Duffy antigen is expressed during erythropoiesis in Duffy-negative individuals.

The erythrocyte silent Duffy blood group phenotype in Africans is thought to confer resistance to Plasmodium vivax blood-stage infection. However, recent studies report P. vivax infections across Africa in Fy-negative individuals. This suggests that the globin transcription factor 1 (GATA-1) SNP underlying Fy negativity does not entirely abolish Fy expression or that P. vivax has developed a Fy-independent red blood cell (RBC) invasion pathway. We show that RBCs and erythroid progenitors from in vitro differentiated CD34 cells and from bone marrow aspirates from Fy-negative samples express a functional Fy on their surface. This suggests that the GATA-1 SNP does not entirely abolish Fy expression. Given these results, we developed an in vitro culture system for P. vivax and show P. vivax can invade erythrocytes from Duffy-negative individuals. This study provides evidence that Fy is expressed in Fy-negative individuals and explains their susceptibility to P. vivax with major implications and challenges for P. vivax malaria eradication.

The fibrinolytic system enables the onset of Plasmodium infection in the mosquito vector and the mammalian host.

Plasmodium parasites must migrate across proteinaceous matrices to infect the mosquito and vertebrate hosts. Plasmin, a mammalian serine protease, degrades extracellular matrix proteins allowing cell migration through tissues. We report that Plasmodium gametes recruit human plasminogen to their surface where it is processed into plasmin by corecruited plasminogen activators. Inhibition of plasminogen activation arrests parasite development early during sexual reproduction, before ookinete formation. We show that increased fibrinogen and fibrin in the blood bolus, which are natural substrates of plasmin, inversely correlate with parasite infectivity of the mosquito. Furthermore, we show that sporozoites, the parasite form transmitted by the mosquito to humans, also bind plasminogen and plasminogen activators on their surface, where plasminogen is activated into plasmin. Surface-bound plasmin promotes sporozoite transmission by facilitating parasite migration across the extracellular matrices of the dermis and of the liver. The fibrinolytic system is a potential target to hamper Plasmodium transmission.

Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient.

BACKGROUND: Plasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosquito proteins and macromolecules present in zygote preparations. Thus, no zygote-specific surface markers have been identified to date. Here, a methodology is developed to obtain large quantities of highly purified zygotes using in vitro culture, including purification methods that include magnetic column cell separation (MACS) followed by Percoll density gradient centrifugation. This straightforward and effective approach provides ample material for studies to enhance understanding of zygote biology and identify novel zygote surface marker candidates that can be tested as transmission blocking vaccine (TBV) candidates. METHODS: Plasmodium falciparum gametocyte cultures were established and maintained from asexual cultures. Gametocytes were matured for 14 days, then transferred into zygote media for 6 h at 27 ± 2 °C to promote gamete formation and fertilization. Zygotes were then purified using a combination of MACS column separation and Percoll density gradient centrifugation. Purity of the zygotes was determined through morphological studies: the parasite body and nuclear diameter were measured, and zygotes were further transformed into ookinetes. Immunofluorescence assays (IFA) were also performed using the ookinete surface marker, Pfs28. RESULTS: After stimulation, the culture consisted of transformed zygotes and a large number of uninfected red blood cells (RBCs), as well as infected RBCs with parasites at earlier developmental stages, including gametes, gametocytes, and asexual stages. The use of two MACS columns removed the vast majority of the RBCs and gametocytes. Subsequent use of two Percoll density gradients enabled isolation of a pure population of zygotes. These zygotes transformed into viable ookinetes that expressed Pfs28. CONCLUSION: The combined approach of using two MACS columns and two Percoll density gradients yielded zygotes with very high purity (45-fold enrichment and a pure population of zygotes [approximately 100%]) that was devoid of contamination by other parasite stages and uninfected RBCs. These enriched zygotes, free from earlier parasites stages and mosquito-derived macromolecules, can be used to further elucidate the biology and developmental processes of Plasmodium.

