Proteomic analysis of tyrosine phosphorylation during human liver transplantation

BACKGROUND: Ischemia-reperfusion (I/R) causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. RESULTS: Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH(2)/SH(3 )adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. CONCLUSION: Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes

Serrate RNA effector molecule (SRRT) is associated with prostate cancer progression and is a predictor of poor prognosis in lethal prostate cancer

Arsenite-resistance protein 2, also known as serrate RNA effector molecule (ARS2/SRRT), is known to be involved in cellular proliferation and tumorigenicity. However, its role in prostate cancer (PCa) has not yet been established. We investigated the potential role of SRRT in 496 prostate samples including benign, incidental, advanced, and castrate-resistant patients treated by androgen deprivation therapy (ADT). We also explored the association of SRRT with common genetic aberrations in lethal PCa using immunohistochemistry (IHC) and performed a detailed analysis of SRRT expression using The Cancer Genome Atlas (TCGA PRAD) by utilizing RNA-seq, clinical information (pathological T category and pathological Gleason score). Our findings indicated that high SRRT expression was significantly associated with poor overall survival (OS) and cause-specific survival (CSS). SRRT expression was also significantly associated with common genomic aberrations in lethal PCa such as PTEN loss, ERG gain, mutant TP53, or ATM. Furthermore, TCGA PRAD data revealed that high SRRT mRNA expression was significantly associated with higher Gleason scores, PSA levels, and T pathological categories. Gene set enrichment analysis (GSEA) of RNAseq data from the TCGA PRAD cohort indicated that SRRT may play a potential role in regulating the expression of genes involved in prostate cancer aggressiveness. Conclusion: The current data identify the SRRT's potential role as a prognostic for lethal PCa, and further research is required to investigate its potential as a therapeutic target.Prostate Cancer Foundation Young Investigator Award ; Prostate Cancer Canada ; Canadian Cancer Society (CCS

Characterization of greater middle eastern genetic variation for enhanced disease gene discovery

The Greater Middle East (GME) has been a central hub of human migration and population admixture. The tradition of consanguinity, variably practiced in the Persian Gulf region, North Africa, and Central Asia1-3, has resulted in an elevated burden of recessive disease4. Here we generated a whole-exome GME variome from 1,111 unrelated subjects. We detected substantial diversity and admixture in continental and subregional populations, corresponding to several ancient founder populations with little evidence of bottlenecks. Measured consanguinity rates were an order of magnitude above those in other sampled populations, and the GME population exhibited an increased burden of runs of homozygosity (ROHs) but showed no evidence for reduced burden of deleterious variation due to classically theorized ‘genetic purging’. Applying this database to unsolved recessive conditions in the GME population reduced the number of potential disease-causing variants by four- to sevenfold. These results show variegated genetic architecture in GME populations and support future human genetic discoveries in Mendelian and population genetics

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Transcriptional profiling of the human liver during the reperfusion phase of transplantation

