Inaugurating High-Throughput Profiling of Extracellular Vesicles for Earlier Ovarian Cancer Detection.

Detecting early cancer through liquid biopsy is challenging due to the lack of specific biomarkers for early lesions and potentially low levels of these markers. The current study systematically develops an extracellular-vesicle (EV)-based test for early detection, specifically focusing on high-grade serous ovarian carcinoma (HGSOC). The marker selection is based on emerging insights into HGSOC pathogenesis, notably that it arises from precursor lesions within the fallopian tube. This work thus establishes murine fallopian tube (mFT) cells with oncogenic mutations and performs proteomic analyses on mFT-derived EVs. The identified markers are then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood of tumor-bearing mice, mFT-EV markers increase with tumor initiation, supporting their potential use in early cancer detection. A pilot clinical study (n = 51) further narrows EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. The combined expression of these markers distinguishes HGSOC from non-cancer with 89% sensitivity and 93% specificity. The same markers are also effective in classifying three groups (non-cancer, early-stage HGSOC, and late-stage HGSOC). The developed approach, for the first time inaugurated in fallopian tube-derived EVs, could be a minimally invasive tool to monitor women at high risk of ovarian cancer for timely intervention.

Profiling extracellular vesicles in circulation enables the early detection of ovarian cancer.

Ovarian cancer is a heterogeneous group of tumors in both cell type and natural history. While outcomes are generally favorable when detected early, the most common subtype, high-grade serous carcinoma (HGSOC), typically presents at an advanced stage and portends less favorable prognoses. Its aggressive nature has thwarted early detection efforts through conventional detection methods such as serum CA125 and ultrasound screening and thus inspired the investigation of novel biomarkers. Here, we report the systematic development of an extracellular-vesicle (EV)-based test to detect early-stage HGSOC. Our study is based on emerging insights into HGSOC biology, notably that it arises from precursor lesions within the fallopian tube before traveling to ovarian and/or peritoneal surfaces. To identify HGSOC marker candidates, we established murine fallopian tube (mFT) cells with oncogenic mutations in Brca1/2, Tp53 , and Pten genes, and performed proteomic analyses on mFT EVs. The identified markers were then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood samples of tumor-bearing mice, mFT-EV markers increased with tumor initiation, supporting their potential use in early cancer detection. A pilot human clinical study ( n = 51) further narrowed EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. Combined expression of these markers achieved high OvCa diagnostic accuracy (cancer vs. non-cancer) with a sensitivity of 0.89 and specificity of 0.93. The same five markers were also effective in a three-group classification: non-cancer, early-stage (I & II) HGSOC, and late-stage (III & IV) HGSOC. In particular, they differentiated early-stage HGSOC from the rest with a specificity of 0.91. Minimally invasive and repeatable, this EV-based testing could be a versatile and serial tool for informing patient care and monitoring women at high risk for ovarian cancer.

Pan-caspase inhibition during normothermic machine perfusion of discarded livers mitigates ex situ innate immune responses.

Access to liver transplantation is limited by a significant organ shortage. The recent introduction of machine perfusion technology allows surgeons to monitor and assess ex situ liver function prior to transplantation. However, many donated organs are of inadequate quality for transplant, though opportunities exist to rehabilitate organ function with adjunct therapeutics during normothermic machine perfusion. In this preclinical study, we targeted the apoptosis pathway as a potential method of improving hepatocellular function. Treatment of discarded human livers during normothermic perfusion with an irreversible pan-caspase inhibitor, emricasan, resulted in significant mitigation of innate immune and pro-inflammatory responses at both the transcriptional and protein level. This was evidenced by significantly decreased circulating levels of the pro-inflammatory cytokines, interleukin-6, interleukin-8, and interferon-gamma, compared to control livers. Compared to emricasan-treated livers, untreated livers demonstrated transcriptional changes notable for enrichment in pathways involved in innate immunity, leukocyte migration, and cytokine-mediated signaling. Targeting of unregulated apoptosis may represent a viable therapeutic intervention for immunomodulation during machine perfusion.

Activation of autophagy during normothermic machine perfusion of discarded livers is associated with improved hepatocellular function.