Multimodal Endoscopic Assessment Guides Treatment Decisions for Rectal Early Neoplastic Tumors.

BACKGROUND: There is a trend toward organ conservation in the management of rectal tumors. However, there is no consensus on standardized investigations to guide treatment. OBJECTIVE: We report the value of multimodal endoscopic assessment (white light, magnification chromoendoscopy and narrow band imaging, selected colonoscopic ultrasound) for rectal early neoplastic tumors to inform treatment decisions. DESIGN: This was a retrospective study. SETTING: The study was conducted in a tertiary referral unit for interventional endoscopy and early colorectal cancer. PATIENTS: A total of 296 patients referred with rectal early neoplastic tumors were assessed using standardized multimodal endoscopic assessment and classified according to risk of harboring invasive cancer. MAIN OUTCOME MEASURES: Sensitivity, specificity, positive and negative predictive values of multimodal endoscopic assessment, and previous biopsy to predict invasive cancer were calculated and treatment outcomes reported. RESULTS: After multimodal endoscopic assessment, lesions were classified as invasive cancer, at least deep submucosal invasion (n = 65); invasive cancer, superficial submucosal invasion or high risk of covert cancer (n = 119); or low risk of covert cancer (n = 112). Sensitivity, specificity, positive predictive values, and negative predictive values of multimodal endoscopic assessment for diagnosing invasive cancer, deep submucosal invasion, were 77%, 98%, 93%, and 93%. The combined classification of all lesions with invasive cancer or high risk of covert cancer had a negative predictive value of 96% for invasive cancer on final histopathology. Sensitivity of previous biopsy was 37%. A total of 47 patients underwent radical surgery and 33 transanal endoscopic microsurgery. No patients without invasive cancer were subjected to radical surgery; 222 patients initially underwent endoscopic resection. Of the 203 without deep submucosal invasion, 95% avoided surgery and were free from recurrence at last follow-up. LIMITATIONS: This was a retrospective study from a tertiary referral unit. CONCLUSIONS: Standardized multimodal endoscopic assessment guides rational treatment decisions for rectal tumors resulting in organ-conserving treatment for all patients without deep submucosal invasive cancer. See Video Abstract at http://links.lww.com/DCR/B133. LA EVALUACIÓN ENDOSCÓPICA MULTIMODAL COMO GUÍA DE DECISIONES EN EL TRATAMIENTO DE TUMORES RECTALES NEOPLÁSICOS PRECOCES: La tendencia actual es la preservación del órgano en el manejo de los tumores de rectao. Sin embargo, no hay consenso sobre las investigaciones estandar para guiar dicho tratamiento.Presentamos los valores de la evaluación endoscópica multimodal (luz blanca, cromoendoscopia de aumento, imagen de banda estrecha y ecografía colonoscópica seleccionada) para tumores rectales neoplásicos tempranos y así notificar las decisiones sobre el tratamiento.Estudio retrospectivo.El estudio se realizó en una unidad de referencia terciaria para endoscopia intervencionista y cáncer colorrectal temprano.Se evaluaron 296 pacientes referidos con tumores neoplásicos precoces de recto mediante una evaluación endoscópica multimodal estandarizada y se clasificaron de acuerdo al riesgo de albergar un cáncer invasivo.Se calcularon la sensibilidad, la especificidad, los valores predictivos positivos y negativos de la evaluación endoscópica multimodal y la biopsia previa para predecir el cáncer invasivo y se notificaron los resultados para el tratamiento.Después de la evaluación endoscópica multimodal, las lesiones se clasificaron como: cáncer invasive (al menos invasión submucosa profunda n = 65); cáncer invasive (invasión submucosa superficial o alto riesgo de cáncer encubierto n = 119) y finalmente aquellos de bajo riesgo de cáncer encubierto (n = 112). La sensibilidad, la especificidad, los valores predictivos positivos y negativos de la evaluación endoscópica multimodal para el diagnóstico de cáncer invasivo, la invasión submucosa profunda fueron 77%, 98%, 93% y 93% respectivamente. La clasificación combinada de todas las lesiones con cáncer invasivo o de alto riesgo de cáncer encubierto tuvo un VPN del 96% para el cáncer invasivo en la histopatología final. La sensibilidad fué de 37% en todas las biopsias previas. 47 pacientes fueron sometidos a cirugía radical, 33 por microcirugía endoscópica transanal. Ningún paciente sin cáncer invasivo fue sometido a cirugía radical. Inicialmente, 222 pacientes fueron sometidos a resección endoscópica. De los 203 sin invasión submucosa profunda, el 95% evitó la cirugía y no tuvieron recurrencia en el último seguimiento.Estudio retrospectivo de una unidad de referencia terciaria.La evaluación endoscópica multimodal estandarizada guía las decisiones racionales de tratamiento para los tumores rectales que resultan en un tratamiento conservador de órganos para todos los pacientes sin cáncer invasivo submucoso profundo. Consulte Video Resumen en http://links.lww.com/DCR/B133.

