Enzyme-catalysed polymer cross-linking: Biocatalytic tools for chemical biology, materials science and beyond.

Intermolecular cross-linking is one of the most important techniques that can be used to fundamentally alter the material properties of a polymer. The introduction of covalent bonds between individual polymer chains creates 3D macromolecular assemblies with enhanced mechanical properties and greater chemical or thermal tolerances. In contrast to many chemical cross-linking reactions, which are the basis of thermoset plastics, enzyme catalysed processes offer a complimentary paradigm for the assembly of cross-linked polymer networks through their predictability and high levels of control. Additionally, enzyme catalysed reactions offer an inherently 'greener' and more biocompatible approach to covalent bond formation, which could include the use of aqueous solvents, ambient temperatures, and heavy metal-free reagents. Here, we review recent progress in the development of biocatalytic methods for polymer cross-linking, with a specific focus on the most promising candidate enzyme classes and their underlying catalytic mechanisms. We also provide exemplars of the use of enzyme catalysed cross-linking reactions in industrially relevant applications, noting the limitations of these approaches and outlining strategies to mitigate reported deficiencies.

Associations between antibiotic prescriptions and recurrent urinary tract infections in female college students.

Urinary tract infections (UTIs) are common among college-aged women and often recur. Some antibiotics recommended to treat UTIs trigger dysbiosis of intestinal and vaginal microbiomes - where uropathogens originate, though few studies have investigated associations between these therapies with recurrent infections. We retrospectively analysed the electronic medical records of 6651 college-aged women diagnosed with a UTI at a US university student health centre between 2006 and 2014. Women were followed for 6 months for incidence of a recurrent infection. In a secondary analysis, associations in women whose experienced UTI recurrence within 2 weeks were also considered for potential infection relapse. Logistic regression was used to assess associations between infection recurrence or relapse and antibiotics prescribed, in addition to baseline patient characteristics including age, race/ethnicity, region of origin, year of encounter, presence of symptomology, pyelonephritis, vaginal coinfection and birth control consultation. There were 1051 instances of infection recurrence among the 6620 patients, indicating a prevalence of 16%. In the analysis of patient characteristics, Asian women were statistically more likely to experience infection recurrence whereas African American were less likely. No significant associations were identified between the antibiotic administered at the initial infection and the risk of infection recurrence after multivariable adjustment. Treatment with trimethoprim-sulphamethoxazole and being born outside of the USA were significantly associated with increased odds of infection relapse in the multivariate analysis. The results of the analyses suggest that treatment with trimethoprim-sulphamethoxazole may lead to an increased risk of UTI relapse, warranting further study.

Methylome analysis of extreme chemoresponsive patients identifies novel markers of platinum sensitivity in high-grade serous ovarian cancer.

