposed children. In Model 1, we adjusted for maternal age , parity , sex of children , maternal education , maternal marital status , and calendar year . In Model 2, we adjusted for the same variables but excluding children with any congenital malformations. In Model 3, we not only excluded children with any congenital malformations but also extended the adjustment to include gestational age in weeks, birth weight, and birth weight squared as continuous variables.The statistical analyses were done using Stata11 .In the final study population, children with missing values on parity, gestational age, birth weight, maternal education, parental marital status, or paternal diabetes were excluded  and 1,78The prevalence of congenital malformation was increased in singletons exposed to maternal type 1 diabetes, maternal type 2 diabetes, and gestational diabetes when compared to unexposed singletons among singletons born from 1987 to 2008 . This inAn increased risk of malignant neoplasm was found in singletons exposed to maternal type 2 diabetes compared to unexposed singletons and the risk remained high after excluding singletons with congenital malformations. The HRs were attenuated a little when analyses were done among all singletons exposed to any types of maternal diabetes . A detaiAn increased risk for disease of the circulatory system was found in singletons exposed to maternal type 1 diabetes, maternal type 2 diabetes, and gestational diabetes but these HRs were attenuated when children with congenital malformations were excluded . SimilarAn increased risk for malignant neoplasm was found in children prenatally exposed to maternal type 2 diabetes, and the increased risk remained after excluding children with congenital malformations. An increased risk of disease of the circulatory system was found not only in children exposed to maternal diabetes but also in children exposed to paternal type 2 diabetes.A higher prevalence of congenital malformations was found in children prenatally exposed to maternal diabetes, which is in line with previous studies. We found an increased risk of malignant neoplasm in children prenatally exposed to maternal type 2 diabetes and the associations seemed not to be modified by higher birth weight or preterm birth since the results did not change much when we adjusted for these factors. A previous study reported a higher rate of hospitalization due to neoplasms in children born to mothers with diabetes  Intrauterine exposure to maternal diabetes is associated with childhood overweight, obesity, metabolic syndrome Although type 1 diabetes and type 2 diabetes have common features in terms of increased level of glucose, triglycerides, and many amino acids in the maternal circulation, their genetic background and ability to modify the intrauterine environment probably differ. We used a large population-based cohort including all children born in Denmark and had up to 30 years of almost complete follow-up. The completeness of the registration of diabetes, malignant neoplasm, and diseases of the circulatory system is high since health services, including antenatal care and hospitalizations, are free of charge for all citizens in Denmark. However, some women have type 2 diabetes without knowing it This study suggests that susceptibility to malignant neoplasm and congenital malformation may be results of fetal programming induced by maternal diabetes. The risk of disease of circulatory system may be related to genetic factors or other time stable family factors as well as fetal programming. Congenital malformation may act as one of the potential pathways of associations between diabetic intrauterine environment and risk of diseases of the circulatory system in children prenatally exposed to maternal diabetes. We had insufficient follow time to examine risk of atherosclerosis and other aging related cardiovascular diseases. Paternal diabetes had no association with congenital malformation measured as prevalence at birth.Table S1A detailed list of the ICD codes and number of malignant neoplasm in children exposed to parental type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes (GD).(DOCX)Click here for additional data file.Table S2A detailed list of the ICD codes and number of disease of circulatory system in children exposed to parental type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes (GD).(DOCX)Click here for additional data file.


Lycium barbarum (LBP) are neuroprotective for retinal ganglion cells (RGCs) in different animal models. Protecting RGCs from secondary degeneration is a promising direction for therapy in glaucoma management. The complete optic nerve transection (CONT) model can be used to study primary degeneration of RGCs, while the partial optic nerve transection (PONT) model can be used to study secondary degeneration of RGCs because primary degeneration of RGCs and secondary degeneration can be separated in location in the same retina in this model; in other situations, these types of degeneration can be difficult to distinguish. In order to examine which kind of degeneration LBP could delay, both CONT and PONT models were used in this study. Rats were fed with LBP or vehicle daily from 7 days before surgery until sacrifice at different time-points and the surviving numbers of RGCs were evaluated. The expression of several proteins related to inflammation, oxidative stress, and the c-jun N-terminal kinase (JNK) pathways were detected with Western-blot analysis. LBP did not delay primary degeneration of RGCs after either CONT or PONT, but it did delay secondary degeneration of RGCs after PONT. We found that LBP appeared to exert these protective effects by inhibiting oxidative stress and the JNK/c-jun pathway and by transiently increasing production of insulin-like growth factor-1 (IGF-1). This study suggests that LBP can delay secondary degeneration of RGCs and this effect may be linked to inhibition of oxidative stress and the JNK/c-jun pathway in the retina.Our group has shown that the polysaccharides extracted from  Glaucoma has been considered to be a neurodegenerative disease characterized by optic nerve (ON) atrophy and irreversible loss of retinal ganglion cells (RGCs) Several animal models have been used in the study of glaucoma, including complete optic nerve transection (CONT), acute and chronic ocular hypertension models and the ON crush model. However, it is difficult to distinguish primary degeneration from secondary degeneration in these commonly used models because each involves insult to all RGCs Lycium