The study prospectively assessed the functional impairments and rehabilitation needs of Africans admitted to a regional trauma center. It also acts as a pilot study to demonstrate the practical use of the Language Independent Functional Evaluation (L.I.F.E.) software in an acute hospital setting.A 5 page questionnaire was used to gather demographic data , cause of disability/injury, severity of disability or functional impairment, and rehabilitation treatment received . Functional status on discharge was evaluated with the L.I.F.E. scale.84 consecutive consenting subjects were recorded. The predominant disability/injury of respondents involved the lower extremities (70%), followed by upper extremities (23%). The mechanisms of injury were largely related to auto accidents (69%). Falls made up 17% of these injuries and 14% were related to violence. Eleven subjects had disability measured using L.I.F.E and all were classified as having major disabilities. Only 14 patients (17%) received any rehabilitation therapy which consisted of only physical therapy provided at a frequency of once a day for less than one week duration.This study found that most persons admitted to a sophisticated trauma unit in Ghana are discharged without adequate rehabilitation services, and that the level of disability experienced by these people can be measured, even while they are still sick and in the hospital, using L.I.F.E. The implications are clear: African trauma systems must measure the long term outcomes from their treatments and provide the inpatient medical rehabilitation services that are a standard of care for trauma victims elsewhere in the world. Around the world, the number of persons living with some form of disability is estimated at 650 million, of which 80% live in low-resource countries . It has Perhaps the most important modifiable risk factor for this increase in disability is trauma. Most cases of trauma in low-resource countries result from automobile accidents. High income countries own 89% of the world's automobiles and yet suffer only 24% of auto accident deaths .Rehabilitation after trauma is a serious and rising problem for low-resource countries, yet it has not been widely studied. Since disability tends to be environmentally and regionally influenced, research into this topic needs to be done at locations where resources are representative but optimal. Ghana serves as an important model for exploring issues of trauma rehabilitation, because although it is a low-resource country, it has highly sophisticated trauma care at a few university-associated hospitals. In contrast to high-level trauma care, and typical of all other African countries, medical rehabilitation resources are almost non-existent . The stuFinally, measurement of disability and rehabilitation needs is difficult in Africa because of language and literacy barriers. A recent development, the Language Independent Functional Evaluation (L.I.F.E.), may be a useful tool for scientists and clinicians to evaluate the functional needs of persons in African hospitals . This coThe current study prospectively assesses the functional impairments and rehabilitation needs of Africans admitted to a regional trauma center. It also acts as a pilot study to demonstrate the practical use of the L.I.F.E. in an acute hospital setting.We designed a cross-sectional study of hospital inpatients. All patients age 18 and older admitted to the trauma ward who were medically stable and cognitively intact to provide informed consent participated in the interview process. These inclusion criteria maximize the range of participants in terms of demographics, injury/disability type, functional impairments, and appropriateness for rehabilitation. Patients with thoracic and abdominal trauma were excluded from the study to ensure that patients were appropriate to participate in rehabilitation.Participation was voluntary, without compensation. Written consent was obtained based on literacy level. Patient who were illiterate gave consent with thumbprints, a substitute for signature in Ghana. Patient privacy was protected during collection and processing of data. A random number was assigned to each patient and the data was collected anonymously. Clearance for this work was obtained from the Committee on Human Research, Publications and Ethics, Kwame Nkrumah University of science and Technology (KNUST) and Komfo Anokyi Teaching Hospital (KATH), Kumasi, Ghana; Medical College of Wisconsin Institutional Review Board, Milwaukee, WI, USA; and the Johns Hopkins University Institutional Review Board, Baltimore, MD, USA.The study was carried out at Komfo Anokye Teaching Hospital (KATH) in Kumasi, the capital of the Ashanti region of Ghana. Kumasi has an estimated population of 650,000. KATH isData collection was conducted from April 14th 2009 to April 28th 2009. Patients were interviewed in English or in their native language, Akan, by investigator AC who is a native speaker. A 5-page questionnaire in English with Ashanti vernacular translation was administered to participants. The questionnaire covered demographic data , cause of disability/injury, severity of disability or functional impairment, and rehabilitation treatment received .A scale developed by Mock et al.  was usedFunctional status on discharge was evaluated with Language Independent Functional Evaluation (L.I.F.E.) software . L.I.F.EThe Medicare model set by the Center for Medicare and Medicaid Services (CMS) for determining rehabilitation needs in the United States was used to determine which patients would qualify for acute inpatient rehabilitation services if available. The Medicare criteria for medical rehabilitation are based on an extensive evidence-base demonstrating the cost-effectiveness of rehabilitation interventions.The admission criteria are based on type of injury, admission evaluation process, and the amount of therapy a person receives in acute inpatient rehabilitation facility. Major multiple trauma is one of the 13 primary diagnoses required to comprise 75% of the admissions to acute rehabilitation facilities in the US. To qualify for an admission to acute inpatient rehabilitation, patients must require and be able to benefit from at least three hours of rehabilitation therapy including physical therapy, occupational therapy or speech therapy. Patients must have a total of 15 hours of therapy over a week, usually three hours daily Monday through Friday. Recreational therapy, music therapy, respiratory therapy, neuropsychology or cognitive therapy can be used to satisfy the requirement for patients to receive intensive rehabilitation therapy .Eighty four consecutive consenting subjects were recorded. Sixty six percent of the respondents were male. The majority (63%) of the respondents had high school education or less. More than one half of the respondents resided in rural areas (55%) and the majority (95%) lived in single story house. Most respondents were between the ages of 18-44 (67%). Seven patients were from senior wards vs. 77 from regular floors.The predominant disability/injury of the respondents involved the lower extremities (70%), followed by upper extremities (23%). The mechanisms of injury were largely related to auto accidents (69%). Falls made up 17% of these injuries and 14% were related to gunshot wounds or assaults.Based on the Mock classification, mobility restrictions accounted for 70% of disabilities, minor disability 16%, self-care limitation 11%, and 2% had major disability.The rehabilitation services received by these patients are described in Of the 84 respondents, only fifteen received some assistive device for temporary use during hospitalization. These consist of five crutches, three wheelchairs, two orthosis (knee brace), one walker and four straight canes. These assistive devices were shared between patients. For example, patients needing wheelchair transport to the bathroom had to wait until one was available. All those who received the equipment said it was helpful. Only 8% of the respondents said a device was prescribed for them upon discharge from the hospital. Fifty percent of patients who received therapy were given follow up therapy upon discharge. More than 90% of patients had some support system to take care of them at home upon discharge. Unfortunately 92% of caregivers received no training in the hospital and have no training in care for the patients, patient prognosis, or injury complications.We measured disability at discharge using L.I.F.E. on 11 subjects (13%). All patients evaluated with L.I.F.E. had significant disabilities as compared to 18% identified as having minor disability by the Mock scale . Among tSubjectively, the first author, an American-trained native Ghanaian physiatrist, found that most of the patients in the study had no idea of what rehabilitation medicine meant. The lack of knowledge about rehabilitation medicine was not related to age, sex, rural vs. urban, or literacy level. It became clear from the beginning that physical therapy, occupational therapy and speech therapy were unknown to these populations even when a translation like “someone who comes and stretch you” was used. This was how physical therapy was described and perceived in the vernacular, however the very concept that injured limbs benefit from rehabilitation was not understood among these trauma victims.This is the first study to objectively review the rehabilitation needs in a tertiary care trauma center in Africa. The results showed that the great majority of people admitted to the trauma service in Ghana did not receive any rehabilitation services, and the few that did received inadequate services. We also demonstrated that L.I.F.E. can be a viable option to measure function among African hospital patients.We were struck by the fact that all patients recruited in the study from the trauma units would have met criteria for acute inpatient rehabilitation admission in the United States. Even patients with non-weight bearing status might have had an early rehabilitation phase involving maintaining joint ROM (range of motion), muscle strength, sitting balance among others. After the medical diagnoses and functional impairments were categorized, a model for determining rehabilitation needs was applied. This model demonstrated that Medicare (the US government's insurance program for people above 65 years of age) would approve and finance rehabilitation care for all the cases studied . MedicarThere was also a lack of appropriate assistive devices in the hospital for patient use and they were rarely prescribed upon discharge. The need for assistive devices is based on the investigators’ proposal that issuance of a specific assistive device  would immediately improve function by one point in one of the basic functional abilities outlined in the L.I.F.E., and would be necessary for more than 1 month.To our knowledge, the only review of rehabilitation in Ghana was recently published by Tinney et al. . They haThere is research on the subject of injury-related disability and orthopedic care in Ghana but few authors explore rehabilitation care. Ghana has more than 1 million people with disabilities , of whomThe absence of medical rehabilitation at KATH is likely the result of lack of knowledge. In Ghana the Essential Trauma Care projects have been important in improving trauma care . KATH, fThe self-report of disability by the person receiving acute trauma care is of note. Most research into the needs of people with disability focuses on people living in the community ,12,24. TThe Mock scale was used to classify all patients admitted, and L.I.F.E was used on 11 patients who were being discharge home during the time of the study. The current experience shows the viability of the L.I.F.E. in the environment of a trauma center where English is not always spoken and the literacy level is low. While global disability scales such as that proposed by Mock have an advantage in terms of simplicity, it is obvious in our research-that they are not detailed enough to show clinicians and policymakers the functional impact of injuries or the potential requirements for medical rehabilitation interventions. Though only 2% and 16% had classification of major and minor disability respectively, based on the Mock scale, there were substantial functional impairments and limitation in the group, as outlined by L.I.F.E. The Mock scale fails to identify impairments that can significantly impact a person's ability to function in society, carry on their occupation, social life, and ADLs.Our results demonstrate that, most people on these wards had significant trauma, leading predictably to substantial disability. However few of them received any rehabilitation services at the level considered appropriate in other systems of care, and there was no possibility of providing the multidisciplinary rehabilitation inpatient services that are standard of care for trauma systems elsewhere, since there are no professionals with the appropriate expertise.The core problem is that trauma policy is biased by the use of short term success metrics such as mortality and hospital length of stay. In contrast the real cost of trauma should be calculated from a lifetime of significant untreated disability. Trauma centers that do not have comprehensive rehabilitation have been shown to have worst outcomes –31. FailAn overwhelming literature supports multidisciplinary, physiatrist-driven inpatient hospital rehabilitation programs as the standard of care for significant trauma ranging from amputation to spinal cord injury. In a study of 124,421 persons admitted to trauma centers across Washington State, USA over a 13 year period, Davidson et al. evaluated the risk of death after hospitalization. The hazards ratio (compared to those who went home without need for any assistance) was 2.88 for those who went to physiatrist-directed rehabilitation, 3.78 for those who went home with assistance, but not inpatient rehabilitation, and 8.92 for those who were admitted to sub-acute facilities without comprehensive rehabilitation .As proposed by Kosar, et al (2010), an integrated multi-trauma rehabilitation service approach is more cost-effective than conventional multi-trauma care service. Their integrated model encompasses early rehabilitation including: 1) early involvement of the physiatrist in the hospital; 2) shorter stay in hospital with early transfer of multi-trauma patients to specialized trauma rehabilitation units; 3) injury-specific physical and psychosocial treatment; 4) individual goal settings 5) psychological and social counseling; and 6) integrated and coordinated treatments between trauma surgeons and rehabilitation physicians .The lack of post-trauma rehabilitation in Africa can be directly tied to the absence of physiatrists on the continent. Since most African physicians and policymakers have never met a physiatrist, they often presume that rehabilitation is equal to physiotherapy. Yet most components of medical rehabilitation fall outside of the expertise of physical therapists. African therapists  are at a distinct disadvantage when advocating for interventions ranging from spasticity injections to antidepressants to swallowing evaluations. In the short term therapists and trauma surgeons can patch the gap, but in the long term they need to advocate for institutions of higher education in Africa to train the physicians and other specialists who can bring rehabilitation services up to standard.These findings need to be interpreted in light of the methodology used. Only a small number of subject participated in L.I.F.E data collection. The study was done at one medical center, with short duration of data collection, limiting generalizability. However, findings at KATH can be extrapolated to other parts of Ghana, since the demographics, causes of injury, and availability of rehabilitation services are not likely to be different. In fact, many parts of Africa have fewer rehabilitation resources and less sophisticated trauma care. Thus these findings likely represent a best-case scenario.KATH being the only tertiary hospital in the upper half of Ghana, recruited participants from a wide population group. Members from most major tribes in Ghana  were represented in our study population. This by no means is suggestive that the study evaluated all the tribes or languages in Ghana but the above represents languages spoken from all corners of the country. Patient from different wards, i.e. general trauma floor and senior wards represented a wider socioeconomic range. Prospective studies of trauma patients from admission to discharge can help us better characterize trauma-care cost as compared to costs in optimized trauma-care systems with early rehabilitation while taking into account the cost of lost productivity.This study found that most persons admitted to a sophisticated trauma unit in Ghana are discharged after receiving little or no rehabilitation services, and that L.I.F.E can be a useful tool to measure the level of disability experienced by trauma patients even while they are still sick and in the hospital. The implications are clear: African trauma systems must measure long-term treatment outcomes and provide inpatient medical rehabilitation services that are standard of care for trauma victims elsewhere in the world.