Liver transplantation is hampered by a severe shortage of donor organs. Normothermic machine perfusion (NMP) of donor livers allows dynamic preservation in addition to viability assessment before transplantation. Little is known about the injury and repair mechanisms induced during NMP. To investigate these mechanisms, we examined gene and protein expression changes in a cohort of discarded human livers, stratified by hepatocellular function, during NMP. Six human livers acquired through donation after circulatory death (DCD) underwent 12 h of NMP. Of the six livers, three met predefined criteria for adequate hepatocellular function. We applied transcriptomic profiling and protein analysis to evaluate temporal changes in gene expression during NMP between functional and nonfunctional livers. Principal component analysis segregated the two groups and distinguished the various perfusion time points. Transcriptomic analysis of biopsies from functional livers indicated robust activation of innate immunity after 3 h of NMP followed by enrichment of prorepair and prosurvival mechanisms. Nonfunctional livers demonstrated delayed and persistent enrichment of markers of innate immunity. Functional livers demonstrated effective induction of autophagy, a cellular repair and homeostasis pathway, in contrast to nonfunctional livers. In conclusion, NMP of discarded DCD human livers results in innate immune-mediated injury, while also activating autophagy, a presumed mechanism for support of cellular repair. More pronounced activation of autophagy was seen in livers that demonstrated adequate hepatocellular function.NEW & NOTEWORTHY We demonstrate that ischemia-reperfusion injury occurs in all livers during NMP, though there are notable differences in gene expression between functional and nonfunctional livers. We further demonstrate that activation of the liver's repair and homeostasis mechanisms through autophagy plays a vital role in the graft's response to injury and may impact liver function. These findings indicate that liver autophagy might be a key therapeutic target for rehabilitating the function of severely injured or untransplantable livers.

Plasma Metabolite Markers of Parkinson's Disease and Atypical Parkinsonism.

Differentiating between Parkinson's disease (PD) and the atypical Parkinsonian disorders of multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) is difficult clinically due to overlapping symptomatology, especially at early disease stages. Consequently, there is a need to identify metabolic markers for these diseases and to develop them into viable biomarkers. In the present investigation, solution nuclear magnetic resonance and mass spectrometry metabolomics were used to quantitatively characterize the plasma metabolomes (a total of 167 metabolites) of a cohort of 94 individuals comprising 34 PD, 12 MSA, and 17 PSP patients, as well as 31 control subjects. The distinct and statistically significant differences observed in the metabolite concentrations of the different disease and control groups enabled the identification of potential plasma metabolite markers of each disorder and enabled the differentiation between the disorders. These group-specific differences further implicate disturbances in specific metabolic pathways. The two metabolites, formic acid and succinate, were altered similarly in all three disease groups when compared to the control group, where a reduced level of formic acid suggested an effect on pyruvate metabolism, methane metabolism, and/or the kynurenine pathway, and an increased succinate level suggested an effect on the citric acid cycle and mitochondrial dysfunction.

Deep mining of oxysterols and cholestenoic acids in human plasma and cerebrospinal fluid: Quantification using isotope dilution mass spectrometry.

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.

Targeted Multiple Reaction Monitoring Analysis of CSF Identifies UCHL1 and GPNMB as Candidate Biomarkers for ALS.

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) share some common molecular deficits including disruption of protein homeostasis leading to disease-specific protein aggregation. While insoluble protein aggregates are the defining pathological confirmation of diagnosis, patient stratification based on early molecular etiologies may identify distinct subgroups within a clinical diagnosis that would respond differently in therapeutic development programs. We are developing targeted multiple reaction monitoring (MRM) mass spectrometry methods to rigorously quantify CSF proteins from known disease genes involved in lysosomal, ubiquitin-proteasomal, and autophagy pathways. Analysis of CSF from 21 PD, 21 ALS, and 25 control patients, rigorously matched for gender, age, and age of sample, revealed significant changes in peptide levels between PD, ALS, and control. In patients with PD, levels of two peptides for chromogranin B (CHGB, secretogranin 1) were significantly reduced. In CSF of patients with ALS, levels of two peptides from ubiquitin carboxy-terminal hydrolase like protein 1 (UCHL1) and one peptide each for glycoprotein non-metastatic melanoma protein B (GPNMB) and cathepsin D (CTSD) were all increased. Analysis of patients with ALS separated into two groups based on length of survival after CSF sampling revealed that the increases in GPNMB and UCHL1 were specific for short-lived ALS patients. While analysis of additional cohorts is required to validate these candidate biomarkers, this study suggests methods for stratification of ALS patients for clinical trials and identifies targets for drug efficacy measurements during therapeutic development.