barbarum has been used as an “upper class herb” for hundreds of years in the Oriental world. It was used for the treatment of the diseases related to vision, the “kidney” and the “liver” Lycium barbarum (LBP) reduce the death of cultured cortical neurons challenged by beta-amyloid, glutamate and Homocysteine The aims of this experiment were to confirm whether ON section caused retinal oxidative stress, to investigate the presence of retinal inflammation after ON section and to determine which kind of degeneration LBP could delay and which mechanism(s) might be involved in any neuroprotective effects of LBP, and we were largely successful in these aims.The use of animals followed the requirements of the Cap. 340 Animals (Control of Experiments) Ordinance and Regulations in Hong Kong. All the experimental and animal handling procedures were approved by the Faculty Committee on the Use of Live Animals in Teaching and Research in The University of Hong Kong (CULATR #1850-09 and #1996-09).ad libitum. The preparation of LBP was as previously described Adult female Sprague Dawley rats (10–12 weeks of age weighing 250–280 g) were used in this study. The rats were housed in a temperature-controlled room subjected to a 12-hour light/12-hour dark cycle and supplied with food and water To investigate if the degeneration speeds were similar between superior and inferior retinas after CONT, the rats without treatment with PBS or LBP were sacrificed either 1 week or 2 weeks after CONT (n = 5 at either time-point). To evaluate the effects of LBP on the survival of RGCs after ON injury, the procedure was as described in in vivo. The death of cells in ganglion cell layer (GCL) was studied using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL assay) and protein expression was examined with Western-blot analysis at 12 hours, 1 day, 4 days and 1 week after PONT; there was no drug treatment .Retrograde labelling of RGCs was achieved using Fluoro-Gold (FG) from the stump of the ON after CONT 2. For TUNEL assay, the rats were sacrificed by injecting overdoses of Phenobarbital followed by perfusion with 0.9% NaCl and 4% paraformaldehyde (PFA).Protein expression after LBP or PBS treatment was also studied with Western-blot analysis both 1 day and 1 week after PONT . The rats for RGC counting (both after FG and DiI labeling) and Western-blot analysis were sacrificed using inhalation of COAnesthesia and the CONT procedure were conducted as previously described The method published by Fitzgerald et al. was adopted Optic nerves were collected, post-fixed in 4% PFA for 60 minutes and then placed into 30% sucrose in 0.1 M phosphate buffer solution overnight until they sank. They were then embedded into optimal cutting temperature embedding compound and sectioned longitudinally. The sections were mounted on slides, rinsed 3 times with 0.01 M PBS and observed using fluorescence microscopy.2) in each sector were captured along the median line, starting from the optic disc to the edges at 500-µm intervals under a fluorescence microscope at 400×magnification 2).After sacrifice, retinas were collected and post-fixed in 4% PFA for 60 minutes. Retinas were divided into the superior and inferior halves and each half was separated into three roughly equal sectors before being flat-mounted as the temporal, middle and nasal sectors . Eight pTUNEL staining was previously believed to detect apoptosis only, but more recently it has been shown to detect necrosis and other types of cell death as well After sacrifice, the inferior retinas were collected in PBS on ice. The procedure including the use of lysis buffer, secondary antibody and the developing reagents were as previously described t-test was used for comparisons of two groups. For more than two groups, one-way ANOVA was used for multiple comparisons followed by Dunn’s or Student-Newman-Keuls method as post hoc tests. Data were analyzed statistically with the Sigmastat software . The P = 0.05 level was considered to be statistically significant.Student’s 2; the normal superior retinas: 2046.5±92.4 RGCs/mm2; and the normal inferior retinas: 2144.4±89.8 RGCs/mm2. There was no difference between the superior and inferior retinas . The surviving RGC densities decreased significantly in the expected areas after both CONT and PONT in animals treated with PBS or LBP .The average densities of FG-labeled RGCs in the normal retinas were as follows: the whole retinas: 2088.1±64.4 RGCs/mmt-test, P>0.05, One PBS group and three LBP groups with different dosages  were examined 1 week after CONT. No significant difference between the PBS group and any LBP group was detected; in addition, no significant difference among the three dosages of LBP was seen . However, there were no changes at 12 hours, 1 day and 4 days. The positive staining was shown in the nuclei, which was confirmed by counter-staining with DAPI .The protein level of TNF-α did not increase after PONT in the inferior retinas . The expAfter LBP treatment, the expression of MnSOD increased significantly 1 day after PONT . On the After CONT, most RGCs died rapidly from primary degeneration. After PONT, more RGCs die from secondary degeneration at a later time-window in addition to primary degeneration In the PONT model, the increasing expression of MnSOD or SOD2, which was demonstrated by immunohistochemistry (IHC), was used as an indicator of oxidative stress in vitroJNKs are the kinases involved in both apoptotic and non-apoptotic cell death in situ hydrogels containing Leu-Ile. This is an inducer for neurotrophic factors, which increase the expression of BDNF in the retina and promote RGC survival after ON injury BDNF belongs to the neurotrophin family and is expressed both in SC in vivoin vivo; diffusion to neighboring tissue was obvious in fixed tissues but not for the tissues in vivo. Therefore, we did an in vivo study where diffusion of DiI was limited. Our results also showed that the axonal transport of DiI was limited to the dorsal region in the ON sections from the labeled animals and where diffusion was insignificant.It is known that DiI could be transported by either active processes or by diffusion Our results confirmed the neuroprotective effects of LBP for RGCs and showed the possible mechanism. The future target of our study is to provide the basis for the use of LBP in clinical conditions. The electroretinogram is used widely by ophthalmologists and optometrists for the diagnosis of retinal diseases and can evaluate the retinal function by measuring the electrical responses of various cell types