TMPRSS2/ERG rearrangement, PTEN gene deletion, and androgen receptor (AR) gene amplification have been observed in various stages of human prostate cancer. We hypothesized that using these markers as a combined panel would allow better differentiation between low-risk and high-risk prostate cancer. We analyzed 110 primary prostate cancer samples, 70 metastatic tumor samples from 11 patients, and 27 xenograft tissues derived from 22 advanced prostate cancer patients using fluorescence in situ hybridization (FISH) analysis with probes targeting the TMPRSS2/ERG, PTEN, and AR gene loci. Heterogeneity of the aberrations detected was evaluated. Genetic patterns were also correlated with transcript levels. Among samples with complete data available, the three-marker FISH panel detected chromosomal abnormalities in 53% of primary prostate cancers and 87% of metastatic (Met) or castration-resistant (CRPC) tumors. The number of markers with abnormal FISH result had a different distribution between the two groups (P<0.001). At the patient level, Met/CRPC tumors are 4.5 times more likely to show abnormalities than primary cancer patients (P<0.05). Heterogeneity among Met/CRPC tumors is mostly inter-patient. Intra-patient heterogeneity is primarily due to differences between the primary prostate tumor and the metastases while multiple metastatic sites show consistent abnormalities. Intra-tumor variability is most prominent with the AR copy number in primary tumors. AR copy number correlated well with the AR mRNA expression . Especially among TMPRSS2:ERG fusion-positive CRPC tumors, AR mRNA and ERG mRNA levels are strongly correlated . Overall, the three-marker FISH panel may represent a useful tool for risk stratification of prostate cancer patients. ETS gene rearrangements in prostate cancers has led to studies evaluating the functional role of ETS genes in the pathogenesis of this disease and as diagnostic and prognostic biomarkers. The most common type of ETS rearrangement, the fusion of androgen-regulated TMPRSS2 with the oncogenic ERG is detected in approximately half of prostate tumors but none of benign glands TMPRSS2:ERG fusion have yielded inconsistent results TMPRSS2 is regulated by androgens, and the androgen receptor (AR) is often amplified in patients treated with androgen deprivation therapy PTEN deletion, another common aberration in prostate cancer, was correlated with the expression of downstream p-Akt and associated with cancer-specific mortality ETS gene rearrangements were shown to cooperate with PTEN deletion and impact prostate cancer prognosis PTEN deletion was shown to suppress androgen-responsive gene expression by modulating AR transcription factor activity. Also, PTEN and AR expression has been shown to inversely correlate in prostate cancer The discovery of recurrent TMPRSS2 and/or ERG rearrangements, AR gene amplification, and PTEN deletion in both primary and CRPC prostate cancer samples and compared the prevalence, concurrence, and interaction of these three markers. With the reference of mRNA expression data generated from matching tumor samples from the same patient, we also demonstrated how FISH findings correlated with changes in gene expression. Intra- and inter-patient tumor heterogeneity was also analyzed.A critical clinical question concerns identifying characteristics of newly diagnosed prostate cancers that will distinguish aggressive from indolent behavior. The molecular heterogeneity of prostate cancers suggests that individual biomarkers may not be sufficient, and that multiple genetic markers may better associate with outcome. In the present study, we used a three-marker fluorescence in situ hybridization (FISH) panel to detect The study was approved by the Institutional Review Boards (IRB) of the Fred Hutchinson Cancer Research Center and the University of Washington Medical Center. IRB waived the need for written consent for this study because only de-identified materials were used, which were from the University of Washington Urology tissue bank.TMPRSS2/ERG FISH data, 65 patients with AR FISH data and 42 patients with PTEN FISH data. Metastatic tumor samples (n = 70) were collected at UW from autopsies performed within 2 to 4 hours of death of 11 CRPC patients under the rapid autopsy program TMPRSS2/ERG FISH data, 65 tumors from 11 patients with AR FISH data, and 62 tumors from 11 patients with PTEN FISH data.De-identified archived untreated primary prostate cancer samples (n = 110) were obtained from the University of Washington (UW) and Virginia Mason Hospital in Seattle. A total of 83 primary tumors generated analyzable data for at least one FISH marker in the panel, including 69 patients with TMPRSS2/ERG FISH data, 26 tumors from 21 patients with AR FISH data, and 25 tumors from 21 patients with PTEN FISH data. Together, combining metastatic patient tumors and xenografts derived from advanced-stage prostate cancer patients, the current study evaluated a total of 94 tumors from 32 patients.Prostate cancer xenografts (LuCaP lines) were originally isolated from various organs of advanced patients TMPRSS2/ERG rearrangement was assessed using our novel 4-color FISH assay as described separately TMPRSS2:ERG” refers to the presence of fusion of the two genes. “TMPRSS2/ERG rearrangement” refers to various subtypes of rearrangement of either or both genes as specified in the Results section. FISH analysis of AR gene amplification was performed using the SpectrumOrange AR (Xq12) probe combined with the SpectrumGreen labeled ChrX centromere (Xp11.1-q11.1) CEP X probe as the control . PTEN gene deletion was examined using the PTEN/CEP10 dual-color FISH Probe set , including the SpectrumOrange labeled PTEN (10q23) probe and the SpectrumGreen labeled Chr10 centromere (10p11.1–10q11.1) CEP 10 probe.AR gain and PTEN deletion were assessed by counting the number of gene signal and the corresponding centromere signal per nucleus. AR gain was defined as an average copy number of AR per nuclei equal or higher than 2. True AR gene amplification was defined as the ratio of the total number of AR signals divided by the total number of the X-chromosome centromere equal or greater than 2. Samples with PTEN heterozygous deletion had a ratio of the total number of PTEN signals divided by the total number of CEP10 signals equal or below 0.75. A PTEN/CEP10 ratio equal or below 0.2 is considered homozygous PTEN deletion. For patient-level analyses of CRPC patients with multiple tumors, expression by a given marker was considered abnormal if the aberration was seen in at least one tumor.For each sample, a range of 25 to 50 intact and non-overlapping interphase nuclei were enumerated manually using a 100×oil immersion lens on a Zeiss Z1 microscope . 2 scaled and mean-centered across each gene.Agilent 44 K whole human genome expression oligonucleotide microarrays  were used to profile prostate cancer xenografts and human castration-resistant soft tissue metastases of prostate. Freshly frozen xenografts were processed to extract total RNA which was amplified one round; patient samples were laser-capture microdissected and amplified two rounds as described previously AR and PTEN for patients with metastatic disease, hypothesizing that prostate tumors could differ from contemporaneous metastatic lesions. Linear mixed models with random patient effects were fitted to non-prostate tumors, and a 95% confidence interval calculated for subject-specific P-values were two-sided; statistical analyses were conducted using SAS/STAT software, version 9.3 .To complement the comparisons of archived primary tumor with a separate cohort of patients with metastatic disease, we examined within-patient heterogeneity of The three-marker FISH panel  used in AR, PTEN or TMPRSS2/ERG; 11 (32%) were abnormal by one marker only. Six patients’ tumors (18%) were detected abnormal by two markers, including 3 with TMPRSS2:ERG fusion and homozygous PTEN deletion, 2 with TMPRSS2:ERG fusion and heterozygous PTEN deletion, and 1 with non-fusion alternative rearrangement along with heterozygous PTEN deletion. None of the patients were abnormal by all three markers because there was no detectable AR abnormality when the cutoff for AR gain was set to > = 2.0 AR per nucleus, an arbitrarily determined stringent cutoff. Two patients would be classified as mild AR gain if using AR copy number per nuclei > = 1.5 as the cutoff value, established as mean+3SD based on enumeration results on normal prostate epithelial cells from 18 different samples.Of the 34 primary tumors in which all 3 markers could be assessed, 16 (47%) exhibited no aberrations involving TMPRSS2/ERG and AR FISH and 5 (17%) by TMPRSS2/ERG and PTEN. Eight patients (27%) were abnormal by all three tests.Of the 30 Met/CRPC patients/xenografts with FISH results from all three markers, 4 (13%) had no abnormal marker values. Five (17%) were shown as abnormal by one marker only; 13 (43%) were detected as abnormal by two markers, including 8 (27%) shown as abnormal by TMPRSS2 and/or ERG were detected in 14 patients (47%), including 5 (17%) with the typical single TMPRSS2:ERG fusion, 5 (17%) with dual or complex TMPRSS2:ERG fusion, and 4 (13%) with alternative rearrangements without fusion. Copy number increase (CNI) of chromosome 21 was observed in 10 patients (33%) using the TMPRSS2/ERG FISH probes. AR gain in one or more lesions was observed in 18 patients (60%), including 6 (20%) that resulted from gain of the X-chromosome and 12 (40%) with true AR gene amplification (AR/X > = 2). Deletion of PTEN was detected in 15 patients (50%), including 5 with homozygous deletion.We further evaluated subtypes of genetic aberrations detected by each marker in the Met/CRPC cohort  B–D. ReaP<0.001). AR gain, including moderate gain , and the combination of TMPRSS2/ERG and PTEN alterations  were also significantly more common in the Met/CRPC tumors.A Wilcoxon rank sum test suggested that the Met/CRPC cohort (n = 30) generally had more alterations detected by FISH than the cohort of primary cancers (N = 34) . This difference appeared to be due to the CNI aberration rather than the TMPRSS2:ERG fusion itself; the percentage of patients with fusion or alternative rearrangement remains similar, but the percentage of patients with dual fusion as opposed to single fusion is clearly greater in the Met/CRPC category than in the primary tumor group (P = 0.05). While nearly all primary cancer patients showed normal AR status, over 70% of Met/CRPC patients demonstrated various degrees of AR gene copy number gain . Of note, for patient-level assessments there was a hierarchy, so if one lesion was heterozygous and the other homozygous, the patient level was considered homozygous.The investigation of individual markers reflected unique trends of changes of each genetic abnormality during the progression of prostate cancer. About 80% of Met/CRPC samples were identified by nificant   also showed an abnormal TMPRSS2/ERG FISH result (PTEN deletion (93%) also showed abnormalities in TMPRSS2/ERG FISH analysis. In Met/CRPC patients, abnormal TMPRSS2/ERG