BACKGROUND: Despite an early response to platinum-based chemotherapy in advanced stage high-grade serous ovarian cancer (HGSOC), the majority of patients will relapse with drug-resistant disease. Aberrant epigenetic alterations like DNA methylation are common in HGSOC. Differences in DNA methylation are associated with chemoresponse in these patients. The objective of this study was to identify and validate novel epigenetic markers of chemoresponse using genome-wide analysis of DNA methylation in extreme chemoresponsive HGSOC patients. METHODS: Genome-wide next-generation sequencing was performed on methylation-enriched tumor DNA of two HGSOC patient groups with residual disease, extreme responders (≥18 months progression-free survival (PFS), n = 8) and non-responders (≤6 months PFS, n = 10) to platinum-based chemotherapy. DNA methylation and expression data of the same patients were integrated to create a gene list. Genes were validated on an independent cohort of extreme responders (n = 21) and non-responders (n = 31) using pyrosequencing and qRT-PCR. In silico validation was performed using publicly available DNA methylation (n = 91) and expression (n = 208) datasets of unselected advanced stage HGSOC patients. Functional validation of FZD10 on chemosensitivity was carried out in ovarian cancer cell lines using siRNA-mediated silencing. RESULTS: Integrated genome-wide methylome and expression analysis identified 45 significantly differentially methylated and expressed genes between two chemoresponse groups. Four genes FZD10, FAM83A, MYO18B, and MKX were successfully validated in an external set of extreme chemoresponsive HGSOC patients. High FZD10 and MKX methylation were related with extreme responders and high FAM83A and MYO18B methylation with non-responders. In publicly available advanced stage HGSOC datasets, FZD10 and MKX methylation levels were associated with PFS. High FZD10 methylation was strongly associated with improved PFS in univariate analysis (hazard ratio (HR) = 0.43; 95% CI, 0.27-0.71; P = 0.001) and multivariate analysis (HR = 0.39; 95% CI, 0.23-0.65; P = 0.003). Consistently, low FZD10 expression was associated with improved PFS (HR = 1.36; 95% CI, 0.99-1.88; P = 0.058). FZD10 silencing caused significant sensitization towards cisplatin treatment in survival assays and apoptosis assays. CONCLUSIONS: By applying genome-wide integrated methylome analysis on extreme chemoresponsive HGSOC patients, we identified novel clinically relevant, epigenetically-regulated markers of platinum-sensitivity in HGSOC patients. The clinical potential of these markers in predictive and therapeutic approaches has to be further validated in prospective studies.

Studying platinum sensitivity and resistance in high-grade serous ovarian cancer: Different models for different questions.

High-grade serous ovarian cancer (HGSOC) has the highest mortality rate among all gynecological cancers. Patients are generally diagnosed in an advanced stage with the majority of cases displaying platinum resistant relapses. Recent genomic interrogation of large numbers of HGSOC patient samples indicated high complexity in terms of genetic aberrations, intra- and intertumor heterogeneity and underscored their lack of targetable oncogenic mutations. Sub-classifications of HGSOC based on expression profiles, termed 'differentiated', 'immunoreactive', 'mesenchymal' and 'proliferative', were shown to have prognostic value. In addition, in almost half of all HGSOC patients, a deficiency in homologous recombination (HR) was found that potentially can be targeted using PARP inhibitors. Developing precision medicine requires advanced experimental models. In the current review, we discuss experimental HGSOC models in which resistance to platinum therapy and the use of novel therapeutics can be carefully studied. Panels of better-defined primary cell lines need to be established to capture the full spectrum of HGSOC subtypes. Further refinement of cell lines is obtained with a 3-dimensional culture model mimicking the tumor microenvironment. Alternatively, ex vivo ovarian tumor tissue slices are used. For in vivo studies, larger panels of ovarian cancer patient-derived xenografts (PDXs) are being established, encompassing all expression subtypes. Ovarian cancer PDXs grossly retain tumor heterogeneity and clinical response to platinum therapy is preserved. PDXs are currently used in drug screens and as avatars for patient response. The role of the immune system in tumor responses can be assessed using humanized PDXs and immunocompetent genetically engineered mouse models. Dynamic tracking of genetic alterations in PDXs as well as patients during treatment and after relapse is feasible by sequencing circulating cell-free tumor DNA and analyzing circulating tumor cells. We discuss how various models and methods can be combined to delineate the molecular mechanisms underlying platinum resistance and to select HGSOC patients other than BRCA1/2-mutation carriers that could potentially benefit from the synthetic lethality of PARP inhibitors. This integrated approach is a first step to improve therapy outcomes in specific subgroups of HGSOC patients.

Access to Care and Health Care Utilization Among Patients With Nephrolithiasis.