Defining fallopian tube-derived miRNA cancer signatures.

BACKGROUND: MicroRNAs have recently emerged as promising circulating biomarkers in diverse cancer types, including ovarian cancer. We utilized conditional, doxycycline-induced fallopian tube (FT)-derived cancer models to identify changes in miRNA expression in tumors and plasma, and further validated the murine findings in high-grade ovarian cancer patient samples. METHODS: We analyzed 566 biologically informative miRNAs in doxycycline-induced FT and metastatic tumors as well as plasma samples derived from murine models bearing inactivation of Brca, Tp53, and Pten genes. We identified miRNAs that showed a consistent pattern of dysregulated expression and validated our results in human patient serum samples. RESULTS: We identified six miRNAs that were significantly dysregulated in doxycycline-induced FTs (P < .05) and 130 miRNAs differentially regulated in metastases compared to normal fallopian tissues (P < .05). Furthermore, we validated miR-21a-5p, miR-146a-5p, and miR-126a-3p as dysregulated in both murine doxycycline-induced FT and metastatic tumors, as well as in murine plasma and patient serum samples. CONCLUSIONS: In summary, we identified changes in miRNA expression that potentially accompany tumor development in murine models driven by commonly found genetic alterations in cancer patients. Further studies are required to test both the function of these miRNAs in driving the disease and their utility as potential biomarkers for diagnosis and/or disease progression.

Nanoformulation of Talazoparib Delays Tumor Progression and Ascites Formation in a Late Stage Cancer Model.

Talazoparib, a potent PARP inhibitor, induces synthetic lethality in BRCA-deficient cancers making it an attractive candidate for ovarian cancer treatment. However, its potency lends itself to side effects associated more closely with traditional chemotherapeutics than other clinically approved PARP inhbitors. We sought to formulate Talazoparib in a nanoparticle delivery system, which allows the drug to be administered intraperitoneally. This was done to specifically target peritoneal dissemination of late stage metastatic ovarian cancer and increase talazoparib's therapeutic efficacy while minimizing toxic side effects. NanoTalazoparib was developed and characterized with regard to its size, loading, and surface charge. Talazoparib and NanoTalazoparib were tested on a panel of murine and human BRCA cell lines and the dose response was compared to Olaparib's, the currently used PARP inhibitor. Therapeutic efficacy was tested in vivo in a Brca peritoneal cancer model that mimics late stage disseminated disease. NanoTalazoparib has a diameter of about 70 nm with a neutral surface charge and ~75% encapsulation efficiency, which slowly releases the drug over several hours. Dose response analysis indicated that the murine cell lines with conditional BRCA1/2, PTEN, and TP53 deletions had the lowest IC50s. NanoTalazoparib administered on a schedule of three doses weekly slowed disease progression and resulted in significantly less mice with ascites at the end point compared to controls. These results indicate that the slow release nanoformulation, NanoTalazoparib, effectively delivers PARP inhibitor therapy to the peritoneal cavity for disseminated cancer treatment. The ability to decrease ascites formation with the introduction of intraperitoneal NanoTalazoparib suggests this treatment may be an effective way to treat ovarian cancer-associated ascites and slow disease progression.

Serum Metabolite Markers of Dementia Through Quantitative NMR Analysis: The Importance of Threonine-Linked Metabolic Pathways.

There is a great need for diagnostic biomarkers of impending dementia. Metabolite markers in blood have been investigated in several studies, but inconclusive findings encourage further investigation, particularly in the pre-diagnostic phase. In the present study, the serum metabolomes of 110 dementia or pre-diagnostic dementia individuals and 201 healthy individuals matched for age, gender, and education were analyzed by nuclear magnetic resonance spectroscopy in combination with multivariate data analysis. 58 metabolites were quantified in each of the 311 samples. Individuals with dementia were discriminated from controls using a panel of seven metabolites, while the pre-diagnostic dementia subjects were distinguished from controls using a separate set of seven metabolites, where threonine was a common significant metabolite in both panels. Metabolite and pathway alterations specific for dementia and pre-diagnostic dementia were identified, in particular a disturbed threonine catabolism at the pre-diagnostic stage that extends to several threonine-linked pathways at the dementia stage.