FISH results were also more prevalent among patients demonstrating gain of AR, and vice versa  with TMPRSS2/ERG abnormalities also demonstrated AR gain. Detailed FISH results on all metastatic samples from each CRPC patient are summarized in Data of the entire panel across different individuals showed that prostate cancer patients with ERG FISH . Among tH result . Among tce versa . SixteenAR copy number , prostate tumors did not differ from metastatic lesions in abnormal vs normal marker signals, but were outside of the 95% confidence interval for the subject-specific average based on linear mixed models fit to metastatic lesions.The 3-marker FISH analyses yielded two observations from patients with metastatic prostate cancer: (1) within the same patient, aberrations in metastatic tumors were generally consistent across tumors; (2) several primary prostate tumors of CRPC patients exhibited a profile distinct from distant metastatic sites. FISH analyses for y number  and PTEN10 ratio  showed dAR and 0.82 (95% CI 0.62–0.92) for PTEN/CEP10. However, when the entire panel was evaluated for each individual, 4  out of 8 patients (50%) with data available from all three markers in the local prostate tumors showed different profiles between the primary and metastatic tumors. Further comparison using data of individual markers showed different levels of deviation of the primary from metastatic tumors. Of the 8 patients with available TMPRSS2/ERG FISH results on prostate site tumors, 3 (37.5%) had results in the prostate different from those in other metastatic sites  out of 10 patients (30%) showed results in the prostate that deviated from extra-prostatic tumors. The assessment of PTEN deletion showed that 2 (#3 and #11) out of 9 patients (22.2%) demonstrated different PTEN FISH results between prostate site and metastatic tumors. In patient #11, all extra-prostatic metastasis showed homozygous PTEN deletion, while prostate tumors showed normal PTEN results. Similarly, the panel data for xenografts also indicated that xenograft lines derived from the same patient tend to show the same genomic abnormality  for average ic sites . Interesormality .AR copy number for prostate site tumors. Linear mixed models predicted AR copy number at the cell level by tumor type, with random patient effects. AR per cell, and for the covariance parameter estimates that show how within-tumor variation and measurement error differ between tumor types. Prostate site tumors had the highest estimated within-person AR standard deviation (1.43 AR per cell). Several prostate tumors and lymph node metastasis had some unusually high counts that may have contributed to the estimate. For the PTEN/CEP10 ratio, the covariance estimates were also found to be heterogeneous by tumor type , but within-patient prostate PTEN/CEP10 intratumoral heterogeneity was not different from that of metastasis . 26 = 412, P<0.001), and a model that distinguished between prostate tissue and other lesions .We next sought to evaluate variation in genomic alterations detected by the FISH panel in individual cells comprising a primary or metastatic tumor. We found substantial intratumor variation in In order to investigate the functional relationship of genetic aberrations detected by our panel, we correlated our FISH findings with gene expression data from 91 matching Met/CRPC samples, including 65 patient tumors and 26 xenografts .AR copy number, determined by FISH, with the AR transcript abundance, determined by cDNA microarray, from the same tumor sample. We observed a wide range of AR expression in Met/CRPC tumors (AR per nucleus and the level of relative AR mRNA were positively correlated with rho = 0.52 (P<0.001) , samples with AR gain (n = 48), including gain of X (n = 17) and AR amplification (n = 31) expressed AR mRNA at 2.5±0.3-fold (Mean±S.E.) higher than the median, while tumors without AR gain (n = 40) had AR mRNA level as 0.7±0.2-fold comparing to the median . When tumors with AR gain were further divided into groups of gain of X (n = 17) vs AR gene amplification (n = 31), our data showed that AR mRNA was expressed at a similar level between the two .We first compared C tumors . The aveP<0.001) . When noTMPRSS2:ERG fusion on ERG mRNA levels and evaluated whether ERG expression also associated with the AR abundance in Met/CRPC tumors  expressed significantly higher ERG mRNA at 910.8±3.2 fold relative to probe median . Copy number increase (CNI) of ERG (or of both TMPRSS2 and ERG without fusion) did not associate with higher ERG mRNA expression.We then assessed the effect of  n = 80, . Fusion-ERG expression in the context of a TMPRSS2:ERG fusion is regulated by AR activity, we evaluated the effect of AR on ERG expression in Met/CRPC tumors with and without TMPRSS2:ERG fusion. While both the fusion-positive and fusion-negative samples showed a significant correlation between AR mRNA and ERG mRNA expression, this correlation appeared stronger in fusion-positive samples  than in fusion-negative samples . This correlation was further confirmed by a dichotomized comparison of ERG expression levels for the 38 fusion-positive samples between low- and high- AR mRNA expression groups using the median probe intensity as a divider. The low AR expressing tumors (n = 20) expressed ERG at 3.6±1.1 fold relative to the probe median, while the high AR expressing tumors (n = 18) expressed ERG at 21.2±5.8 fold of probe median (P<0.01).As TMPRSS2:ERG fusion in prostate cancer, we employed the strategy of a three-marker FISH panel to detect well documented prostate cancer DNA aberrations, including TMPRSS2/ERG rearrangements, AR copy number gain, and PTEN deletion. This panel clearly detected a significant number of genetic abnormalities in prostate carcinomas, 53% in primary tumors and 87% in Met/CRPCs. At the individual tumor level, the odds of a Met/CRPC tumor being abnormal were 4.5 times greater than that for a primary tumor. Collectively, if aberrations in these genomic loci associate with aggressive tumor behavior, then this three-marker FISH panel may be a useful tool in distinguishing high-risk patients from low-risk ones at diagnosis or in repeat assessments using active surveillance strategies. In addition, this approach may be particularly useful in characterization of circulating and disseminated tumor cells (CTC/DTC) as using fewer cells for analysis and getting data on three specific markers would be a significant advantage. The utility of these three markers is further supported by findings from a recent study using whole exome and transcriptome sequencing technologies AR and PTEN had the highest level of copy number gains and losses, respectively, in prostate cancer, especially CRPC. Their integrated genomic approach also demonstrated the interplay of these genomic alterations with TMPRSS2/ERG rearrangements. For each individual marker, our study detected similar abnormality rates as reported in the literature. For rearrangements of TMPRSS2 and/or ERG, previous findings showed ERG rearrangements in 30–50% of localized prostate cancers TMPRSS2 and/or ERG simultaneously in a single hybridization, we found the similar prevalence for TMPRSS2:ERG fusion, as well as non-fusion alternative rearrangements in 10–12% patients in both groups. However, dual/complex TMPRSS2:ERG fusion, which has been shown to associate with poor survival, occurs with a substantially greater frequency in Met/CRPC patients (17%) than in primary cancer patients (3%). Similarly, copy number increase (CNI) of TMPRSS2 and ERG without fusion was more frequent in Met/CRPC patients (33%) than in primary cancer patients (3%), suggesting increased genetic instability as the disease progresses, which was also observed in our studies of disseminated tumor cells obtained from prostate cancer patients Because of the controversial prognostic utility of PTEN loss ranging from less than 20% to nearly 70% in early stage prostate cancer PTEN deletion. Setting the cutoffs  as 10% for homozygous and 40% for heterozygous deletion, Reid and colleagues identified 17% of untreated primary prostate cancers exhibiting heterozygous or homozygous deletion of PTENPTEN deletion in 39% and 5% prostate cancer patients, respectively PTEN deletion in 21% of primary cancer patients and 47% of the CRPC patients based on the average ratio of PTEN/CEP 10 signals. Similar to previous findings PTEN deletion tumors also tended to harbor TMPRSS2/ERG abnormalities . These data argue for using the absolute AR copy number alone to define AR gain/amplification in FISH studies regardless of the AR/X ratio. Consequently, using the cutoff of 2.0 for AR gain would mean that a tumor would be considered abnormal for AR gain when AR copy number is at least doubled  in 100% of the cells, which might be too stringent a criterion and explains why AR gain was never reported previously in primary prostate cancer. Our experimental cutoff based on signal patterns seen in a series of normal controls was 1.48. Therefore, we used the 1.50 cutoff for moderate AR gain in AR gain in 50% of the cells would be considered abnormal. With this cutoff, we observed 6% of primary patients and 77% of Met/CRPC patients with AR gain. This should be a better definition for AR gain and may allow identification of primary prostate cancer patients with high risk for disease progression. Supporting evidence came from LuCaP 96 (AR gain (1.52 AR per nucleus). Its castration-resistant derivative line LuCaP 96CR showed clear AR amplification (5.72 AR per nucleus). The original patient indeed had aggressive disease and died from prostate cancer. The caveat, however, is that the xenograft data may not faithfully represent the original tumor genomics due to potential selection pressures over time on the xenograft specimens.Our findings on ding 2.0 . There wA levels . We alsoLuCaP 96 , a xenoget al. identified a strong association between ERG protein staining using immunohistochemistry and the TMPRSS2:ERG fusion status defined by FISH ERG mRNA expression was significantly correlated with the presence of TMPRSS2:ERG fusion. We also demonstrated a strong positive correlation between AR copy number gain and increased level of AR mRNA expression, supporting previous studies which showed higher levels of AR protein expression in prostate tumors with AR gene amplification than tumors without AR amplification AR mRNA, we found higher AR mRNA levels in tumors with simple gain of X-chromosome, the amplitude of which could not be differentiated from tumors with true AR gene amplification. The similarity in AR mRNA levels in these two groups may in part be due to the nature of transcriptome array analyses, where the quantification of very high levels of AR mRNA reaches a plateau.Chaux PTEN deletion and TMPRSS2/ERG rearrangements in animal models PTEN gene deletion and deregulation of p-AKT as well as AR protein expression in advanced localized prostate cancer TMPRSS2:ERG fusion and PTEN gene deletion in prostate cancer TMPRSS2/ERG abnormalities in tumors with either PTEN deletion or AR gain Click here for additional data file.


