Mimicking continuous capture chromatography for virus clearance using a single chromatography column model.

Continuous chromatography is increasingly being used across the biotechnology industry due to its economic advantages. For adoption in commercial manufacturing, also models for virus clearance studies must be available. It is demonstrated how for a virus clearance study for a multispecific antibody, the continuous protein A capture chromatography process, being run on multiple interconnected columns, can be mimicked with only a single column. With this mimicking small-scale model, resources and complexity can be minimized, when conducting virus clearance studies at a contract research organization (CRO) lab. Obtained log reduction values (LRV) for murine leukemia virus (xMuLV) and minute virus of mice (MVM) virus, used as model viruses, are comparable to batch protein A chromatography and results described by other groups. The feasibility of this mimicking small-scale model helps to further reduce barriers to adoption when implementing continuous chromatography.

Polymeric delivery systems for nucleic acid therapeutics: Approaching the clinic.

Gene therapy using nucleic acids has many clinical applications for the treatment of diseases with a genetic origin as well as for the development of innovative vaccine formulations. Since nucleic acids in their free form are rapidly degraded by nucleases present in extracellular matrices, have poor pharmacokinetics and hardly pass cellular membranes, carrier systems are required. Suitable carriers that protect the nucleic acid payload against enzymatic attack, prolong circulation time after systemic administration and assist in cellular binding and internalization are needed to develop nucleic acid based drug products. Viral vectors have been investigated and are also clinically used as delivery vehicles. However, some major drawbacks are associated with their use. Therefore there has been substantial attention on the use of non-viral carrier systems based on cationic lipids and polymers. This review focuses on the properties of polymer-based nucleic acid formulations, also referred as polyplexes. Different polymeric systems are summarized, and the cellular barriers polyplexes encounter and ways to tackle these are discussed. Finally attention is given to the clinical status of non-viral nucleic acid formulations.

Utility of Serum miR-371a-3p in Predicting Relapse on Surveillance in Patients with Clinical Stage I Testicular Germ Cell Cancer.

BACKGROUND: Optimal management of clinical stage I (CSI) testicular cancer is controversial due to lack of robust prognostic factors; miRNA-371a-3p holds promise as a biomarker, although its clinical utility for identifying patients at risk of relapse is unknown. OBJECTIVE: To explore the association between serum miR-371a-3p and CSI surveillance relapse. DESIGN, SETTING, AND PARTICIPANTS: Serial banked sera from 151 CSI (101 seminomas and 50 nonseminomatous germ cell tumors [NSGCTs]) samples from our Princess Margaret active surveillance cohort were tested. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Using the ampTSmiR test, miR-371a-3p was assayed. Multivariate logistic regression was used to assess the association between postorchiectomy miRNA and relapse. RESULTS AND LIMITATIONS: Thirty-four (23%) patients relapsed. There was no association between postorchiectomy miR-371a-3p (2.43 vs 2.74, p = 0.31) or percent decline from before to after orchiectomy (95.8% vs 93.1%, p = 0.14) and relapse. After adjustment for clinical prognostic factors, there remained no association between postorchiectomy miR-371a-3p and relapse (seminoma: odds ratio [OR] 1.33, 95% confidence interval [CI] 0.87-2.02, p = 0.18; NSGCT: OR 0.45, 95% CI 0.21-1.00, p = 0.05). Postorchiectomy miR-371a-3p levels rose as the date of miRNA assessment approached relapse. At relapse, serum markers alpha-fetoprotein and human chorionic gonadotropin were normal in 62%; yet, miR-371a-3p was elevated in 32/34 (94.1%). The magnitude of miR-371a-3p elevation at relapse correlated with disease burden (N1/M0 122.5 vs N2-N3/M0: 521.1; p = 0.05). Limitations include small numbers of relapses and variable time points of serum collection. CONCLUSIONS: In our cohort of CSI testis cancer patients on surveillance, postorchiectomy miR-371a-3p levels were not associated with relapse, suggesting that miR-371a-3p may not be a useful biomarker for guiding adjuvant therapy. Our data suggest that miR-371a-3p holds potential as an early relapse marker and warrants a prospective study, as this may allow a window for less morbid relapse therapy. PATIENT SUMMARY: The promising novel blood biomarker for testis cancer miR-371a-3p may not provide information at testicle removal, but serial monitoring may lead to earlier detection of relapse.

Prediction of relapse in stage I testicular germ cell tumor patients on surveillance: investigation of biomarkers.