Intraperitoneal delivery of NanoOlaparib for disseminated late-stage cancer treatment.

BACKGROUND: PARP inhibitors, such as Olaparib, have advanced the treatment of ovarian cancer by providing patients with an effective and molecularly-targeted maintenance therapy. However, all orally-administered drugs, including Olaparib, must undergo first-pass metabolism. In contrast, a nanoparticle delivery system has the advantage of administering Olaparib directly into the peritoneal cavity for local treatment. Consequently, we sought to optimize the sustained-release formulation NanoOlaparib, previously deemed effective as an intravenous solid tumor treatment, for the local treatment of disseminated disease via intraperitoneal (i.p.) therapy. METHODS: The tumor cell line 404, which was derived from a Brca2 -/-, Tp53 -/-, Pten -/- genetically engineered mouse model, exhibited high sensitivity to Olaparib in vitro. It was chosen for use in developing an i.p. spread xenograft for testing nanotherapy efficacy in vivo. NanoOlaparib as a monotherapy or in combination with cisplatin was compared to oral Olaparib alone or in combination using two different dose schedules. A pilot biodistribution study was performed to determine drug accumulation in various organs following i.p. administration. RESULTS: Daily administration of NanoOlaparib reduced tumor growth and decreased the variability of the treatment response observed with daily oral Olaparib administration. However, systemic toxicity was observed in both the NanoOlaparib and vehicle (empty nanoparticle) treated groups. Scaling back the administration to twice weekly was well tolerated up to 100 mg/kg but reduced the effect on tumor growth. Biodistribution profiles indicated that NanoOlaparib began accumulating in tissues within an hour of administration and persisted for at least 72 hours after a single dose, exiting the peritoneal cavity faster than expected. CONCLUSION: NanoOlaparib must be modified for use against disseminated disease. Future avenues to develop NanoOlaparib as an i.p. therapy include a modified surface-coating to retain it in the peritoneal cavity and prevent entry into systemic circulation, in addition to targeting moieties for localization in tumor cells.

Epigenetic Reprogramming Strategies to Reverse Global Loss of 5-Hydroxymethylcytosine, a Prognostic Factor for Poor Survival in High-grade Serous Ovarian Cancer.

Purpose: A major challenge in platinum-based cancer therapy is the clinical management of chemoresistant tumors, which have a largely unknown pathogenesis at the level of epigenetic regulation.Experimental Design: We evaluated the potential of using global loss of 5-hydroxymethylcytosine (5-hmC) levels as a novel diagnostic and prognostic epigenetic marker to better assess platinum-based chemotherapy response and clinical outcome in high-grade serous tumors (HGSOC), the most common and deadliest subtype of ovarian cancer. Furthermore, we identified a targetable pathway to reverse these epigenetic changes, both genetically and pharmacologically.Results: This study shows that decreased 5-hmC levels are an epigenetic hallmark for malignancy and tumor progression in HGSOC. In addition, global 5-hmC loss is associated with a decreased response to platinum-based chemotherapy, shorter time to relapse, and poor overall survival in patients newly diagnosed with HGSOC. Interestingly, the rescue of 5-hmC loss restores sensitivity to platinum chemotherapy in vitro and in vivo, decreases the percentage of tumor cells with cancer stem cell markers, and increases overall survival in an aggressive animal model of platinum-resistant disease.Conclusions: Consequently, a global analysis of patient 5-hmC levels should be included in future clinical trials, which use pretreatment with epigenetic adjuvants to elevate 5-hmC levels and improve the efficacy of current chemotherapies. Identifying prognostic epigenetic markers and altering chemotherapeutic regimens to incorporate DNMTi pretreatment in tumors with low 5-hmC levels could have important clinical implications for newly diagnosed HGSOC disease. Clin Cancer Res; 24(6); 1389-401. ©2017 AACR.

Metabolite quantification by NMR and LC-MS/MS reveals differences between unstimulated, stimulated, and pure parotid saliva.