Mutation of Lys65 to Ala, Glu and Gln significantly attenuated, whereas mutation of Lys65 to Arg strongly augmented α2A-AR expression at the cell surface. Consistent with the effects on the cell-surface expression of α2A-AR, mutation of Lys65 to Ala and Arg produced opposing effects on α2A-AR-mediated ERK1/2 activation. Furthermore, confocal microscopy revealed that the α2A-AR mutant K65A displayed a strong intracellular expression pattern and was extensively co-localized with the ER marker DsRed2-ER, suggestive of ER accumulation. These data provide the first evidence indicating an important function for a single Lys residue on the ICL1 in the ER export and cell-surface expression of α2A-AR. These data also suggest that the ICL1 may possess multiple signals that control the cell-surface targeting of GPCRs via distinct mechanisms.Export from the endoplasmic reticulum (ER) represents an initial step in intracellular trafficking of G protein-coupled receptors (GPCRs). However, the underlying molecular mechanisms remain poorly understood. We have previously demonstrated that a highly conserved Leu residue on the first intracellular loop (ICL1) is required for exit of several GPCRs from the ER. Here we found that, in addition to Leu64 residue in the ICL1, the neighboring positively charged residue Lys65also modulates the cell-surface transport of α As the first step in post-translational biogenesis, the efficiency of ER export of nascent GPCRs plays a crucial role in the regulation of maturation, cell-surface expression, and physiological functions of the receptors G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface receptors and regulate the cellular responses to a broad spectrum of extracellular signals, such as hormones, neurotransmitters, chemokines, proteinases, odorants, light and calcium ions Great progress has been made on the understanding of GPCR export from the ER over the past decade 2-AR subtypes, designated as α2A-AR, α2B-AR, and α2C-AR. It has been known that both α2A-AR and α2B-AR mainly express at the cell surface, whereas α2C-AR cell-surface expression depends on the cell types 6LL motif in the C-terminus, the RRR motif in the third intracellular loop (ICL3), and an isolated Leu residue in the ICL1, which are essential for export trafficking of α2B-AR 2-AR transport, we expanded our studies to define the role of the ICL1 in the cell-surface expression of α2A-AR. Surprisingly we found that, in addition to Leu residue, a neighboring Lys residue specifically modulates the ER export and cell-surface expression of α2A-AR and this function is likely dictated by its positively charged property. These data provide the first evidence indicating that the ICL1 may possess multiple signals that use distinct mechanisms to control the processing of α2A-AR.There are three α3H]-RX821002 (specific activity = 50 Ci/mmol) was from Perkin Elmer Life Sciences. All other materials were obtained as described previously Antibodies against ERK1/2 and phospho-ERK1/2 were from Cell Signaling Technology . The ER marker pDsRed2-ER was purchased from BD Biosciences . Prolong antifade reagent with DAPI was obtained from Invitrogen Life Technologies . UK14,304 was from Sigma . -RX821002 as described previously 3H]-RX821002 in a total of 300 µl for 60 min at room temperature. In the initial experiments, the cells were incubated with increasing concentrations of [3H]-RX821002 (from 0.3125 to 20 nM) to generate ligand dose-dependent binding curves. Because ligand binding to the receptors is almost saturated at a concentration of 20 nM, this concentration was then used to measure the cell-surface expression of the receptors. The binding was terminated and excess radioligand eliminated by washing the cells twice with ice-cold DMEM. The retained radioligand was extracted by digesting the cells in 1 M NaOH for 2 h at room temperature. The liquid phase was collected and suspended in 4 ml of Ecoscint A scintillation fluid . The amount of radioactivity retained was measured by liquid scintillation spectrometry. The non-specific binding of α2-AR was determined in the presence of rauwolscine (10 µM). All radioligand binding assays were performed in triplicate.Cell-surface expression of α6 cells/ml. Total GFP fluorescence was then measured on a flow cytometer  as described previously (38). For measurement of the cell-surface expression of α2A-AR, HEK293 cells were cultured on 6-well dishes and transfected with 1 µg of HA-tagged α2A-AR for 24 h. The cells were then collected, suspended in PBS containing 1% FBS and incubated with high affinity anti-HA-fluorescein (3F10) at a final concentration of 2 µg/ml at 4°C for 60 min. After washing for 2×0.5 ml PBS containing 1% FBS, the cells were re-suspended and the fluorescence was analyzed as described above.For measurement of total receptor expression, HEK293 cells cultured on 6-well dishes were transiently transfected with 1 µg of GFP-tagged receptors for 24 h. The cells were collected, washed twice with PBS and re-suspended at a density of 8×10For fluorescence microscopic analysis of receptor subcellular distribution, HEK293 and HeLa cells were grown on coverslips pre-coated with poly-L-lysine in 6-well plates and transfected with 100 ng of GFP-tagged receptors. For co-localization of GFP-tagged receptors with the ER marker DsRed2-ER, HEK293 cells grown on coverslips were transfected with 100 ng of GFP-tagged receptors and 100 ng of pDsRed2-ER. The cells were fixed with 4% paraformaldehyde-4% sucrose mixture in PBS for 15 min and the coverslips were mounted with prolong antifade reagent containing DAPI. Images were captured using a Zessis confocal microscope (LSM510) equipped with a 63x objective (NA = 1.3). The colocalization of the receptor with the ER marker DsRed2-ER was determined by Pearson’s coefficient using the ImageJ JaCoP plug-in as described 2A-AR or its mutants as described above. After 24 h transfection, the cells were starved for at least 3 h and then stimulated with different concentrations of UK14,304  for 5 min. Stimulation was terminated by addition of 150 µl of ice-cold cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate and protease inhibitor cocktail (Roche). After solubilizing the cells on ice for 20 min, 10 µl of total cell lysates was separated by 12% SDS-PAGE and ERK1/2 activation was determined by measuring the levels of phosphorylation of ERK1/2 with phospho-specific ERK1/2 antibodies by immunoblotting HEK293 cells were cultured in 6-well dishes and transfected with 1 µg of αp<0.05 was considered as statistically significant. Data are expressed as the mean ± S.E.Differences were evaluated using one-way ANOVA and post-hoc Tukey’s test, and 2-AR subtype among different species but different between three α2-AR subtypes. The ICL1 of α2A-AR, α2B-AR and α2C-AR have the sequence of ALK, SLR and ALR, respectively (2B-AR 2A-AR. We first measured the saturation binding of the radioligand [3H]-RX821002 to α2A-AR in intact live HEK293 cells. The ligand dose-dependently bound to α2A-AR and the binding was close to saturation at 20 nM (The amino acid sequences of the ICL1 are highly conserved in each αectively . We haveat 20 nM .2A-AR and its mutants L64A, K65A and LK-AA were transiently expressed in HEK293 cells and their cell-surface expression at steady state was measured by intact cell ligand binding using [3H]-RX821002 at 20 nM. Consistent with the remarkable inhibitory effect of mutation of Leu48 on α2B-AR cell-surface expression, mutation of Leu64 markedly reduced the cell-surface number of α2A-AR by 87%. Surprisingly, in contrast to mutation of Arg49 which did not have significant effects on α2B-AR cell-surface expression, mutation of Lys65 to Ala significantly attenuated α2A-AR expression at the cell surface by 52%. Double mutation of Leu48/Arg49 in α2B-AR and Leu64/Lys65 α2A-AR almost abolished their cell-surface transport (2A-AR binding to the ligand, α2A-AR and its mutants were tagged with HA at their N-termini and their cell-surface expression was measured by flow cytometry following staining with anti-HA antibodies in nonpermeabilized cells. The cell-surface expression of the mutants L64A, K65A and LK-AA was reduced by 81, 58 and 93%, respectively, as compared with their wild-type counterpart (Wild-type αransport . To exclnterpart .2A-AR and α2B-AR and their mutants tagged with GFP at their C-termini were transiently expressed in HEK293 cells and their overall expression was determined by flow cytometry measuring the GFP signal. In contrast to the cell-surface expression, these mutations did not significantly alter the overall expression of α2A-AR (2B-AR (2A-AR and α2B-AR.To determine if these mutations could alter the total expression of the receptors, αf α2A-AR  and α2B-R (2B-AR . These d2A-AR, the subcellular distribution of GFP-tagged α2A-AR and its mutants in HEK293 cells was visualized by confocal microscopy. As expected, wild-type α2A-AR was robustly expressed at the cell surface. Mutation of Leu64 and Lys65 to Ala individually or in combination caused a remarkable accumulation of α2A-AR in the perinuclear region (To further confirm the inhibitory effect of mutation of Leu64 and Lys65 on the cell-surface transport of αr region .2A-AR is cell-type specific, GFP-tagged α2A-AR and its mutants were transiently expressed in HeLa cells. Similar to the results obtained in HEK293 cells, the α2A-AR mutants L64A, K65A and LK-AA displayed a strong intracellular distribution pattern in HeLa cells (2A-AR cell-surface expression in different cell types.To determine if the effect of the mutations on the subcellular distribution of αLa cells . These d2A-AR cell-surface expression. To define the possible underlying molecular mechanisms, we first determined the role of its positively charged property. Lys65 was mutated to the same charged Arg residue, opposite charged Glu residue and non-charged Gln residue and the effects of these mutations on α2A-AR expression at the cell surface were defined by intact cell ligand binding and subcellular distribution analysis. Similar to its Ala mutation, mutation of Lys65 to Glu and Gln inhibited α2A-AR expression at the cell surface by 67 and 36%, respectively, as measured by intact cell ligand binding (2A-AR by 42% (2A-AR and its mutants K65R, K65E and K65Q (Our preceding data have revealed that Lys65 plays an important role in modulating α binding . More inR by 42% . Similarand K65Q .2A-AR and its mutant K65R strongly expressed at the cell surface, whereas the mutants K65E and K65Q were largely accumulated inside the cell, unable to transport to the cell surface in both HEK293 and HeLa cells (Consistent with the measurement of the cell-surface expression by ligand binding and flow cytometry, analysis of the subcellular distribution of the receptors showed that wild-type αLa cells .2A-AR by using the agonist-mediated activation of ERK/12 as readout. HEK293 cells were transiently transfected with α2A-AR and its mutants K65R and K65A and their abilities to activate ERK1/2 in response to stimulation with the α2-AR agonist UK14,304 were compared. ERK1/2 were activated by UK14,304 in a dose-dependent fashion in cells expressing α2A-AR and the dose-dependent activation of ERK1/2 was clearly inhibited in cells transfected with the mutant K65A (2A-AR (2A-AR. These data also suggest that Lys65 in the ICL1 modulates not only receptor trafficking but also receptor signaling.We then sought to determine if the mutation of Lys65 could alter the function of αant K65A . In contA (2A-AR . These d2A-AR transport, GFP-tagged α2A-AR and its mutants K65A and K65R were co-localized with different intracellular markers. The mutant K65A was extensively co-localized with the ER marker DsRed2-ER (2A-AR and its mutant K65R did not clearly co-localize with DeRed2-ER (2A-AR and the mutant K65R (2A-AR out of the ER.To define the intracellular compartment where the residue Lys65 influences αsRed2-ER , but noteRed2-ER . To quanant K65R . These d2B-AR 2A-AR export from the ER and transport to the cell surface. Thus, this isolated Leu may provide a common signal directing anterograde transport of multiple nascent GPCRs.The molecular mechanisms underlying export from the ER and subsequent transport to the cell surface of the GPCR superfamily remain poorly elucidated. It has been significant efforts to define the structural determinants for GPCR export and several highly conserved hydrophobic and charged sequences which are required for GPCR export from the ER to the cell surface have been identified 2A-AR. First, intact cell ligand binding and flow cytometry to quantify the cell-surface receptors showed that mutation of Lys65 to Ala reduced α2A-AR cell-surface number by more than 50%. Second, the significant reduction of receptor expression at the cell surface was supported by direct visualization of intracellular localization of the mutated receptors. As revealed by confocal microscopy, GFP-tagged K65A was extensively expressed inside the cell which is in contrast to robust cell-surface expression of its wild-type counterpart. Third, the function of Lys65 in controlling α2A-AR transport is also supported by receptor-mediated signal propagation as measured by ERK1/2 activation. The reduction of ERK1/2 activation in cells expressing K65A as compared with cells expressing wild-type α2A-AR could be simply due to less expression of the mutated α2A-AR at the cell surface, which are available for binding to the ligand, indicating that mutation of Lys65 not only reduced the cell-surface expression of α2A-AR but also attenuated receptor-mediated signal propagation. These data strongly indicate that, in addition to Leu64, Lys65 in the ICL1 also plays a crucial role in regulating cell-surface transport of α2A-AR. By contrast, mutation of the positively charged residue at the same position, Arg49, did not influence the cell-surface transport of α2B-AR. These data demonstrated for the first time that a single positively charged residue on the short ICL1 is involved in the regulation of export trafficking of GPCRs in a receptor subtype-specific fashion.The most important finding described in this manuscript is that different positively charged residues on the ICL1 have differential impacts on the cell-surface transport of distinct GPCRs. During the studies on the function of the ICL1, we have surprisingly found that mutation of Lys65 produced a marked inhibitory effect on the cell-surface transport of α2A-AR transport at the level of the ER. We found that the α2A-AR mutant K65A was extensively co-localized with the ER marker DsRed2-ER, suggesting that the mutant was unable to exit from the ER where they are synthesized. Therefore, these studies provide another novel regulatory mechanism for the ER export of nascent α2A-AR. As this