Liver transplantation continues to be the only remedy for end-stage liver disease. Moreover, the number of recipients far exceeds the number of donors and patients die on waiting lists. Unfortunately, not all grafts survive the process of transplantation and marginal livers are discarded, as they would not tolerate the stresses of ischemia-reperfusion and therefore would not survive the process of transplantation. If we are to resolve these problems, and decrease the chasm between the donor and recipient numbers, we need to characterize how a normal liver survives the process of transplantation.We have developed a protocol that allows us to characterize the normal liver's response to transplantation. Indeed, a liver that endures the process of transplantation must be able to limit the amount of damage caused by the various stresses related to cold ischemia and warm oxygenated reperfusion. Unfortunately, the process of transplantation is not easily amenable to the reduction much less the elimination of these stresses. Therefore, a means of investigating what happens to the liver during the process of transplantation was needed that would take into account the global effect of these variables on the graft's survival.The underlying hypothesis of my thesis is that the surviving liver invokes protective mechanisms to moderate the damage that could occur as a result of transplantation in part by regulating the level and type of expressed genes. Using microarray technology, we determined the identity of the mRNAs that revealed a degree of regulation, either up- or down-regulation during the reperfusion phase of transplantation. Furthermore, because ischemia precedes reperfusion, the process of reperfusion per se includes all of the stresses associated with ischemia, e.g. all reperfused livers were ischemia preconditioned. Thus, to conduct our analysis biopsy specimens were taken at three time-points during the peri-reperfusion phase of the operation. Our methodology not only permitted us to identify regulated genes, it also allowed us to control for recipient blood-borne contribution of messenger RNA. Because the last biopsy specimen was taken 1h post-reperfusion, our list was comprised of immediate early genes.Of the other immediate-early genes that were on our list, we found an up-regulated gene that coded for map kinase phosphatase-1 (MKP-1). Immunohistochemistry preformed on frozen human liver sections revealed expression of MKP-1 in hepatocytes. MKP-1 is a phosphatase is best known for JNK-1, p38MAPK and ERK1/2/5 dephosphorylation. Using transplantation relevant stresses in vitro, and HepG2 as a cellular model for hepatocytes, we characterized mkp-1 mRNA regulation. Furthermore, using the same protocol and MKP-1 shRNA expressed in HepG2, we found that a lack of MKP-1 protein expression increased apoptosis. The second gene, als2, we investigated was slightly down-regulated and coded for a protein called alsin. The latter is a RhoGEF, a guanine nucleotide exchange factor that activates Rac1, Rab5 and Ran. We proceeded to characterize alsin mRNA regulation in vitro using ischemia-reperfusion relevant stresses, as we did for MKP-1. Finally, we are seeking to determine if or how the Rho proteins (Rac1, Rab5 or Ran) are implicated during the "reperfusion" phase of the operation. All in all, our results indicate a hepatic coping mechanism invoked for the purpose of reducing the damage caused by the trauma of ischemia and reperfusion.La transplantation du foie demeure le seul remède pour les maladies hépatiques mortelles. Malheureusement le nombre de donneurs d'organe est inférieur au nombre de patients. Cette situation s'aggrave lorsque l'on considère qu'un certain nombre de foies transplantés ne survivent pas et que ce ne sont pas tous les foies qui s'avèrent utilisables pour la transplantation. En effet, ces foies de « qualité inférieure » ne survivraient pas au traumatismes de l'ischémie et de la réperfusion donc ils sont refusés. Actuellement il n'existe pas de solution pour pallier ce nombre de foies à transplanter. frNous avons développé une approche permettant de caractériser les mécanismes par lesquels un foie normal survit au processus de la transplantation. Un tel foie doit être en mesure de diminuer ou du moins de contrôler les effets toxiques associés aux stress d'ischémie froide et de la réperfusion tiède oxygénée. Malheureusement, le processus de transplantation ne peut être manipulé facilement de façon à réduire ou à éliminer les variables qui lui sont associées. Compte tenu ces observations, une analyse détaillée de ce phénomène doit tenir compte de l'effet global de ces variables sur la survie de la greffe.Une hypothèse fondamentale de mon travail de thèse présuppose qu'un foie qui survit au processus de la transplantation doit être en mesure de contrôler les effets toxiques des stress associés à ce processus. Cette régulation peut être accomplie en partie par des mécanismes de contrôle de l'expression des gènes. En utilisant des biopuces à ADN, nous avons déterminé l'identité des ARN messagers régulés positivement ou négativement au cours de la phase de réperfusion de la transplantation. Cette phase de la transplantation est ciblée pour notre étude, puisque que le processus d'ischémie précède la réperfusion, le processus de réperfusion englobe tous les stress associés à l'ischémie. Afin d'accomplir notre analyse, des biopsies hépatiques ont été prélevées à trois intervalles de temps différents. Notre protocole expérimental nous a permis non seulement d'établir une liste de gènes qui étaient régulés à la hausse ou à la baisse, une heure après le ré-établissement du flot sanguin, mais aussi nous a permis d'éliminer les ARN messagers provenant du sang du receveur. Étant donné que la dernière biopsie prélevée était à une heure après la réperfusion du foie par la veine porte hépatique, notre liste de gènes fut composé de gènes immédiats précoces.Parmi les gènes identifiés, le messager codant pour la map kinase phosphatase-1 (MKP-1), était régulé à la hausse. En utilisant des coupes de foie humain, l'immunohistochimie a révélé l'expression hépatocytaire de la protéine MKP-1. Cette protéine déphosphoryle les map kinases JNK-1, p38MAPK et ERK1/2/5. En simulant les stress d'ischémie et de réperfusion sur des cellules d'origine hépatomique, HepG2, nous avons caractérisé la régulation de l'ARN messager de MKP-1. De plus, en utilisant le même protocole nous avons déterminé les conséquences d'une pénurie de la protéine MKP-1 pour les cellules HepG2. Pour ce, nous avons exprimé du « shRNA » dirigé contre l'ARN de MKP-1, dans ces cellules, afin de diminuer l'expression de MKP-1. Ceci a causé une augmentation d'apoptose. Le second gène à l'étude était celui d'als2 qui code pour la protéine alsine, dont l'ARN messager était légèrement diminué en quantité lors de son évaluation par biopuces. L'alsine est une « RhoGEF », une protéine échangeuse de nucléotides, qui active les RhoGTPases Rac1, Rab5 et Ran. Nous avons procédé à la caractérisation de ce gène, en utilisant les conditions expérimentales préétablies avec MKP-1. La dernière étape se résume à déceler si ou comment les protéines RhoGTPases soient Rac1, Rab5 ou Ran, sont impliquées dans la phase de réperfusion du foie. En conclusion, nos résultats indiquent que le foie transplanté utilise différents mécanismes pour contrecarrer les effets des stress d'ischémie et de réperfusion