Saliva is a readily available biofluid that is sensitive to metabolic changes and can be collected through rapid and non-invasive collection procedures, and it shows great promise for clinical metabolomic studies. This work studied the metabolite composition of, and the differences between, saliva samples collected by unstimulated spitting/drooling, paraffin chewing-stimulated spitting, and parotid gland suction using targeted nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for metabolite quantification. As applied here, these two analytical techniques provide complementary metabolite information and together extend the metabolome coverage with robust NMR quantification of soluble metabolites and sensitive targeted LC-MS/MS analysis of bioactive lipids in specific metabolic pathways. The NMR analysis was performed on ultrafiltrated (3kDa cutoff) saliva samples and resulted in a total of 45 quantified metabolites. The LC-MS/MS analysis was performed on both filtered and unfiltered samples and resulted in the quantification of two endocannabinoids (AEA and PEA) and 22 oxylipins, which at present is the most comprehensive targeted analysis of bioactive lipids in human saliva. Important differences in the metabolite composition were observed between the three saliva sample collection methods, which should be taken into consideration when designing metabolomic studies of saliva. Furthermore, the combined use of the two metabolomics platforms (NMR and LC-MS/MS) proved to be viable for research and clinical studies of the salivary metabolome.

When Maxillary Sinusitis Does Not Heal: Findings on CBCT Scans of the Sinuses With a Particular Focus on the Occurrence of Odontogenic Causes of Maxillary Sinusitis.

Objectives: This study sought to investigate the proportion of patients with suspected sinusitis referred for radiological examination who have radiologically verified sinusitis of odontogenic origin and to describe this type of sinusitis. Study Design: This investigation is a retrospective study. Methods: A total of 303 sinus examinations involving cone beam computed tomography (CBCT) performed at Sunderby Hospital, Luleå, Sweden in 2012 were independently reviewed by two radiologists. The number of cases of maxillary sinusitis and the correlation between maxillary sinusitis and odontogenic infections were determined. Results: Overall, 24% of the verified cases of sinusitis were odontogenic. An odontogenic origin was identified in 40% of unilateral maxillary sinusitis cases but only 6% of bilateral maxillary sinusitis cases (p = 0.0015). Forty-nine out of 54 patients with periapical destruction had adjacent mucosal swelling in the maxillary sinus, but only 15 of these patients satisfied the criteria for sinusitis. Conclusion: The present study confirms the close relationship between odontogenic infections and unilateral maxillary sinusitis. Level of Evidence 4.

NMR analysis of the human saliva metabolome distinguishes dementia patients from matched controls.

Saliva is a biofluid that is sensitive to metabolic changes and is straightforward to collect in a non-invasive manner, but it is seldom used for metabolite analysis when studying neurodegenerative disorders. We present a procedure for both an untargeted and targeted analysis of the saliva metabolome in which nuclear magnetic resonance (NMR) spectroscopy is used in combination with multivariate data analysis. The applicability of this approach is demonstrated on saliva samples selected from the 25 year prospective Betula study, including samples from dementia subjects with either Alzheimer's disease (AD) or vascular dementia at the time of sampling or who developed it by the next sampling/assessment occasion five years later, and age-, gender-, and education-matched control individuals without dementia. Statistically significant multivariate models were obtained that separated patients with dementia from controls and revealed seven discriminatory metabolites. Dementia patients showed significantly increased concentrations of acetic acid (fold change (fc) = 1.25, p = 2 × 10(-5)), histamine (fc = 1.26, p = 0.019), and propionate (fc = 1.35, p = 0.002), while significantly decreased levels were observed for dimethyl sulfone (fc = 0.81, p = 0.005), glycerol (fc = 0.79, p = 0.04), taurine (fc = 0.70, p = 0.007), and succinate (fc = 0.62, p = 0.008). Histamine, succinate, and taurine are known to be important in AD, and acetic acid and glycerol are involved in related pathways. Dimethyl sulfone and propionate originate from the diet and bacterial flora and might reflect poorer periodontal status in the dementia patients. For these seven metabolites, a weak but statistically significant pre-diagnostic value was observed. Taken together, we present a robust and general NMR analysis approach for studying the saliva metabolome that has potential use for screening and early detection of dementia.

Computed tomography (CT) in the selection of treatment for root-filled maxillary molars with apical periodontitis.