OBJECTIVE: To characterize the impact of nephrolithiasis diagnosis and treatment on health care utilization and identify predictors of barriers to care in the patient population. METHODS: We conducted a retrospective cohort study using the All of Us Database, a National Institutes of Health database targeting recruitment of underrepresented populations. Patients with a diagnosis of kidney stones were included and matched to a control group. Primary outcomes were patients' self-reported health care access and utilization. Univariable and multivariable regression analyses were performed. RESULTS: 9173 patients with a diagnosis of nephrolithiasis were included and matched to 9173 controls without a diagnosis of nephrolithiasis. Patients with kidney stones were less likely to have had >1 year since last provider visit (1.7% vs 3.8%, P <.001), but did not report increased delays obtaining care (31%), inability to afford care (11.4%), or higher likelihood of skipping medications (12.9%). Among patients with stones, 1208 (13.2%) had been treated surgically. On multivariable analysis, younger age, female sex, lower income, lower education, non-insured status, and lower physical and mental health were all associated with delays obtaining care, difficulty affording care, skipping medications, and/or prolonged time since seeing a provider. CONCLUSION: A diagnosis of nephrolithiasis and subsequent surgical intervention were not associated with an increase in patient-reported barriers to care. However, among patients with nephrolithiasis, younger, comorbid, female patients from lower socioeconomic status are at significant risk of being unable to access and utilize treatment.

The Use of Genetic Testing in Nephrolithiasis Evaluation: A Retrospective Review from a Multidisciplinary Kidney Stone Clinic.

OBJECTIVES: To describe genetic and clinical characteristics for patients undergoing genetic testing at our multidisciplinary kidney stone clinic. METHODS: A retrospective review was performed on patients evaluated in our stone clinic and referred to genetics between 2018 and 2022. Patient demographic, clinical, stone, and genetic data were included. We assessed the specific variants identified in our cohort, both those with a pathogenic association and variants of unknown significance (VUS). RESULTS: Of 825 patients seen in our stone clinic from 2018-2022, 50 patients were referred to genetics. Among these patients, 33/50 (66%) underwent genetic testing and were included in the analysis. Of those who underwent genetic testing, a variant was identified in 19/33 (58%) patients, and 9/33 (27%) of these were a known pathogenic variant. Among patients with a pathogenic variant identified, the majority had a family history of stones (55.6%), calcium-based stones (77.8%), had their first stone prior to age 18 (66.7), were recurrent stone formers (100%), and had been managed medically (88.9%) or surgically (88.9%) prior to testing. When comparing patients with a pathogenic variant, VUS, and no variant, there were no significant differences in demographic or clinical parameters. CONCLUSION: In our stone practice, more than half of patients who underwent genetic testing were found to have a variant. However, the majority of these variants were of unknown significance. Further evaluation regarding how genetic testing can impact nephrolithiasis management is needed.

Aubergine is a component of a nanos mRNA localization complex.

Localization of nanos (nos) mRNA to the posterior pole of the Drosophila oocyte is essential for abdominal segmentation and germline development during embryogenesis. Posterior localization is mediated by a complex cis-acting localization signal in the nos 3' untranslated region that comprises multiple partially redundant elements. Genetic analysis suggests that this signal is recognized by RNA-binding proteins and associated factors that package nos mRNA into a localization competent ribonucleoprotein complex. However, functional redundancy among localization elements has made the identification of individual localization factors difficult. Indeed, only a single direct-acting nos localization factor, Rumpelstiltskin (Rump), has been identified thus far. Through a sensitized genetic screen, we have now identified the Argonaute family member Aubergine (Aub) as a nos localization factor. Aub interacts with nos mRNA in vivo and co-purifies with Rump in an RNA-dependent manner. Our results support a role for Aub, independent of its function in RNA silencing, as a component of a nos mRNA localization complex.

Magnetic Resonance Imaging-targeted Prostate Biopsy Compared with Systematic Prostate Biopsy in Biopsy-naïve Patients with Suspected Prostate Cancer.