Interferon-beta is a potent promoter of nerve growth factor production by astrocytes

Recent clinical evidence has suggested that interferon-$beta$ is efficacious in the treatment of multiple sclerosis, a demyelinating disease that is thought to be immune mediated. The mechanism of its efficacy remains unclear. Possible modes of action include effects of interferon-$beta$ on systemic or CNS immune or non-immune parameters. In the latter context, astrocytes are known to provide trophic support to oligodendrocytes and neurons. Since nerve growth factor (NGF) is reported to cause adult porcine oligodendrocytes to proliferate and extend processes and since these phenotypes can impact favourably on remyelination, we have examined the possibility that interferon-$beta$ could increase the astrocyte production of NGF. Recombinant mouse interferon-$beta$ (rmIFN-$beta$) was added to confluent neonatal mouse astrocyte cultures. Analyses reveal an increase in NGF mRNA and protein elicited by rmIFN-$beta$. This finding may be relevant to interferon-$beta$'s clinical efficacy in multiple sclerosis

Serrate RNA Effector Molecule (SRRT) Is Associated with Prostate Cancer Progression and Is a Predictor of Poor Prognosis in Lethal Prostate Cancer.

Arsenite-resistance protein 2, also known as serrate RNA effector molecule (ARS2/SRRT), is known to be involved in cellular proliferation and tumorigenicity. However, its role in prostate cancer (PCa) has not yet been established. We investigated the potential role of SRRT in 496 prostate samples including benign, incidental, advanced, and castrate-resistant patients treated by androgen deprivation therapy (ADT). We also explored the association of SRRT with common genetic aberrations in lethal PCa using immunohistochemistry (IHC) and performed a detailed analysis of SRRT expression using The Cancer Genome Atlas (TCGA PRAD) by utilizing RNA-seq, clinical information (pathological T category and pathological Gleason score). Our findings indicated that high SRRT expression was significantly associated with poor overall survival (OS) and cause-specific survival (CSS). SRRT expression was also significantly associated with common genomic aberrations in lethal PCa such as PTEN loss, ERG gain, mutant TP53, or ATM. Furthermore, TCGA PRAD data revealed that high SRRT mRNA expression was significantly associated with higher Gleason scores, PSA levels, and T pathological categories. Gene set enrichment analysis (GSEA) of RNAseq data from the TCGA PRAD cohort indicated that SRRT may play a potential role in regulating the expression of genes involved in prostate cancer aggressiveness. Conclusion: The current data identify the SRRTs potential role as a prognostic for lethal PCa, and further research is required to investigate its potential as a therapeutic target

Copy Number Profiles of Prostate Cancer in Men of Middle Eastern Ancestry.