single Lys residue also exists in several other group A GPCRs, including angiotensin II, muscarinic and chemokine receptors 6LL, RRR and YS motifs 2-AR is coordinated by many structural determinants and export motifs located in the intracellular domains of the receptors.It is likely that Lys65 residue modulates α2A-AR is likely dictated by its specific physiochemical and structural features. First, our data demonstrating that mutation of Lys65 to Ala, Glu and Gln significantly inhibited α2A-AR export from the ER and transport to the cell surface suggest that the positively charged property is an important factor determining the function of Lys65 in α2A-AR export. It is possible that Lys65 plays a role in the correct folding or proper assembly of α2A-AR in the ER and thus, its mutation results in the misfolding and defective export of the receptor. It is also possible that Lys65 may mediate α2A-AR interaction with other regulatory proteins and such interactions are crucial for the receptor export from the ER. Consistent with this possibility, a number of accessory proteins directly interact with the intracellular loops of GPCRs to modulate receptor export from the ER to the cell surface 2B-AR interaction with Sec24 isoforms, components of COPII transport vesicles, to control receptor export from the ER The function of Lys65 residue in regulating the ER export and cell-surface expression of α2A-AR. There are at least two possible explanations for the enhancement of the cell-surface expression of α2A-AR induced by mutation of Lys65 to Arg. It is possible that mutation of Lys to Arg stabilizes α2A-AR once transported to the cell surface. As the overall size of the side chain of Arg is larger than that of Lys, it may enhance the possible ionic interactions between the positively charged side chain and some negatively charged components embedded within the plasma membrane. It is also possible that mutation of Lys to Arg may reduce the targeting of the cell-surface α2A-AR to some degradation pathways, such as those mediated by ubiquitination and/or sumoylation which are carried out specifically on Lys residues. However, whether or not Lys65 residue indeed undergoes ubiquitination and/or sumoylation remains unknown. Nevertheless, this study has demonstrated a crucial role for Lys65 residue in regulating the ER export and cell-surface expression of α2A-AR, yet the precise mechanisms of its actions remain to be elucidated.Second, more interestingly, mutation of Lys65 to the same charged Arg residue enhanced the cell-surface number of αen route from the ER through the Golgi is tightly associated with the pathogenesis of a variety of human diseases, including those induced by naturally occurring mutations or truncations of GPCRs, leading to the accumulation of misfolded receptors in the ER It has become increasingly clear that the efficient trafficking and precise positioning to specific functional destination of GPCRs are critical aspects in controlling integrated responses of the cell to hormones. Indeed, defective anterograde transport of GPCRs to the cell surface


Constitutively active androgen receptor variants (AR-V) lacking the ligand binding domain (LBD) may promote the development of castration-resistant prostate cancer (CRPC). The expression of AR-Vs in the clinically most important metastatic site, the bone, has, however, not been well documented. Our aim was therefore to compare levels of AR-Vs in hormone-naive (HN) and CRPC bone metastases in comparison to primary PC and non-malignant prostate tissue, as well as in relation to AR protein expression, whole-genome transcription profiles and patient survival.Hormone-naïve (n = 10) and CRPC bone metastases samples (n = 30) were obtained from 40 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomized men. Levels of full length AR (ARfl) and AR-Vs termed AR-V1, AR-V7, and AR-V567es mRNA were measured with RT-PCR and whole-genome transcription profiles with an Illumina Beadchip array. Protein levels were examined by Western blotting and immunohistochemistry. Transcripts for ARfl, AR-V1, and AR-V7 were detected in most primary tumors and metastases, and levels were significantly increased in CRPC bone metastases. The AR-V567es transcript was detected in 23% of the CRPC bone metastases only. A sub-group of CRPC bone metastases expressed LBD-truncated AR proteins at levels comparable to the ARfl. Detectable AR-V567es and/or AR-V7 mRNA in the upper quartile, seen in 1/3 of all CRPC bone metastases, was associated with a high nuclear AR immunostaining score, disturbed cell cycle regulation and short survival.Expression of AR-Vs is increased in CRPC compared to HN bone metastases and associated with a particularly poor prognosis. Further studies are needed to test if patients expressing such AR-Vs in their bone metastases benefit more from drugs acting on or down-stream of these AR-Vs than from therapies inhibiting androgen synthesis. Androgens regulate normal prostate tissue growth and differentiation, through activation of androgen receptors in epithelial and stroma cells, and also play an important role during all phases of prostate cancer (PC) growth. The standard therapy for patients with advanced PC is consequently to reduce androgens, either by surgical or medical castration. After a period of initial remission tumors eventually relapse, predominantly within the bone, and are then termed castration-resistant PC (CRPC). The mechanisms controlling CRPC growth in bone metastases are however largely unknown and seldom explored by actually examining such metastases in patients.Interestingly, the androgen receptor (AR) is commonly active in CRPC despite castrate levels of circulating testosterone AR gene is composed of eight exons encoding a 110 kDa protein consisting of a NH2 terminal trans-activation domain (NTD), a DNA binding domain (DBD), a hinge region, and the COOH terminal domain (CTD) which is also the ligand-binding domain (LBD) AR gene Structurally the human The aim of this study was therefore to analyze expression of the full length AR (ARfl) and its clinically most abundant splice variants here termed AR-V1, AR-V7 The study was approved by the local ethic review board of Umeå University and participants gave written or verbal consent.Bone metastases were obtained from a series of fresh-frozen and formalin fixed paraffin embedded biopsies collected from patients with PC operated for metastatic spinal cord compression or pathologic fractures at Umeå University Hospital (2003–2009). These patients have been thoroughly described in Representative areas of bone metastases, primary PC and non-malignant prostate tissue were cryo-sectioned into extraction tubes and RNA was isolated using the Trizol protocol . The percentage of tumor cells in the samples varied between 25–95%. The RNA concentrations were quantified by absorbance measurements using a spectrophotometer . The RNA quality was analyzed with the 2100 Bioanalyzer  and verified to have a RNA integrity number ≥6.5′-CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3′ and 5′-AGCTTCTGGGTTGTCTCCTCAGTGG-3′, AR-V1: 5′-CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3′ and 5′-CTGTTGTGGATGAGCAGCTGAGAGTCT-3′, AR-V7: 5′-CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3′ and 5′-TTTGAATGAGGCAAGTCAGCCTTTCT-3′, and AR-V567es: 5′- CCAAGGCCTTGCCTGATTGC and 5′-TTGGGCACTTGCACAGAGAT-3′, resulting in amplicons of 143, 145, respectively 125 bp, as previously described 5′-GTACGCTGTGAAGGCATCAA-3′ and 5′- GTTGGTGTTCATCCGCTTG-3′) amplified a 125 bp fragment. Each sample was run in duplicates and adjusted for corresponding RPL13A levels. Melting curve analysis confirmed the amplified products, which were also analyzed by 1% agarose gel electrophoresis to confirm the expected size (data not shown).Two-hundred (200) ng of RNA were reversed transcribed with Superscript II reverse transcriptase (Invitrogen) in a total volume of 10 µl. The subsequent PCR amplification was performed using the Biorad iQ5 iCycler . Reactions were done in a 20 µl volume using the IQ SYBR Green Supermix (Bio-Rad laboratories). The following primer sets were used; ARfl: Fresh-frozen bone metastases and prostate samples were cryo-sectioned and proteins were extracted from 20 sections (10 µm each) using the AllPrep Mini kit, according to the manufacturer's instructions . Protein concentration was determined by the BCA Protein assay . 22RW1 cells were maintained according to manufacturer's instructions (ATCC) and proteins were extracted as previously described Samples (20 µg protein) were separated by 7.5 SDS-PAGE under reducing conditions and subsequently transferred to PVDF membranes . The membranes were stained with Ponceau red to confirm equal sample loading and transfer (results not shown). Membranes were then blocked in 5% milk followed by anti-AR antibody incubations; N-20  or PG-21  in order to detect the ARfl and AR-Vs, and C-19 (Santa Cruz) in order to detect ARfl but not AR-Vs lacking the LBD. Primary antibodies were diluted in 1% milk/PBST and incubated in 4°C over night. The secondary anti-rabbit IgG  antibody (diluted 1∶20 000 in 2.5% milk) was applied after washing in PBST and incubated for 1 h in RT. Protein expression was visualized after extensive washing using the ECL Advanced detection kit  and quantified with a ChemiDoc scanner and the Quantity One 4 software (Bio-Rad Laboratories).The bone metastases examined in this study have been previously immunostained for the AR, Ki67 (cell proliferation), activated caspase 3 (apoptotic cells) and PSA antigens  followinP<0.05) and defined to have a fold change in-between groups greater than 1.5. Gene ontology analysis was done with the Metacore software .Two-hundred-fifty (250) ng of total RNA per sample was used for cRNA production by the Illumina TotalPrep RNA amplification kit  according to the provided protocol. The quality of cRNA was evaluated using the RNA 6000 pico kit (Agilent Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies). A total of 750 ng cRNA was used for hybridization to a human HT12-v3 Illumina Beadchip gene expression array , including 48803 probes and 37846 annotated genes, according to the manufacturer's protocol. The arrays were scanned and fluorescence signals obtained using the Illumina Bead Array Reader . Array data analysis was performed with GenomeStudio software . Samples were normalized by the cubic spline algorithm and probes with all signals lower than two-times the mean background levels were excluded from the analysis. Differentially expressed genes were identified with the Mann-Whitney differential expression algorithm . A P-value less or equal to 0.05 was considered statistically significant.Transcripts for the normal ARfl and the most abundant AR splice variants known so far P≤0.001, There was a tendency for higher levels of ARfl, AR-V1, and AR-V7 mRNA in HN bone metastases than in primary PC tissue, but the differences were non-significant . Clearlyn = 13, data not shown). This was in contrast to corresponding ratios on the RNA level where the AR-V7 and AR-V567 transcripts represented in median only 0.4%  and 1.0% , respectively, of the ARfl mRNA level, according to difference in RT-PCR ct levels. Our results thus indicate that AR-Vs lacking the LBD domain are possible post-transcriptionally stabilized in CRPC bone metastases in relation to the ARfl.Proteins levels of AR-Vs were examined using Western blot analysis of 13 samples of CRPC bone metastases randomly selected to represent cases with high AR-V7 mRNA levels , cases with low and intermediate levels of AR-V7 mRNA  as well as detectable/non-detectable levels of AR-V567es mRNA . The 22Rn = 66) and in the sub-group of CRPC bone metastases . Bone metastases with detectable AR-V567es mRNA had about 3-fold higher ARfl median mRNA level than the other CRPC metastases (P = 0.004).The AR-V1, AR-V7, and ARfl mRNA levels were correlated in all samples examined .As we have previously found that high AR immunostaining scores were associated with a particularly short survival after metastasis surgery and now that AR-V high patients had a poor prognosis, we wanted to examine if high AR staining score was associated with levels of AR-Vs. The AR-V high metastases all had high AR staining scores in comparison with other CRPC bone metastases which showed variable staining ranging from weak to strong (P = 0.038) and tendencies of having higher fractions of proliferating and apoptotic tumor epithelial cells  than other CRPC metastases  and further included in ontology analysis for evaluation of enriched processes. Many (60) of the differentiating gene products were proposed to be directly interacting via the AR, C-MYC, and CDK1 proteins  probes were differentially expressed between the AR-V high bone metastases and other CRPC bone metastases according to a fold-change of at least 1.5 in-between groups  of the hinge region in exon 4 The enrichment of structurally different, constitutively active AR-Vs during the development of CRPC metastases points out complex and heterogeneous molecular events which by difference means seem to ensure AR activation in the absence of testicular androgens. Patients expressing constitutively active AR-Vs will in the long-term probably not benefit from anti-androgen therapies aiming to reduce steroid synthesis, such as surgical castration, LHRH analogs, or the newly introduced agent abiraterone that inhibits CYP17 and thereby steroid synthesis not only in testis but also in the adrenal glands and in tumor tissue in vitroIn the clinical samples examined in the present study, detectable levels of the AR-V567es and/or high levels of the AR-V7 transcripts were associated with deregulated levels of known AR-controlled genes, but specifically not of some classical androgen regulated genes such as KLK3 (PSA), TMPRSS2, and FKBP5 which were induced by over-expression of AR-V7 or AR-V567es in LNCaP cells In conclusion, LBD-truncated AR-Vs are frequently expressed in bone metastases, with higher levels in CRPC than in HN cases. Furthermore, transcript levels of the constitutively active AR-V7 and AR-V567es seem to be associated with high nuclear AR immunostaining scores, disturbed cell cycle control, and particularly poor prognosis within the group of CRPC patients. This conclusion is however based on a limited number of patients and results must therefore be verified in other studies. If the results hold true patients expressing high levels of AR-V7 and AR-V567es probably will need to be treated with drugs acting on or down-stream of the AR-Vs rather than with drugs inhibiting androgen synthesis.Table S1a between the AR-V highb group and the other CRPC bone metastases.Differentially expressed genes(DOC)Click here for additional data file.