Background: It remains uncertain whether transrectal ultrasound (TRUS)-guided systematic biopsies can be omitted and rely solely on multiparametric magnetic resonance imaging-targeted biopsies (MRI-TBx) in biopsy-naïve men suspected of prostate cancer (PCa). Objective: To compare PCa detection in biopsy-naïve men between systematic biopsy and MRI-TBx. Design setting and participants: A prospective cohort study was conducted in a Dutch teaching hospital. Consecutive patients with suspected PCa, no history of biopsy, and no clinical suspicion of metastasis underwent both TRUS-guided systematic biopsies and MRI-TBx by multiparametric magnetic resonance imaging (mpMRI)-ultrasound fusion, including sham biopsies in case of negative mpMRI. Outcome measurements and statistical analysis: Clinically significant PCa (csPCa), defined as group ≥2 on the International Society of Urological Pathology grading, was detected. Results and limitations: The overall prevalence of csPCa, irrespective of biopsy technique, was 37.4% (132/353) in our population. MRI-TBx were performed in 263/353 (74.5%) patients with suspicious mpMRI (Prostate Imaging Reporting and Data System [PI-RADS] ≥3). The detection rates for csPCa were 39.5% for MRI-TBx and 42.9% for systematic biopsies. The added values, defined as the additional percentages of patients with csPCa detected by adding one biopsy technique, were 8.7% for the systematic biopsies and 5.3% for MRI-TBx. In patients with nonsuspicious mpMRI, five cases (6%) of csPCa were found by systematic biopsies. Conclusions: This study in biopsy-naïve patients suspected for PCa showed that systematic biopsies have added value to MRI-TBx alone in patients with mpMRI PI-RADS >2. Patient summary: We studied magnetic resonance imaging (MRI)-guided prostate biopsy for diagnosing prostate cancer and compared it with the standard method of prostate biopsy. Standard systematic biopsies cannot be omitted in patients with suspicious MRI, as they add to the detection of significant prostate cancer.

Spontaneous membrane-less multi-compartmentalization via aqueous two-phase separation in complex coacervate micro-droplets.

Polyelectrolyte/nucleotide multiphase complex coacervate droplets are produced by internalized aqueous two-phase separation and used for the spatially dependent chemical transfer of sugar molecules, providing a step towards the development of membrane-free "organelles" within coacervate-based protocells.

Prevalence of potential modifiable factors of hypertension in patients with difficult-to-control hypertension.

BACKGROUND: A comprehensive diagnostic evaluation of potential modifiable factors of difficult-to-control hypertension would enable clinicians to target-specific amendable causes. Therefore, we assessed the prevalence of underlying medical conditions, lifestyle factors, and concomitant medication use in an integrated diagnostic evaluation in patients with difficult-to-control hypertension, referred to a tertiary center. METHODS: The study population consisted of 653 patients referred between 2006 and 2016 for difficult-to-control hypertension to the University Medical Center Utrecht. Difficult-to-control hypertension was defined by not reaching blood pressure (BP) goals despite BP-lowering drug use, or high office BP (>160/100 mmHg) without BP-lowering drug use. Patients were evaluated according to a highly standardized protocol including 24-h ambulatory blood measurements after cessation of BP-lowering drugs, 24-h urine sample, and a isotonic (0.9%) saline infusion test. RESULTS: In 621 patients (95%) one or more modifiable factors related to hypertension were identified (mean 2.1, SD 1.1). Obesity-related insulin resistance was the most common underlying medical condition which was diagnosed in 130 patients (20%). Primary aldosteronism was diagnosed in 40 patients (6%) and obstructive sleep apnea in 17 patients (3%). Sodium intake was deemed to high (urinary excretion of >6 g/day) in 433 patients (66%). In total, 283 patients (43%) were physical inactive (<30 min/day, during 5 days/week). Oral contraceptive-related hypertension was diagnosed in 10 women (3% of women). CONCLUSION: In patients with difficult-to-control hypertension there is a high prevalence of potential modifiable factors related to hypertension, highlighting the importance for an integrated diagnostic evaluation.

Direct Transfer of a Mycoplasma mycoides Genome to Yeast Is Enhanced by Removal of the Mycoides Glycerol Uptake Factor Gene glpF.

We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.