Our knowledge of prostate cancer (PCa) genomics mainly reflects European (EUR) and Asian (ASN) populations. Our understanding of the influence of Middle Eastern (ME) and African (AFR) ancestry on the mutational profiles of prostate cancer is limited. To characterize genomic differences between ME, EUR, ASN, and AFR ancestry, fluorescent in situ hybridization (FISH) studies for NKX3-1 deletion and MYC amplification were carried out on 42 tumors arising in individuals of ME ancestry. These were supplemented by analysis of genome-wide copy number profiles of 401 tumors of all ancestries. FISH results of NKX3-1 and MYC were assessed in the ME cohort and compared to other ancestries. Gene level copy number aberrations (CNAs) for each sample were statistically compared between ancestry groups. NKX3-1 deletions by FISH were observed in 17/42 (17.5%) prostate tumors arising in men of ME ancestry, while MYC amplifications were only observed in 1/42 (2.3%). Using CNAs called from arrays, the incidence of NKX3-1 deletions was significantly lower in ME vs. other ancestries (20% vs. 52%; p = 2.3 × 10-3). Across the genome, tumors arising in men of ME ancestry had fewer CNAs than those in men of other ancestries (p = 0.014). Additionally, the somatic amplification of 21 specific genes was more frequent in tumors arising in men of ME vs. EUR ancestry (two-sided proportion test; Q < 0.05). Those included amplifications in the glutathione S-transferase family on chromosome 1 (GSTM1, GSTM2, GSTM5) and the IQ motif-containing family on chromosome 3 (IQCF1, IQCF2, IQCF13, IQCF4, IQCF5, IQCF6). Larger studies investigating ME populations are warranted to confirm these observations

Decreased ATM Protein Expression Is Substantiated with PTEN Loss in Defining Aggressive Phenotype of Prostate Cancer Associated with Lethal Disease

Background: Ataxia Telangiectasia Mutated (ATM) serine/threonine protein kinase is a known tumor suppressor, involved in DNA damage repair. It has prognostic and predictive therapeutic implications and is associated with aggressive prostate cancer (PCa). Objective: To investigate the prognostic value of ATM protein expression in PCa patients and assessed the combined value of ATM, ERG, and PTEN status. Design, setting, and participants: This study consisted of 303 patients with incidental, locally advanced, and castrate-resistant PCa by transurethral resection of the prostate (TURP). Outcome measurements and statistical analysis: TURP samples from 303 PCa patients were assessed by immunohistochemistry (IHC for ATM, ERG, and PTEN. Individual and combined marker status were correlated with International Society of Urological Pathology Gleason grade group, overall survival (OS), and PCa-specific mortality (PCSM). Results and limitations: Decreased ATM expression (negative/weak intensity) occurred in 164/303 (54.1%) patients, and was associated with shorter OS and higher PCSM (p = 0.015 and p = 0.001, respectively). Negative/weak ATM expression was significantly associated with PCSM with a hazard ratio of 2.09 (95% confidence interval 1.34–3.27, p = 0.001). Assessment of Combined ATM/PTEN expression showed improved prognostic power to predict OS and PCSM, independent of Gleason grade groups. Conclusions: Decreased ATM protein expression is associated with poor outcomes in advanced PCa patients. Patients with combined low ATM/PTEN negative expression are at the highest risk for reduced OS and PCSM. Assessing the combined status of ATM/PTEN by IHC in PCa patients may aid in risk stratification relative to OS and PCSM. Moreover, since ATM plays an integral role in DNA damage response pathways, future studies will enhance our understanding of how outcomes of patients with altered ATM and PTEN expression can be improved further with poly-ADP ribose polymerase inhibitors (PARPi), combinations of PARPi and androgen receptor–targeted therapies, as well as platinum-based chemotherapies. Patient summary: Lower ATM intensity is associated with increased cancer-specific mortality in prostate cancer patients. Patients with lower ATM and PTEN negative expression showed decreased overall survival and increased cancer mortality compared with controls