N-fucopentaose III (1) and its corresponding human serum albumin conjugate, is described. Key transformations of the strategy include two highly regio- and stereoselective glycosylations for the construction of disaccharide 10 and pentasaccharide 12, a Birch reduction for deprotection of benzyl ethers, and a UV-promoted radical addition of a thiol to an alkene for modification of the aglycone.A concise synthetic route to an immunomodulatory pentasaccharide, lacto- N-fucopentaose III (LNFPIII), a pentasaccharide containing the LewisX trisaccharide antigen, is an immunomodulatory glycan that is present on schistosome eggs. glycosylation, a key strategy in earlier routes to LNPFIII derivatives,undec-7-ene (DBU). The freshly made donor was then used in a reaction with diol acceptor 3 to give the pentasaccharide 12 in 49 % yield over two steps. The β stereochemistry of the newly formed linkage was established based on the 3JH−1, H−2 (8.5 Hz) and 1JC−1, H−1 (165.5 Hz) values. To confirm the regioselectivity, a small amount of pentasaccharide 12 was acetylated to generate 12′. Upon comparison of 1H NMR spectra of these two compounds, a broad signal at 4.05 ppm in 12 shifted to 5.46 ppm in 12’ and appeared as a doublet of doublets (J=3.6 and 0.6 Hz). The values of these coupling constants indicated that the acetyl group was introduced onto O-4’ of the lactose moiety, confirming that the newly introduced glycosidic linkage was β-(1→3) not β-(1→4).The final planned glycosylation reaction, coupling of trisaccharide anhydride was expl12 with ethylenediamine in n-butanol at 100 °C for 20 h  ester 1 in 83 % overall yield. Conversion of 1 into the corresponding human serum albumin (HSA) conjugate was then done by treatment of HSA with 1 in phosphate buffer (pH 7.5). The MALDI-MS spectrum of the resulting glycoconjugate showed two peaks, one centred around m/z=45 693 and another at m/z=91 377, corresponding to the +2 and +1 charge states of the protein, respectively, both bound to 21 pentasaccharide units.With the pentasaccharide assembled, treatment of h Scheme , followehe alkene was thenl adipate in dimetp-nitrophenol ester (PNP) and the corresponding human serum albumin (HSA) conjugate. The synthetic strategy features two highly regio- and stereoselective glycosylations for the construction of disaccharide 10 and pentasaccharide 12 based on the reactivity difference between two hydroxyl groups in acceptor 6 and 3, respectively. Birch reduction enabled the deprotection of benzyl ethers while leaving the octenyl linker intact, which allowed further functionalization to form PNP ester 1 and, in turn, the HSA conjugate. Studies on the use of this HSA conjugate in animal models of graft survival are ongoing.In conclusion, we have achieved the synthesis of LNPFIII functionalized with reactive General: All reagents were purchased from commercial sources and were used without further purification unless noted. Dry solvents used in reactions were purified by successive passage through columns of alumina and copper under an argon atmosphere. All reactions were carried out under a positive pressure of argon unless otherwise stated, monitored by TLC on silica gel 60 F254 , and the spots were visualized under UV light (254 nm) and/or stained by charring with acidified anisaldehyde solution in EtOH. Column chromatography was performed on silica gel 60 (40–60 μm) or C18 silica gel . 1H NMR spectra were recorded at 500 or 600 MHz, and chemical shifts were referenced to CHCl3 , CD2HOD (3.31 ppm CD3OD), or HOD . 13C NMR spectra were recorded at 126 MHz and chemical shifts were referenced to CDCl3 , CD3OD  or external acetone . Assignments of NMR spectra were made on the basis of 2D experiments , and the stereochemistry of the newly formed glycosidic linkages was confirmed by measuring 1JC−1,H−1 values using an 1H-coupled HSQC experiment. In the data provided below, the resonances on particular residues are indicated by an increasing number of primes (′) moving from the reducing to nonreducing end. For example, in 15 H-1 is H-1 of the Glc residue, H-1′′ is H-1 of the GlcNAc residue, and H-1′′′′ is H-1 of the Fuc residue. Electrospray ionization mass spectra were recorded on an Agilent Technologies 6220 TOF spectrometer on samples dissolved in CH2Cl2 or MeOH. MALDI mass spectra were obtained in the linear positive mode of ionization on a Bruker Daltonics  UltrafleXtreme MALDI TOF/TOF mass spectrometer using sinapinic acid as the matrix. Optical rotations were measured on PerkinElmer 241 polarimeter at 22±2 °C in units of degree mL/(g dm).d-galactopyranosyl-(1→4)-[α-L-fucopyranosyl-(1→3)]-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→3)-β-d-galactopyranosyl-(1→4)-β-d-glucopyranosyl-oxy)octyl]thiol]ethyl]amino]-6-oxo-hexanoic acid6-+ calcd for C54H87N3NaO30S: 1312.4987, found: 1312.4986.p-Tolyl 2,3,4–6-tetra-O-acetyl-β-d-galactopyranosyl-(1→4)--6-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (2): A mixture of thioglycoside 5 , acceptor 10  and powdered 4 Å molecular sieves was suspended in toluene (30 mL) and stirred at RT for 1 h. The solution was cooled to 0 °C, and N-iodosuccinimide  and silver trifluoromethanesulfonate  were added. After stirring at 0 °C for 2 h, Et3N (1 mL) was added, and the mixture was filtered through Celite. The filtrate was concentrated, and the resulting residue was subjected to flash chromatography (2:1 v/v hexane/EtOAc) to afford trisaccharide 2 as a white foam : Rf=0.24 (2:1 v/v hexane/EtOAc); [α]D=+7.1 ; 1H NMR : δ=7.75–7.70 , 7.45–7.01 , 5.44 , 5.26 , 5.04 , 4.84–4.79 , 4.77–4.72 , 4.65–4.60 , 4.58 , 4.53–4.48 , 4.46, 4.28 , 4.18–4.13 , 3.98 , 3.92–3.88 , 3.85–3.80  3.64 , 3.57–3.55 , 2.29 , 2.03 , 2.02 , 1.97 , 1.86 , 1.21 ppm ; 13C NMR : δ 170.0 (C=O), 169.8 (C=O), 168.7 (C=O), 138.9 (Ar), 138.7 (Ar), 138.2 (Ar), 138.1 (Ar), 137.9 (Ar), 134.2 (Ar), 133.3 (Ar), 131.8 (Ar), 129.6 (Ar), 128.6 (Ar), 128.3 (Ar), 128.24 (Ar), 128.17 (Ar), 128.1 (Ar), 128.02 (Ar), 127.97 (Ar), 127.86 (Ar), 127.4 (Ar), 127.2 (Ar), 127.1 (Ar), 127.0 (Ar), 123.7 (Ar), 99.5 (C-1′), 97.6 (C-1′′), 84.4 (C-1), 79.8 (C-3′′), 79.6 (C-5), 77.2 (C-4′′), 75.0 (C-4), 74.5 (C-2′′) 74.2 (OCH2Ph), 73.64 (C-3), 73.58, 73.0, 72.3 (3×OCH2Ph), 71.0 (C-3′), 70.4 (C-5′), 69.0 (C-2′), 67.85 (C-6), 66.76 (C-4′), 66.5 (C-5′′), 60.2 (C-6′), 55.6 (C-2), 21.1, 20.7, 20.62, 20.55, 20.54 ppm ; 16.7 (C-6′′); HRMS (ESI): m/z [M+Na]+ calcd for C69H73NNaO19S: 1274.4390, found: 1274.4385.O-benzyl-β-d-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (3)7-Octen-1-yl 2,6-di-: NaOMe  was added to a solution of 7  in CH2Cl2/MeOH . After stirring at RT overnight, the solution was neutralized with Amberlite IR120 H+ ion-exchange resin. The solution was filtered and the filtrate was concentrated to afford 8 as a white solid . A portion of 8  and p-TsOH (41 mg) were suspended in a mixture of dry N,N-dimethylformamide  and 2,2-dimethoxypropane (24 mL). After stirring at 85 °C for 1.5 h, the solution was cooled to RT, neutralized with Et3N (1 mL), concentrated in vacuo and dried. The resulting residue and BnBr  were dissolved in dry DMF (40 mL), to which NaH  was added in portions at 0 °C, followed by vigorous stirring. After stirring overnight at RT, MeOH (4 mL) was added. The solution was concentrated in vacuo, dissolved in EtOAc (200 mL) and washed with H2O and brine. The organic layer was dried over Na2SO4, filtered and concentrated to dryness. Subsequently, the obtained crude residue was treated with 80 % aq HOAc (160 mL) at 80 °C for 3 h. The solution was concentrated, dissolved in EtOAc (200 mL) and washed with saturated aq NaHCO3, H2O and brine. The