Apparent resistant hypertension and the risk of vascular events and mortality in patients with manifest vascular disease.

OBJECTIVE: Patients with apparent resistant hypertension (aRH) are at increased risk for developing cardiovascular disease. It is unknown if this condition is related to increased cardiovascular risk in patients with clinically manifest vascular disease. METHODS: In 6191 hypertensive patients with clinically manifest vascular disease, we evaluated the risk of subsequent vascular events and mortality between patients with controlled hypertension, uncontrolled hypertension, controlled aRH, and uncontrolled aRH. Controlled aRH was defined as office blood pressure less than 140/90 mmHg while using at least four antihypertensive drugs. Uncontrolled aRH was defined as office blood pressure at least 140/90 mmHg while using three antihypertensive drugs including a diuretic, or at least four antihypertensive drugs. Outcomes of interest were myocardial infarction, stroke, cardiovascular mortality, the composite outcome of cardiovascular events, and all-cause mortality. RESULTS: In total 2564 patients (41%) had controlled hypertension, 3063 patients (49%) had uncontrolled hypertension, 123 patients (2%) had controlled aRH, and 411 patients (7%) had uncontrolled aRH. During 7.1 years of follow-up patients with controlled aRH were at a higher risk of cardiovascular mortality [hazard ratios 1.86; 95% confidence interval (CI) 1.10-3.15], and all-cause mortality (hazard ratios 1.64; 95% CI 1.07-2.52) compared with patients with controlled hypertension. Patients with uncontrolled aRH were at a higher risk of cardiovascular mortality (hazard ratios 1.36; 95% CI 1.01-1.83), and higher risk of all-cause mortality (hazard ratios 1.27; 95% CI 1.01-1.60) compared with patients with controlled hypertension. CONCLUSION: In hypertensive patients with clinically manifest vascular disease, presence of controlled and uncontrolled aRH is related to an increased risk of cardiovascular mortality and all-cause mortality.

Relation between Kidney Length and Cardiovascular and Renal Risk in High-Risk Patients.

BACKGROUND AND OBJECTIVES: Kidney length is often measured during routine abdominal ultrasonography and may be of use to identify patients at high vascular and renal risk. We aimed to explore patient characteristics related to kidney length, from which reference values were derived, and evaluate the relationship between kidney length and the risk of cardiovascular events and ESRD in high-risk patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The study population consisted of 10,251 patients with clinical manifest arterial disease or vascular risk factors included in the Second Manifestations of ARTerial disease (SMART) Study cohort between 1996 and 2014. Linear regression was used to explore patient characteristics of kidney length. The relationship between kidney length and cardiovascular events (myocardial infarction, stroke, and cardiovascular mortality), all-cause mortality, and ESRD was analyzed using Cox regression. Kidney length was analyzed in tertiles, using the second tertile as the reference category. RESULTS: Kidney length was strongly correlated with body surface area (2.04 mm; 95% confidence interval [95% CI], 1.95 to 2.13 per 0.1 m2 increase) and eGFR (1.62 mm; 95% CI, 1.52 to 1.73 per 10 ml/min per 1.73 m2 increase). During the median follow-up of 6.3 years, 1317 patients experienced a cardiovascular event, including 711 myocardial infarctions, 369 strokes, and 735 vascular cause deaths. A total of 1462 patients died of any cause and 52 patients developed ESRD. Irrespective of eGFR, patients in the third tertile of kidney length (11.7-16.1 cm) were at higher risk of cardiovascular mortality (hazard ratio, 1.33; 95% CI, 1.05 to 1.67) and cardiovascular events (hazard ratio, 1.28; 95% CI, 1.09 to 1.50). Patients in the first tertile of kidney length (7.8-10.8 cm) were not at higher risk of cardiovascular adverse events. CONCLUSIONS: Large kidney length is related to higher risk of cardiovascular events and mortality in high-risk patients, irrespective of eGFR. Kidney length may serve as a clinical marker to further identify patients at high cardiovascular risk.