On Perfect Clustering of High Dimension, Low Sample Size Data.

Popular clustering algorithms based on usual distance functions (e.g., the Euclidean distance) often suffer in high dimension, low sample size (HDLSS) situations, where concentration of pairwise distances and violation of neighborhood structure have adverse effects on their performance. In this article, we use a new data-driven dissimilarity measure, called MADD, which takes care of these problems. MADD uses the distance concentration phenomenon to its advantage, and as a result, clustering algorithms based on MADD usually perform well for high dimensional data. We establish it using theoretical as well as numerical studies. We also address the problem of estimating the number of clusters. This is a challenging problem in cluster analysis, and several algorithms are available for it. We show that many of these existing algorithms have superior performance in high dimensions when they are constructed using MADD. We also construct a new estimator based on a penalized version of the Dunn index and prove its consistency in the HDLSS asymptotic regime. Several simulated and real data sets are analyzed to demonstrate the usefulness of MADD for cluster analysis of high dimensional data.

Risk factors for early and late adenoma recurrence after advanced colorectal endoscopic resection at an expert Western center.

BACKGROUND AND AIMS: Few large Western series examine risk factors for recurrence after endoscopic resection (ER) of large (≥20 mm) colorectal laterally spreading tumors. Recurrence beyond initial surveillance is seldom reported, and differences between residual/recurrent adenoma and late recurrence are not scrutinized. We report the incidence of recurrence at successive surveillance intervals, identify risk factors for recurrent/residual adenoma and late recurrence, and describe the outcomes of ER of recurrent adenomas. METHODS: Recurrence was calculated for successive surveillance periods after colorectal ER. Multiple logistic regression was used to identify independent risk factors for recurrent/residual adenoma and late recurrence (≥12 months). RESULTS: Six hundred twenty colorectal ERs were performed, and 456 eligible patients (98%) had completed 3- to 6-month surveillance. Residual/recurrent adenoma (3-6 months) was detected in 8.3%, at 12 months in 6.1%, between 24 and 36 months in 6.4%, and after 36 months in 13.5%. Independent risk factors for residual/recurrent adenoma were piecemeal resection (odds ratio [OR], 13.0; P = .01), adjunctive argon plasma coagulation (OR, 2.4; P = .01), and lesion occupying ≥75% of the luminal circumference (OR, 5.6; P < .001) and for late recurrence were lesion size >60 mm (OR, 6.3; P < .001) and piecemeal resection (OR, 4.4; P = .04). Of 66 patients with recurrence, 5 required surgery, 8 left the treatment pathway, 20 are still receiving ER or surveillance, and 33 had ER with normal subsequent surveillance. CONCLUSIONS: Recurrence occurs at successive periods of surveillance after ER even beyond 3 years. Aside from piecemeal resection, risk factors for residual/recurrent adenoma and late recurrence are different. Recurrence can be challenging to treat, but surgery is rarely required.