organic layer was dried over Na2SO4, filtered, concentrated and the residue subjected to flash chromatography (5:2 v/v hexane/EtOAc) to afford diol 3 as a white solid : Rf=0.40 (2:1 v/v hexane/EtOAc); [α]D=+16.4 ; 1H NMR : δ=7.39–7.22 , 5.81 , 5.02–4.92 , 4.91, 4.72 , 4.82, 4.68 , 4.79 , 4.61 , 4.47–4.44 , 4.41–4.38 , 3.99 , 3.97–3.92 , 3.82 , 3.77 , 3.64–3.58 , 3.54–3.49 , 3.45–3.39 , 3.37–3.35 , 2.48 , 2.41 , 2.06–2.02 3C2HCH=CH2), 1.69–1.62 3C2HCH=CH2), 1.45–1.31 ppm 3C2HCH=CH2); 13C NMR : δ=139.2 (Ar), 139.1 (CH=CH2), 138.7 (Ar), 138.4 (Ar), 138.3 (Ar), 138.0 (Ar), 128.5 (Ar), 128.4 (Ar), 128.32 (Ar), 128.30 (Ar), 128.08 (Ar), 128.06 (Ar), 128.0 (Ar), 127.93 (Ar), 127.86 (Ar), 127.7 (Ar), 127.6 (Ar), 127.57 (Ar), 127.54 (Ar), 127.3 (Ar), 114.3 (CH=CH2), 103.7 (C-1), 102.6 (C-1′), 82.9 (C-3), 81.8, 80.1 , 76.7 (C-4), 75.2 (OCH2Ph), 75.1 (C-5), 74.93, 74.90 (2×OCH2Ph), 73.6 (C-3′), 73.5, 73.2 (2×OCH2Ph), 72.9 (C-5′), 70.0 (octenyl OCH2), 68.8 (C-4′), 68.7 (C-6′), 68.4 (C-6), 33.8, 29.7, 29.0, 28.9, 26.1 ppm (5×octenyl CH2); HRMS (ESI): m/z [M+Na]+ calcd for C55H66NaO11: 925.4497, found: 925.4489.pO-benzyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (6)-Tolyl 6-: Benzylidene acetal 9  and BH3⋅NMe3  were dissolved in THF (26 mL) and cooled to 0 °C, then an ice-cold solution of AlCl3  in THF (10 mL) was added. After stirring at RT for 3 h, the solution was concentrated, dissolved in EtOAc (200 mL) and washed with saturated aq NaHCO3, H2O and brine. The organic layer was dried over Na2SO4, filtered, concentrated and subjected to flash chromatography (hexane/EtOAc 3:4 v/v) to afford diol 6 as a white solid : Rf=0.29 (3:4 v/v hexane/EtOAc); [α]D=+16.3 ; 1H NMR : δ=7.87–7.85 , 7.75–7.74 , 7.40–7.29 , 7.04–7.02 , 5.56 , 4.63, 4.58 , 4.35 , 4.21 , 3.85 , 3.81 , 3.68–3.60 , 2.30 ppm ; 13C NMR : δ=138.2 (Ar), 137.7 (Ar), 134.2 (Ar), 133.3 (Ar), 131.7 (Ar), 129.6 (Ar), 128.5 (Ar), 128.2 (Ar), 127.9 (Ar), 127.8 (Ar), 83.8 (C-1), 77.8 (C-5), 73.8 (OCH2Ph), 73.6 (C-4), 72.8 (C-3), 70.5 (C-6), 55.4 (C-2), 21.1 ppm (ArMe); HRMS (ESI): m/z [M+Na]+ calcd for C28H27NNaO6S: 528.1451, found: 528.1451.O-acetyl-β-d-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-β-d-glucopyranoside (7)7-Octen-1-yl 2,3,4,6-tetra-: d-Lactose  and NaOAc  were heated in Ac2O (80 mL) at 100 °C for 5 h. The solution was allowed to cool to RT and poured into ice water (1 L). The precipitate was filtered and recrystallized from EtOAc/hexane to yield peracetylated lactose as colourless crystals . A portion of this compound , 7-octen-1-ol  and 4 Å molecular sieves was dissolved in CH2Cl2 (80 mL) and stirred at RT for 0.5 h. The solution was cooled to 0 °C, and BF3⋅Et2O  was added. After stirring at RT for 24 h, Et3N (5 mL) was added, and the reaction mixture was filtered through Celite. The filtrate was concentrated and the residue was subjected to flash chromatography (4:3 v/v hexane/EtOAc) to afford 7 as white foam : Rf=0.39 (1:1 v/v hexane/EtOAc); [α]D=−11.6 ; 1H NMR : δ=5.79 , 5.34 , 5.19 , 5.10 , 5.01–4.91 , 4.88 , 4.49–4.46 , 4.45 , 4.15–4.06 , 3.88–3.85 , 3.82 , 3.79 , 3.59 , 3.44 , 2.15 , 2.11 , 2.06–2.03 3C2HCH=CH2), 1.96 , 1.57–1.51 3CH2CH=CH2), 1.39–1.25 ppm 3CH2CH=CH2); 13C NMR : δ=170.4 (C=O), 170.3 (C=O), 170.1 (C=O), 170.0 (C=O), 169.8 (C=O), 169.6 (C=O), 169.1 (C=O), 139.0 (CH=CH2), 114.3 (CH=CH2), 101.1 (C-1′), 100.6 (C-1), 76.4 (C-4), 72.9 (C-3), 72.6 (C-5), 71.8 (C-2), 71.0 (C-3′), 70.7 (C-5′), 70.2 (octenyl OCH2), 69.1 (C-2′), 66.6 (C-4′), 62.1 (C-6), 60.8 (C-6′), 33.7, 29.4, 28.84, 28.77, 25.7 (5×octenyl CH2); 20.9, 20.8, 20.71, 20.66, 20.65 (2C), 20.5 ppm (7×OAc); HRMS (ESI): m/z [M+Na]+ calcd for C34H50NaO18: 769.2889, found: 769.2880.p-Tolyl 2,3,4–6-tetra-O-acetyl-β-d-galactopyranosyl-(1→4)-6-O-benzyl-2-deoxy-2-phthalimido-1-thiol-β-d-glucopyranoside (10): A mixture of trichloroacetimidate 4 , diol 6 (1.76 g 3.48 mmol) and powdered 4 Å molecular sieves was suspended in CH2Cl2 (40 mL) and stirred at RT for 1 h. The solution was cooled to −40 °C, and trimethylsilyl trifluoromethanesulfonate  was added drop wise. After stirring at −40 °C for 2 h, the mixture was allowed to warm to RT. Et3N (1 mL) was added and the mixture was filtered through Celite. The filtrate was concentrated and subjected to flash chromatography (4:3 v/v hexane/EtOAc) to afford 10 as a white foam : Rf=0.39 (1:1 v/v hexane/EtOAc); [α]D=+18.5 ; 1H NMR : δ=7.88–7.82 , 7.75–7.71 , 7.41–7.37 , 7.36–7.30 , 7.02–6.99 , 5.53 , 5.31 , 5.18 , 4.93 , 4.68, 4.52 , 4.50 , 4.39 , 4.20 , 4.05–4.00 , 3.89 , 3.76–3.64 , 2.27 , 2.11 , 2.00 , 1.97 , 1.89 ppm ; 13C NMR : δ=170.4 (C=O), 170.1 (C=O), 169.9 (C=O), 169.2 (C=O), 168.2 (C=O), 167.5 (C=O), 138.3 (Ar), 138.2 (Ar), 134.1 (Ar), 133.7 (Ar), 131.9 (Ar), 131.8 (Ar), 129.6 (Ar), 128.5 (Ar), 127.84 (Ar), 127.81 (Ar), 123.6 (Ar), 123.3 (Ar), 101.6 (C-1′), 83.4 (C-1), 81.8 (C-4), 78.2 (C-5), 73.7 (OCH2Ph), 71.2 (C-5′), 70.87 (C-3), 70.78 (C-3′), 68.7 (C-2′), 68.2 (C-6), 66.8 (C-4′), 61.4 (C-6′), 55.2 (C-2), 21.1, 20.7, 20.6, 20.5, 20.3 ppm ; HRMS (ESI): m/z [M+Na]+ calcd for C42H45NNaO15S: 858.2402, found: 858.2395.O-acetyl-β-d-galactopyranosyl-(1→4)--6-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→3)-2,6-di-O-benzyl-β-d-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (12)7-Octen-1-yl 2,3,4–6-tetra-: N-Bromosuccinimide  was added to a solution of trisaccharide 2  in acetone/H2O  at 0 °C. After stirring at 0 °C for 0.5 h, saturated aq NaHCO3 (2 mL) was added. The solution was concentrated, and the residue was dissolved in EtOAc (80 mL) and washed with H2O and brine. The organic layer was dried over Na2SO4, filtered, concentrated and subjected to flash chromatography (4:5 v/v hexane/EtOAc) to afford 11 as a white solid . A solution of 11  in CH2Cl2 (4 mL) was treated with trichloroacetonitrile  and catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and stirred at RT for 4 h. Concentration and flash chromatography (1:1 v/v hexanes/EtOAc) afforded the trichloroacetimidate that was immediately used in the next step. The trichloroacetimidate , diol 3  and powdered 4 Å molecular sieves were suspended in CH2Cl2 (4 mL) and stirred at RT for 1 h. The solution was then cooled to −30 °C, to which TMSOTf (14 μL) was added. The mixture was allowed to warm to RT, and after stirring at −30 °C for 2 h, Et3N (1 mL) was added, and the mixture was filtered through Celite. The filtrate was concentrated and subjected to flash chromatography (5:2 v/v hexane/EtOAc) to afford 12 as a white foam : Rf=0.46 (5:3 v/v hexane/EtOAc); [α]D=+5.5 ; 1H NMR : δ=7.42–7.07 , 7.01 , 6.79 , 5.78 , 5.32 , 5.25 , 5.03 , 4.97 , 4.93–4.89 , 4.84–4.78 , 4.74–4.65 , 4.60–4.53 , 4.48–4.42 , 4.33 , 4.30–4.25 , 4.21–4.17 , 4.16–4.12 , 4.05 , 3.95 , 3.85–3.81 , 3.75 , 3.71–3.68 , 3.61–3.58 , 3.49 , 3.42 , 3.42–3.35 , 3.32–3.27 , 2.96 , 2.71 , 2.03–1.99 3C2HCH=CH2), 1.96 , 1.85 , 1.62–1.55 3CH2CH=CH2), 1.37–1.26 3CH2CH=CH2), 1.19 ppm ; 13C NMR : δ=170.1 (C=O), 170.0 (C=O), 169.9 (C=O), 168.7 (C=O), 139.10 (Ar), 139.06 (CH=CH2), 138.8 (Ar), 138.75 (Ar), 138.65 (Ar), 138.57 (Ar), 138.56 (Ar), 138.4 (Ar), 138.2 (Ar), 137.6 (Ar), 133.9 (Ar), 131.3 (Ar), 128.7 (Ar), 128.4 (Ar), 128.27 (Ar), 128.25 , 128.16 (Ar), 128.13 (Ar), 128.11 (Ar), 128.02 (Ar), 128.0 (Ar), 127.85 (Ar), 127.83 (Ar), 127.75 (Ar), 127.5 (Ar), 127.4 (Ar), 127.2 , 127.1 (Ar), 127.0 (Ar), 126.6 (Ar), 126.3 (Ar), 123.3 (Ar), 114.2 (CH=CH2), 103.5 (C-1), 102.0 (C-1′), 99.6 (C-1′′′), 99.0 (C-1′′), 97.6 (C-1′′′′), 83.5 (C-3), 82.9 (C-2′), 81.8 (C-2), 79.7 (C-3′′′′), 78.1 (C-3′), 77.2 (C-4′′′′), 75.9 (C-4), 75.4 (OCH2Ph), 75.3 (C-4′′), 75.1 (C-5′′), 74.9 (OCH2Ph), 74.75, 74.72 , 74.21, 74.16, 