Prevalence and clinical characteristics of apparent therapy-resistant hypertension in patients with cardiovascular disease: a cross-sectional cohort study in secondary care.

OBJECTIVES: Our aim was to investigate the prevalence of apparent therapy-resistant hypertension (aTRH) in patients with clinical manifest cardiovascular disease (CVD), and to study clinical characteristics related to aTRH in this population. SETTING: The SMART (Second Manifestations of ARTerial disease) study is a large, single-centre cohort study in secondary care. PARTICIPANTS: Office blood pressure (BP) at inclusion was used to evaluate BP control in 6191 hypertensive patients with clinical manifest (cardio)vascular disease. Therapy-resistant hypertension was defined as BP ≥140/90 mm Hg despite use of antihypertensive drugs from ≥3 drug classes including a diuretic or use of ≥4 antihypertensive drugs irrespective of BP. Logistic regression analysis was used to explore the relationship between clinical characteristics measured at baseline and presence of aTRH. RESULTS: The prevalence of aTRH was 9.1% (95% CI 8.4 to 9.8). Prevalence increased with age and when albuminuria was present and was higher in patients with lower estimated glomerular filtration rate (eGFR). Presence of aTRH was related to diabetes, female sex, duration and multiple locations of vascular disease, body mass index and waist circumference. Carotid intima-media thickness was higher (0.99±0.28 vs 0.93±0.28 mm) and ankle-brachial index lower (1.07±0.20 vs 1.10±0.19) in patients with aTRH compared with patients without aTRH. CONCLUSION: aTRH is prevalent in patients with clinical manifest CVD and is related to clinical factors known to be related with increased vascular risk, and with lower eGFR.

Safety of Temporary Discontinuation of Antihypertensive Medication in Patients With Difficult-to-Control Hypertension.

Successful control of blood pressure relies on identification of secondary causes and contributing factors of hypertension. As antihypertensive medication can interfere with diagnostic investigations, temporary discontinuation of medication is advised. However, there are concerns about the safety of temporary discontinuation of antihypertensive medication in patients with difficult-to-control hypertension. We assessed the occurrence of adverse cardiovascular and cerebrovascular events potentially attributable to temporary discontinuation of antihypertensive medication between February 2010 and March 2016 (n=604) in our Analysis of Complicated Hypertension screening program. A reference group (n=604) was extracted from the SMART study (Second Manifestations of Arterial Disease) cohort (comprising a similar cohort at our hospital in whom medication was not stopped) and individually matched for blood pressure, age, sex, and history of cardiovascular disease. Discontinuation of medication was well tolerated; 62% reported no complaints, 24% had mild discomfort that could be left untreated, and 14% experienced complaints that required prescription of antihypertensive escape medication. Three major adverse events were observed in the Analysis of Complicated Hypertension group between discontinuation of medication and 30 days after restart of medication (event rate=31.2 events per 1000 patient-year). In the reference cohort, 5 cardiovascular events were observed during a similar follow-up period (event rate=51.2 events per 1000 patient-year). In conclusion, discontinuation of antihypertensive medication for the diagnostic evaluation of hypertension does not increase the acute risk of cardiovascular events when performed in a well-controlled setting in specialized hospitals with appropriate protocols for monitoring safety.

Genome-wide methylation profiling of ovarian cancer patient-derived xenografts treated with the demethylating agent decitabine identifies novel epigenetically regulated genes and pathways.