Endoscopic resection of colorectal circumferential and near-circumferential laterally spreading lesions: outcomes and risk of stenosis.

PURPOSE: Almost any colorectal superficial neoplastic lesion can be treated by endoscopic resection (ER) but very little is known about outcomes of ER leaving circumferential or near-circumferential mucosal defects. We report the outcomes of ER leaving ≥ 75% circumferential mucosal defects performed in a western expert centre. METHODS: Five hundred eighty-seven ERs of large colorectal lesions ≥ 20 mm were grouped according to the extent of the mucosal defect and comparisons made between those with < 75% and ≥ 75% defects. Independent predictors of stenosis were identified. RESULTS: Forty-seven patients had ER leaving ≥ 75% circumference defect, most located at or distal to the rectosigmoid, with ≥ 90% defects in 5 and 100% in 11. There were no significant colonic muscle injuries in patients with ≥ 75% defect and no differences in post-procedure bleeding (OR 1.6, 95% CI 0.2-13.7, p = 0.64) between patients with ≥ 75% and < 75% defects. Stenosis developed in 9 patients. ≥ 90% circumference defect was the only independent risk factor for stenosis (OR 286, p < 0.001). Three of 4 patients with asymptomatic stenosis had successful expectant management. The remainder were treated with dilatation. Recurrence was more likely in those with ≥ 75% defect (OR 7.9, 95% CI 3.8-16.4, p < 0.001) but was managed with further ER in all but 2 cases. CONCLUSION: ER of colorectal lesions resulting in defects ≥ 75% of the luminal circumference is challenging but safe and effective when performed in an expert centre. The only independent predictor of stenosis is ≥ 90% circumference defect but some patients improve with expectant management; therefore, pre-emptive intervention may not be warranted.

PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro.

BACKGROUND: Despite the importance of the Plasmodium berghei oocyst capsule protein (PbCap380) in parasite survival, very little is known about the orthologous Plasmodium falciparum capsule protein (PfCap380). The goal of this work was to study the growth of P. falciparum oocysts using PfCap380 as a developmental marker. METHODS: To study P. falciparum oocyst development using both in vivo (mosquito-derived) and in vitro (culture-derived) growth conditions, antibodies (polyclonal antisera) were raised against PfCap380. For studies on in vivo oocysts, mature P. falciparum gametocytes were fed to Anopheles stephensi mosquitoes. For studies on in vitro parasites, P. falciparum gametocytes were induced and matured for subsequent ookinete production. Ookinetes were purified and then tested for binding affinity to basal lamina components and transformation into early oocysts, which were grown on reconstituted basal lamia coated wells with novel oocyst media. To monitor in vivo oocyst development, immunofluorescence assays (IFA) were performed using anti-PfCap380 antisera on Pf-infected mosquito midguts. IFA were also performed on culture-derived oocysts to follow in vitro oocyst development. RESULTS: The anti-PfCap380 antisera allowed detection of early midgut oocysts starting at 2 days after gametocyte infection, while circumsporozoite protein was definitively observed on day 6. For in vitro culture, significant transformation of gametocytes to ookinetes (24%) and of ookinetes to early oocysts (85%) was observed. After screening several basal lamina components, collagen IV provided greatest binding of ookinetes and transformation into early oocysts. Finally, PfCap380 expression was observed on the surface of culture-derived oocysts but not on gametocytes or ookinetes. CONCLUSIONS: This study presents developmental monitoring of P. falciparum oocysts produced in vivo and in vitro. The anti-PfCap380 antisera serves as an important reagent for developmental studies of oocysts from the mosquito midgut and also from oocyst culture using in vitro methodology. The present data demonstrate that PfCap380 is a useful marker to follow the development and maturation of in vivo and in vitro produced oocysts as early as 2 days after zygote formation. Further in vitro studies focused on oocyst and sporozoite maturation will support the manufacturing of whole sporozoites for malaria vaccines.