73.8, 73.4, 73.0, 72.9 (6×OCH2Ph), 72.64, 72.60 , 72.4 (OCH2Ph), 71.0 (C-3′′′), 70.5 (C-5′′′), 69.9 (octenyl OCH2), 69.1 (C-2′′′), 68.5, 68.0, 67.9 , 67.5 (C-4′), 66.8 (C-4′′′), 66.6 (C-5′′′′), 60.3 (C-6′′′), 56.2 (C-2′′), 33.7, 29.7, 28.9, 28.8, 26.0 (5×octenyl CH2), 20.7, 20.62, 20.56, 20.5 (4×OAc), 16.8 ppm (C-6′′′′); HRMS (ESI): m/z [M+Na]+ calcd for C117H131NNaO30: 2052.8648, found: 2052.8612.O-acetyl-β-d-galactopyranosyl-(1→4)--6-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→3)-4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (12′)7-Octen-1-yl 2,3,4–6-tetra-: A solution of 12  in pyridine (1 mL) and Ac2O  was stirred overnight at RT, concentrated in vacuo, and the residue subjected to flash chromatography (5:2 v/v hexane/EtOAc) to afford 12’ as a white foam : Rf=0.45 (5:3 v/v hexane/EtOAc); [α]D=+4.2 ; 1H NMR : δ=7.45–7.09 , 7.02–7.01 , 6.87–6.85 , 5.80 , 5.46 , 5.28 , 5.26 , 5.04 , 5.00–4.97 , 4.95–4.92 , 4.90 , 4.86–4.83 , 4.81, 4.57 , 4.78 , 4.75 , 4.69–4.63 , 4.55 , 4.47 , 4.43–4.40 , 4.30–4.26 , 4.23–4.15 , 4.00 , 3.97 , 3.93 , 3.89–3.83 , 3.77 , 3.63–3.60 , 3.56–3.52 , 3.47–3.41 , 3.38 , 3.33–3.29 , 2.98–2.96 , 2.09 , 2.05 , 2.03–2.02 3C2HCH=CH2), 1.97 , 1.85 , 1.63–1.59 3CH2CH=CH2), 1.39–1.29 3CH2CH=CH2), 1.21 ppm ; 13C NMR : δ=170.02 (C=O), 170.01 (C=O), 169.96 (C=O), 169.86 (C=O), 168.8 (C=O), 139.12 (Ar), 139.05 (CH=CH2), 138.89 (Ar), 138.69 (Ar), 138.68 (Ar), 138.4 (Ar), 138.28 (Ar), 138.27 (Ar), 138.23 (Ar), 138.19 (Ar), 133.8 (Ar), 131.3 (Ar), 128.5 (Ar), 128.31 (Ar), 128.26 (Ar), 128.15 (Ar), 128.13 (Ar), 128.09 (Ar), 128.01 (Ar), 127.94 (Ar), 127.92 (Ar), 127.90 (Ar), 127.83 (Ar), 127.79 , 127.78 (Ar), 127.53 (Ar), 127.48 (Ar), 127.41 (Ar), 127.3 (Ar), 127.13 (Ar), 127.11 (Ar), 127.00 (Ar), 126.9 (Ar), 126.4 (Ar), 123.3 (Ar), 114.2 (CH=CH2), 103.6 (C-1), 102.0 (C-1′), 99.5 (C-1′′′), 99.1 (C-1′′), 97.2 (C-1′′′′), 82.6 (C-3), 81.6, 78.84, 78.79 , 79.7 (C-3′′′′), 77.2 (C-4′′′′), 75.7 (C-4), 75.4 (C-5′′), 75.2 (OCH2Ph), 74.96 (C-4′′), 74.92 (OCH2Ph), 74.7, 74.5 , 74.20, 74.15, 73.6, 73.50, 73.0, 72.7 (6×OCH2Ph), 72.6 (C-5′), 72.4 (OCH2Ph), 72.0 (C-3′′), 71.0 (C-3′′′), 70.4 (C-5′′′), 69.94 (C-4′), 69.90 (octenyl OCH2), 69.0 (C-2′′′), 68.3, 67.7, 67.6 , 66.8 (C-4′′′), 66.4 (C-5′′′′), 60.2 (C-6′′′), 56.6 (C-2′′), 33.7, 29.7, 28.9, 28.8, 26.0 (5×octenyl CH2), 20.83, 20.75, 20.64, 20.56, 20.54 (5×OAc), 16.7 ppm (C-6′′′′); HRMS (ESI): m/z [M+Na]+ calcd for C119H133NNaO31: 2094.8754, found: 2094.8751.d-galactopyranosyl-(1→4)--6-O-benzyl-2-deoxy-2-acetamido-β-d-glucopyranosyl-(1→3)-2,6-di-O-benzyl-β-d-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (13)7-Octen-1-yl β-: A solution of pentasaccharide 12  in n-butanol (15 mL) was treated with ethylenediamine , followed by stirring at 100 °C for 20 h. The solution was concentrated in vacuo to dryness. The crude residue was dissolved in CH2Cl2/MeOH , to which Ac2O (1 mL) and Et3N (1 mL) were added. After stirring at RT for 5 h, the solution was concentrated, and the residue subjected to flash chromatography (3:2 v/v toluene/acetone). Further purification by C18 chromatography (1:1 v/v MeOH/H2O→MeOH) afforded 13 as a white foam . Rf=0.51 (4:5 v/v toluene/acetone); [α]D=−19.6 ; 1H NMR : δ=7.42–7.19 , 5.80 , 5.67 , 5.16 , 5.05 , 5.00–4.97 , 4.94–4.87 , 4.74–4.70 , 4.66–4.63 , 4.60–4.55 , 4.53, 4.47 , 4.44–4.38 , 4.35–4.32 , 4.29 , 4.12–4.08 , 4.05–4.01 , 3.94–3.86 , 3.78–3.76 , 3.74–3.65 , 3.62–3.58 , 3.54–3.46 , 3.44–3.33 , 3.30–3.27 , 2.99–2.97 , 2.72 , 2.05–2.01 3C2HCH=CH2), 1.64–1.62 3CH2CH=CH2), 1.40–1.32 3CH2CH=CH2), 1.11 ppm ; 13C NMR : δ=170.9 (C=O), 139.2 (Ar), 139.1 (CH=CH2), 139.0 (Ar), 138.7 (Ar), 138.44 (Ar), 138.43 (Ar), 138.38 (Ar), 138.2 (Ar), 137.5 (Ar), 128.6 (Ar), 128.51 (Ar), 128.47 (Ar), 128.31 (Ar), 128.27 (Ar), 128.25 (Ar), 128.20 (Ar), 128.1 (Ar), 128.04 , 128.01 (Ar), 127.91 (Ar), 127.86 (Ar), 127.71 (Ar), 127.67 (Ar), 127.66 (Ar), 127.63 (Ar), 127.53 (Ar), 127.51 (Ar), 127.46 (Ar), 127.39 (Ar), 127.37 (Ar), 127.2 (Ar), 114.2 (CH=CH2), 103.6 (C-1), 102.2 (C-1′), 100.1 (C-1′′′), 99.7 (C-1′′), 98.0 (C-1′′′′), 82.9 (C-3), 82.3 (C-2′), 81.9 (C-2), 79.4, 79.0 , 77.2 (C-4′′′′), 76.6, 76.5, 76.4, 76.0, 75.3 (OCH2Ph), 75.14, 75.08, 74.95 (OCH2Ph), 74.93 (OCH2Ph), 74.8, 74.7 (OCH2Ph), 74.1 (OCH2Ph), 73.7, 73.6, 73.4, 73.2 (3×OCH2Ph), 72.9, 72.4 (OCH2Ph), 71.7, 70.0, 69.9, 68.9, 68.4 , 69.3, 67.9, 67.5 (C-5′′′′), 63.0 (C-6′′′), 57.5 (C-2′′), 33.7, 29.7, 28.95, 28.86, 26.0 (5×octenyl CH2), 22.9 (NHAc), 16.7 ppm (C-6′′′′); HRMS (ESI): m/z [M+Na]+ calcd for C103H123NNaO25: 1796.8276, found: 1796.8254.d-galactopyranosyl-(1→4)-[α-L-fucopyranosyl-(1→3)]-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→3)-β-d-galactopyranosyl-(1→4)-β-d-glucopyranoside (14)7-Octen-1-yl β-: Sodium was added to freshly collected liquid ammonia (∼8 mL) at −78 °C until the blue colour of the solution persisted. A solution of 13  in THF (4 mL) and MeOH (30 μL) was added drop wise at −78 °C. After 2 h, MeOH (5 mL) was added and the solution was concentrated in vacuo to dryness. The residue was dissolved in MeOH (30 mL), neutralized with Amberlite IR120 H+ ion-exchange resin, filtered and concentrated. The crude residue was purified by C18 chromatography using gradient elution (H2O→3:7 v/v MeOH/H2O) to give 14 as a white solid : [α]D=−45.6 ; 1H NMR : δ=5.91 , 5.11 , 5.06–5.02 , 4.97–4.95 , 4.82 , 4.70 , 4.46 , 4.45 , 4.42 , 4.14 , 3.96–3.84 , 3.79–3.55 , 3.48 , 3.28 , 2.07–2.03 3C2HCH=CH2), 2.01 , 1.63–1.59 3CH2CH=CH2), 1.41–1.30 3CH2CH=CH2), 1.16 ppm ; 13C NMR : δ=175.7 (C=O), 141.3 (CH=CH2), 115.0 (CH=CH2), 103.9 (C-1), 103.5, 103.0, 102.8 , 99.6 (C-1′′′′), 83.1, 79.4, 76.1, 75.9, 75.8, 75.7 (2C), 75.4, 74.1, 73.8, 73.5, 72.9, 72.0, 71.7, 70.9, 70.2, 69.33, 69.28, 68.7, 67.7 (C-5′′′′), 62.5, 61.9, 61.1, 60.6 , 57.0 (C-2′′), 34.0 (octenyl CH2), 29.6 (octenyl CH2), 29.0 , 25.8 (octenyl CH2), 23.2 (NHAc), 16.3 ppm (C-6′′′′); HRMS (ESI): m/z [M+Na]+ calcd for C40H69NNaO25: 986.4051, found: 986.4047.d-galactopyranosyl-(1→4)-[α-L-fucopyranosyl-(1→3)]-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→3)-β-d-galactopyranosyl-(1→4)-β-d-glucopyranoside (15)8-[(2-Aminoethyl)thiol]-1-octyl β-: Compound 14  and cysteamine hydrochloride  were dissolved in dry MeOH (0.5 mL) in a quartz tube. The solution was degassed and the tube was filled with argon. After irradiation with UV light for 2.5 h, the solution was concentrated and subjected to C18 chromatography using gradient elution (0.5 % aq AcOH→3:7 v/v MeOH/0.5 % aq AcOH) to afford the corresponding amine salt 15 . [α]D=−41.1 ; 1H NMR : δ=5.11 , 4.83–4.80 , 4.70 , 4.46 , 4.45 , 4.42 , 4.14 , 3.96–3.84 , 3.79–3.55 , 3.48 , 3.29–3.26 , 3.20 , 2.83 , 2.58 6CH2O), 2.01 , 1.63–1.56 4C2HCH2S), 1.38–1.31 4CH2CH2S), 1.16 ppm ; 13C NMR : δ=175.7 (C=O), 103.9 (C-1), 103.5, 103.0, 102.8 , 99.6 (C-1′′′′), 83.1, 79.4, 76.1, 75.90, 75.86, 75.7 (2C), 75.5, 74.1, 73.8, 73.5, 72.9, 72.0, 71.7, 70.9, 70.2, 69.34, 69.27, 68.7, 67.7 (C-5′′′′), 62.5, 61.9, 61.1, 60.6 , 57.0 (C-2′′), 39.4 (SCH2CH2N), 31.7 (SCH2(CH2)6CH2O), 29.7 (SCH2(CH2)6CH2O), 29.5 (SCH2CH2N), 29.3, 29.1(2C), 28.8, 25.9 (5×SCH2(CH2)6CH2O), 23.2 (NHAc), 16.3 ppm (C-6′′′′); HRMS (ESI): m/z [M+H]+ calcd for C42H77N2O25S: 1041.4531, found: 1041.4517.Preparation of HSA conjugate: Compound 1 (1.5 mg) was dissolved in DMF (15 μL) and injected into a solution of human serum albumin  in phosphate buffer . The reaction was left at RT for one day, and the mixture was dialyzed against deionized H2O (5×4 L). A white solid was obtained after lyophilization. The degree of incorporation of the pentasaccharide into the glycoconjugate was determined to be 21 by MALDI-TOF MS.