BACKGROUND: In high-grade serous ovarian cancer (HGSOC), intrinsic and/or acquired resistance against platinum-containing chemotherapy is a major obstacle for successful treatment. A low frequency of somatic mutations but frequent epigenetic alterations, including DNA methylation in HGSOC tumors, presents the cancer epigenome as a relevant target for innovative therapy. Patient-derived xenografts (PDXs) supposedly are good preclinical models for identifying novel drug targets. However, the representativeness of global methylation status of HGSOC PDXs compared to their original tumors has not been evaluated so far. Aims of this study were to explore how representative HGSOC PDXs are of their corresponding patient tumor methylome and to evaluate the effect of epigenetic therapy and cisplatin on putative epigenetically regulated genes and their related pathways in PDXs. METHODS: Genome-wide analysis of the DNA methylome of HGSOC patients with their corresponding PDXs, from different generations, was performed using Infinium 450 K methylation arrays. Furthermore, we analyzed global methylome changes after treatment of HGSOC PDXs with the FDA approved demethylating agent decitabine and cisplatin. Findings were validated by bisulfite pyrosequencing with subsequent pathway analysis. Publicly available datasets comprising HGSOC patients were used to analyze the prognostic value of the identified genes. RESULTS: Only 0.6-1.0 % of all analyzed CpGs (388,696 CpGs) changed significantly (p < 0.01) during propagation, showing that HGSOC PDXs were epigenetically stable. Treatment of F3 PDXs with decitabine caused a significant reduction in methylation in 10.6 % of CpG sites in comparison to untreated PDXs (p < 0.01, false discovery rate <10 %). Cisplatin treatment had a marginal effect on the PDX methylome. Pathway analysis of decitabine-treated PDX tumors revealed several putative epigenetically regulated pathways (e.g., the Src family kinase pathway). In particular, the C-terminal Src kinase (CSK) gene was successfully validated for epigenetic regulation in different PDX models and ovarian cancer cell lines. Low CSK methylation and high CSK expression were both significantly associated (p < 0.05) with improved progression-free survival and overall survival in HGSOC patients. CONCLUSIONS: HGSOC PDXs resemble the global epigenome of patients over many generations and can be modulated by epigenetic drugs. Novel epigenetically regulated genes such as CSK and related pathways were identified in HGSOC. Our observations encourage future application of PDXs for cancer epigenome studies.

Biobanking of patient and patient-derived xenograft ovarian tumour tissue: efficient preservation with low and high fetal calf serum based methods.

Using patient-derived xenografts (PDXs) for preclinical cancer research demands proper storage of tumour material to facilitate logistics and to reduce the number of animals needed. We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%. Corresponding cells from mouse replaced human tumour stromal and endothelial cells in second generation PDXs as demonstrated with mouse-specific vimentin and CD31 immunohistochemical staining. For biobanking purposes two cryopreservation methods, a fetal calf serum (FCS)-based (95%v/v) "FCS/DMSO" protocol and a low serum-based (10%v/v) "vitrification" protocol were tested. After primary cryopreservation, tumour take rates were 38% and 67% using either the vitrification or FCS/DMSO-based cryopreservation protocol, respectively. Cryopreserved tumour tissue of established PDXs achieved take rates of 67% and 94%, respectively compared to 91% using fresh PDX tumour tissue. Genotyping analysis showed that no changes in copy number alterations were introduced by any of the biobanking methods. Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues. However, FCS/DMSO-based cryopreservation is more successful. Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.

Total nitrogen vs. amino-acid profile as indicator of protein content of beef.

In most cited food composition studies and tables, the proximate system measures protein as total nitrogen (N) (determined by Kjeldahl or Dumas method) multiplied by a specific factor. A factor of 6.25 is used for determining total protein from total N (Jones, Munsey, & Walker, 1942). Although more expensive, it is considered more accurate to base protein content of foods on amino acid data (Greenfield & Southgate, 2003). A study on the nutrient composition of beef analysed the full amino-acid profile of fifteen retail cuts from three age groups and six fat codes, as well as determined total nitrogen content to determine proximate protein composition. For all cuts, the correlation coefficient of total amino acids to protein (N×6.25) was 0.635. This indicates a poor correlation for predicting actual protein content (as determined by total amino acid count), based on the nitrogen factor of 6.25. On average, the sum of amino acids per cut amounted to 91% of total determined protein (N×6.25) for the same cut.

Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster.

Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. DOI: http://dx.doi.org/10.7554/eLife.01179.001.