The Development of Whole Sporozoite Vaccines for Plasmodium falciparum Malaria.

Each year malaria kills hundreds of thousands of people and infects hundreds of millions of people despite current control measures. An effective malaria vaccine will likely be necessary to aid in malaria eradication. Vaccination using whole sporozoites provides an increased repertoire of immunogens compared to subunit vaccines across at least two life cycle stages of the parasite, the extracellular sporozoite, and intracellular liver stage. Three potential whole sporozoite vaccine approaches are under development and include genetically attenuated parasites, radiation attenuated sporozoites, and wild-type sporozoites administered in combination with chemoprophylaxis. Pre-clinical and clinical studies have demonstrated whole sporozoite vaccine immunogenicity, including humoral and cellular immunity and a range of vaccine efficacy that depends on the pre-exposure of vaccinated individuals. While whole sporozoite vaccines can provide protection against malaria in some cases, more recent studies in malaria-endemic regions demonstrate the need for improvements. Moreover, challenges remain in manufacturing large quantities of sporozoites for vaccine commercialization. A promising solution to the whole sporozoite manufacturing challenge is in vitro culturing methodology, which has been described for several Plasmodium species, including the major disease-causing human malaria parasite, Plasmodium falciparum. Here, we review whole sporozoite vaccine immunogenicity and in vitro culturing platforms for sporozoite production.

Antibodies as epidemiological markers of genetically modified crop exposure: detection of Cry1Ab-specific IgG.

There has been an increasing concern with the safety of genetically modified (GM) crops. An important modification of GM crops is the expression of Bacillus thuringiensis (Bt) protein, Cry1Ab. Animal exposure to Cry1Ab indicates that the protein is safe, but that it is immunogenic. Whether Cry1Ab is a human immunogen and whether antibody response to this protein can serve as a marker of high exposure to GM crops is unknown. Here we develop an enzyme-linked immunosorbent assay to detect the presence of Cry1Ab-specific IgG in ~100 individuals living in each of three countries that have varied exposure to GM crops (Papua New Guinea (PNG), low exposure; Kenya, moderate exposure; and the USA, high exposure). Cry1Ab-specific IgG antibodies were detected in individuals living in each region (8%, the USA; 3%, PNG; and 2%, Kenya). Thus, individuals develop anti-Cry1Ab antibodies at a frequency that roughly correlates with the exposure to GM crops expressing this protein.

Relative telomere lengths in tumor and normal mucosa are related to disease progression and chromosome instability profiles in colorectal cancer.

Telomeric dysfunction is linked to colorectal cancer (CRC) initiation. However, the relationship of normal tissue and tumor telomere lengths with CRC progression, molecular features and prognosis is unclear. Here, we measured relative telomere length (RTL) by real-time quantitative PCR in 90 adenomas (aRTL), 419 stage I-IV CRCs (cRTL) and adjacent normal mucosa (nRTL). Age-adjusted RTL was analyzed against germline variants in telomere biology genes, chromosome instability (CIN), microsatellite instability (MSI), CpG island methylator phenotype (CIMP), TP53, KRAS, BRAF mutations and clinical outcomes. In 509 adenoma or CRC patients, nRTL decreased with advancing age. Female gender, proximal location and the TERT rs2736100 G allele were independently associated with longer age-adjusted nRTL. Adenomas and carcinomas exhibited telomere shortening in 79% and 67% and lengthening in 7% and 15% of cases. Age-adjusted nRTL and cRTL were independently associated with tumor stage, decreasing from adenoma to stage III and leveling out or increasing from stage III to IV, respectively. Cancer MSI, CIMP, TP53, KRAS and BRAF status were not related to nRTL or cRTL. Near-tetraploid CRCs exhibited significantly longer cRTLs than CIN- and aneuploidy CRCs, while cRTL was significantly shorter in CRCs with larger numbers of chromosome breaks. Age-adjusted nRTL, cRTL or cRTL:nRTL ratios were not associated with disease-free or overall survival in stage II/III CRC. Taken together, our data show that both normal mucosa and tumor RTL are independently associated with CRC progression, and highlight divergent associations of CRC telomere length with tumor CIN profiles.

BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling.

OBJECTIVE: Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival. DESIGN: Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-κB complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5-aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated. RESULTS: We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-κB p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK-3ß and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells. CONCLUSIONS: Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy.

Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival.

BACKGROUNDS: Spontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms. METHODS: PCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis. RESULTS: The 25.4 kDa native PCMT from C. utilis was found to have a Km of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro. GENERAL SIGNIFICANCE: Identification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.

'Little old lady's hernia' causing small bowel obstruction in a man: a case report with a review of literature on the pathophysiology of obturator hernias.

Commonly known as 'little old lady's hernia', obturator hernias are usually seen in frail, octogenarian multiparous women reporting non-specific nausea and vomiting, abdominal pain and anteromedial thigh pain. They are exceedingly rare; even less frequently are they diagnosed preoperatively, with the vast majority being found incidentally at laparotomy for small bowel obstruction. This case report describes an atypical presentation of a 'little old lady's hernia' in a man, in whom, thanks to high degree of clinical suspicion, an incarcerated obturator hernia was diagnosed preoperatively and treated successfully.

Multiple pathways for Plasmodium ookinete invasion of the mosquito midgut.

Plasmodium ookinete invasion of the mosquito midgut is a crucial step of the parasite life cycle but little is known about the molecular mechanisms involved. Previously, a phage display peptide library screen identified SM1, a peptide that binds to the mosquito midgut epithelium and inhibits ookinete invasion. SM1 was characterized as a mimotope of an ookinete surface enolase and SM1 presumably competes with enolase, the presumed ligand, for binding to a putative midgut receptor. Here we identify a mosquito midgut receptor that binds both SM1 and ookinete surface enolase, termed "enolase-binding protein" (EBP). Moreover, we determined that Plasmodium berghei parasites are heterogeneous for midgut invasion, as some parasite clones are strongly inhibited by SM1 whereas others are not. The SM1-sensitive parasites required the mosquito EBP receptor for midgut invasion whereas the SM1-resistant parasites invaded the mosquito midgut independently of EBP. These experiments provide evidence that Plasmodium ookinetes can invade the mosquito midgut by alternate pathways. Furthermore, another peptide from the original phage display screen, midgut peptide 2 (MP2), strongly inhibited midgut invasion by P. berghei (SM1-sensitive and SM1-resistant) and Plasmodium falciparum ookinetes, suggesting that MP2 binds to a separate, universal receptor for midgut invasion.

Purification, characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae.

BACKGROUND: In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS). METHODS: The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant. RESULTS: The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30°C and 7.0 respectively. The Km Vmax and Kcat against S-adenosyl-l-methionine (AdoMet) were 4.95µM, 3.2U/mg and 6.4s(-1) respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a Ki as 10.9µM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kan(r) exhibited almost 50% reduction in intracellular trehalose concentration. CONCLUSION: A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae. GENERAL SIGNIFICANCE: This is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.

Enzymatic and regulatory attributes of trehalose-6-phosphate phosphatase from Candida utilis and its role during thermal stress.

Trehalose-6-phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti-stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43-fold purification. The protein sequence, determined by N-terminal sequencing and MALDI-TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length gene sequence of TPP of C. utilis was identified using rapid amplification of cDNA ends-PCR reaction (RACE-PCR). The gene was cloned and expressed in Escherichia coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steady-state fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. Km was determined to be ~0.8 mM. Optimum temperature and pH were found to be 30 °C and 8.5, respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylation-mediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations toward understanding the physico-chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalose synthesis and survivability of the organism under thermal stress was established.