OBJECTIVES: The aims of this study were to evaluate whether the use of CT facilitates agreement among endodontists in selecting treatments for root-filled maxillary molars with apical periodontitis and to assess the efficacy of CT in choosing a treatment for such teeth. METHODS: 39 root-filled maxillary molars from 34 patients with suspected apical periodontitis were independently evaluated by 4 endodontists and 1 postgraduate student (decision-makers). Treatment decisions were made based on intra-oral radiographs and a fictive clinical history. After 1-3 months, the same decision-makers repeated the examination of the same teeth but with additional information from a CT examination. Agreement between decision-makers with or without the availability of the CT results was measured with Cohen's kappa coefficient. Differences in selected treatments with or without accessibility to the CT results were plotted for the same endodontists using descriptive statistics. RESULTS: The agreement in assessments among endodontists was slight or fair before the CT results were available (range: 0.081-0.535). No increase was observed after reviewing the CT results (range: 0.116-0.379). After the use of CT, the treatment plan was changed 38-76% of the time by all decision-makers, and the changes affected 57.8% of the cases in the study. CONCLUSIONS: The endodontists in this study exhibited a low degree of agreement when choosing a treatment for root-filled maxillary molars with apical periodontitis. A CT examination of the investigated teeth did not result in a significantly higher degree of agreement, and CT frequently contributed to a shift in the selected therapy.

NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation.

Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol/water (MeOH/H2O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H2O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. The human milk metabolomes at the early (9-24 days after delivery) and late (31-87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant.

Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae.

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

The promise and challenge of ovarian cancer models.

The complexity and heterogeneity of ovarian cancer cases are difficult to reproduce in in vitro studies, which cannot adequately elucidate the molecular events involved in tumor initiation and disease metastasis. It has now become clear that, although the multiple histological subtypes of ovarian cancer are being treated with similar surgical and therapeutic approaches, they are in fact characterized by distinct phenotypes, cell of origin, and underlying key genetic and genomic alterations. Consequently, the development of more personalized treatment methodologies, which are aimed at improving patient care and prognosis, will greatly benefit from a better understanding of the key differences between various subtypes. To accomplish this, animal models of all histotypes need to be generated in order to provide accurate in vivo platforms for research and the testing of targeted treatments and immune therapies. Both genetically engineered mouse models (GEMMs) and xenograft models have the ability to further our understanding of key mechanisms facilitating tumorigenesis, and at the same time offer insight into enhanced imaging and treatment modalities. While genetic models may be better suited to examine oncogenic functions and interactions during tumorigenesis, patient-derived xenografts (PDXs) are likely a superior model to assess drug efficacy, especially in concurrent clinical trials, due to their similarity to the tumors from which they are derived. Genetic and avatar models possess great clinical utility and have both benefits and limitations. Additionally, the laying hen model, which spontaneously develops ovarian tumors, has inherent advantages for the study of epithelial ovarian cancer (EOC) and recent work champions this model especially when assessing chemoprevention strategies. While high-grade ovarian serous tumors are the most prevalent form of EOC, rarer ovarian cancer variants, such as small cell ovarian carcinoma of the hypercalcemic type and transitional cell carcinoma, or non-epithelial tumors, including germ cell tumors, will also benefit from the generation of improved models to advance our understanding of tumorigenic mechanisms and the development of selective therapeutic options.

NMR metabonomics of cerebrospinal fluid distinguishes between Parkinson's disease and controls.

This study assesses if nuclear magnetic resonance (NMR) metabonomics can discriminate between Parkinson's disease (PD) patients and control subjects, and consequently identify metabolic markers for the disease. One-dimensional (1)H NMR spectroscopy was used for quantitative analysis of metabolites in the cerebrospinal fluid (CSF) from 10 PD patients and 10 control individuals, together with uni- and multivariate statistical analysis to discriminate between the groups and to identify significantly altered metabolite concentrations. In total 60 metabolites were identified and of those 38 were quantified in all CSF samples. An overall lowering of metabolite content was observed in PD patients compared to control subjects (fold change of 0.85±0.30). Multivariate statistics reveal significant changes (ǀw*ǀ>0.2) among nine metabolites (alanine, creatinine, dimethylamine, glucose, lactate, mannose, phenylalanine, 3-hydroxyisobutyric acid and 3-hydroxyisovaleric acid). Three of these (alanine, creatinine and mannose) are identified as significantly changed also by univariate statistics (p<0.00132, Bonferroni corrected). Panels with all or a selected set of these metabolites were successfully used for discriminating between the two groups. In conclusion, NMR metabonomics can readily determine metabolite concentrations in CSF, identify putative biomarkers that distinguish between the PD patients and control subjects, and thus potentially become a tool for diagnostic purposes.