Analysis of colorectal cancers in British Bangladeshi identifies early onset, frequent mucinous histotype and a high prevalence of RBFOX1 deletion.

BACKGROUND: Prevalence of colorectal cancer (CRC) in the British Bangladeshi population (BAN) is low compared to British Caucasians (CAU). Genetic background may influence mutations and disease features. METHODS: We characterized the clinicopathological features of BAN CRCs and interrogated their genomes using mutation profiling and high-density single nucleotide polymorphism (SNP) arrays and compared findings to CAU CRCs. RESULTS: Age of onset of BAN CRC was significantly lower than for CAU patients (p=3.0 x 10-5) and this difference was not due to Lynch syndrome or the polyposis syndromes. KRAS mutations in BAN microsatellite stable (MSS) CRCs were comparatively rare (5.4%) compared to CAU MSS CRCs (25%; p=0.04), which correlates with the high percentage of mucinous histotype observed (31%) in the BAN samples. No BRAF mutations was seen in our BAN MSS CRCs (CAU CRCs, 12%; p=0.08). Array data revealed similar patterns of gains (chromosome 7 and 8q), losses (8p, 17p and 18q) and LOH (4q, 17p and 18q) in BAN and CAU CRCs. A small deletion on chromosome 16p13.2 involving the alternative splicing factor RBFOX1 only was found in significantly more BAN (50%) than CAU CRCs (15%) cases (p=0.04). Focal deletions targeting the 5' end of the gene were also identified. Novel RBFOX1 mutations were found in CRC cell lines and tumours; mRNA and protein expression was reduced in tumours. CONCLUSIONS: KRAS mutations were rare in BAN MSS CRC and a mucinous histotype common. Loss of RBFOX1 may explain the anomalous splicing activity associated with CRC.

HSP27 expression in primary colorectal cancers is dependent on mutation of KRAS and PI3K/AKT activation status and is independent of TP53.

Colorectal adenomas display features of senescence, but these are often lost upon progression to carcinoma, indicating that oncogene induced senescence (OIS) could be a roadblock in colorectal cancer (CRC) development. Heat shock proteins (HSPs) have been implicated in the prognosis of CRC and HSP based therapy is a current interest for drug development. Recent cell culture studies have suggested that in the absence of a TP53 mutation, OIS mediated by PI3K/AKT activation can be circumvented by high expression of HSPs. Furthermore, while PI3K/AKT activation and KRAS mutations are independent inducers of OIS, PI3K/AKT activation can suppress KRAS-induced OIS when both are present in cultured cells. As KRAS mutations, PI3K/AKT activation and TP53 mutations are all common features of CRC, it is possible that the requirement for HSP to inhibit OIS in CRC is dependent on the mutation spectrum of a tumour. However, work on HSP that utilised mutation profiled human tumour tissues has been limited. Here, we characterised the expression of two major HSP proteins (HSP27 and 72) by immunohistochemistry (IHC), the mutation status of TP53, KRAS and PIK3CA genes by direct sequencing and the activation status of AKT by IHC in a cohort of unselected primary CRC (n=74). We compare our data with findings generated from cell-based studies. Expression of HSP27 and HSP72 was correlated to clinicopathological and survival data but no significant association was found. We also established the mutation status of TP53, KRAS and PIK3CA genes and the activation status of AKT in our CRC panel. We did not detect any associations between HSP27 or HSP72 expression with TP53 mutation status. However, HSP27 expression in CRCs was strongly associated with the co-presence of wildtype KRAS and activated PI3K/AKT (p=0.004), indicating a possible role of HSP27 in overcoming PI3K/AKT induced OIS in tumours. Our studies suggest a role for using archival tissues in validating hypotheses generated from cell culture based investigations.