BACKGROUND: Better biomarkers for assessing risk of relapse in stage I testicular germ cell tumor patients are needed, to complement classical histopathological variables. We aimed to assess the prognostic value of previously suggested biomarkers, related to proliferation (MIB-1 and TEX19) and to immune microenvironment (CXCL12, CXCR4, beta-catenin and MECA-79) in a surveillance cohort of stage I testicular germ cell tumor patients. METHODS: A total of 70 patients were included. Survival analyses were performed, including Cox regression models. RESULTS: Patients with vascular invasion and elevated human chorionic gonadotropin levels showed significantly poorer relapse-free survival in multivariable analysis (hazard ratio = 2.820, 95% confidence interval 1.257-6.328; hazard ratio = 3.025, 95% confidence interval 1.345-6.808). Patients with no vascular invasion but with MIB-1 staining in > 50% tumor cells showed significantly shorter relapse-free survival (p = 0.042). TEX19 nuclear immunoexpression was confirmed in spermatogonial cells, and weak cytoplasmic immunoexpression was depicted in 15/70 tumors, not significantly impacting survival. CXCL12 immunoexpression in tumor cells did not associate with relapse, but non-seminoma patients exhibiting vascular invasion and CXCL12-positive stromal/inflammatory cells showed significantly improved relapse-free survival (p = 0.015). Exclusively nuclear immunoexpression of CXCR4 associated with better relapse-free survival (p = 0.032), but not after adjusting for vascular invasion. Patients with higher beta-catenin scores showed a tendency for poorer relapse-free survival (p = 0.056). MECA-79 immunoexpression was absent. CONCLUSIONS: The informative protein biomarkers (i.e., MIB-1, CXCL12, beta-catenin, and possibly CXCR4) may prove useful for risk-stratifying patients if validated in larger, multicentric and well-defined studies. Currently, classical histopathological features of testicular germ cell tumors remain key for relapse prediction.

NOTCH inhibition promotes bronchial stem cell renewal and epithelial barrier integrity after irradiation.

Hyperactivity of the NOTCH pathway is associated with tumor growth and radiotherapy resistance in lung cancer, and NOTCH/γ-secretase inhibitors (GSIs) are a potential therapeutic target. The therapeutic outcome, however, is often restricted by the dose-limiting toxicity of combined treatments on the surrounding healthy tissue. The NOTCH signaling pathway is also crucial for homeostasis and repair of the normal airway epithelium. The effects of NOTCH/γ-secretase inhibition on the irradiation of normal lung epithelium are unknown and may counteract antitumor activity. Here we, therefore, investigated whether normal tissue toxicity to radiation is altered upon NOTCH pathway inhibition. We established air-liquid interface pseudostratified and polarized cultures from primary human bronchial epithelial cells and blocked NOTCH signaling alone or after irradiation with small-molecule NOTCH inhibitor/GSI. We found that the reduction in proliferation and viability of bronchial stem cells (TP63+) in response to irradiation is rescued with concomitant NOTCH inhibition. This correlated with reduced activation of the DNA damage response and accelerated repair by 24 hours and 3 days postirradiation. The increase in basal cell proliferation and viability in GSI-treated and irradiated cultures resulted in an improved epithelial barrier function. Comparable results were obtained after in vivo irradiation, where the combination of NOTCH inhibition and irradiation increased the percentage of stem cells and ciliated cells ex vivo. These encourage further use of normal patient tissue for toxicity screening of combination treatments and disclose novel interactions between NOTCH inhibition and radiotherapy and opportunities for tissue repair after radiotherapy.

Identification and Validation Model for Informative Liquid Biopsy-Based microRNA Biomarkers: Insights from Germ Cell Tumor In Vitro, In Vivo and Patient-Derived Data.

Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scenario, contradicting the presumed role of miR-375 as marker of teratoma histology in liquid biopsy setting. Our findings indicate other putative microRNAs (miR-885-5p, miR-448 and miR-197-3p) fulfilling this clinical need. The identification model is informative to identify the best candidate microRNAs to pursue in a clinical setting.

XIST-Promoter Demethylation as Tissue Biomarker for Testicular Germ Cell Tumors and Spermatogenesis Quality.

BACKGROUND: The event of X chromosome inactivation induced by XIST, which is physiologically observed in females, is retained in testicular germ cell tumors (TGCTs), as a result of a supernumerary X chromosome constitution. X chromosome inactivation also occurs in male germline, specifically during spermatogenesis. We aimed to analyze the promoter methylation status of XIST in a series of TGCT tissues, representative cell lines, and testicular parenchyma. METHODS: Two independent cohorts were included, comprising a total of 413 TGCT samples, four (T)GCT cell lines, and 86 testicular parenchyma samples. The relative amount of methylated and demethylated XIST promoter fragments was assessed by quantitative methylation-specific PCR (qMSP) and more sensitive high-resolution melting (HRM) methylation analyses. RESULTS: Seminomas showed a lower amount of methylated XIST fragments as compared to non-seminomas or normal testis (p < 0.0001), allowing for a good discrimination among these groups (area under the curve 0.83 and 0.81, respectively). Seminomas showed a significantly higher content of demethylated XIST as compared to non-seminomas. The percentage of demethylated XIST fragment in cell lines reflected their chromosomal constitution (number of extra X chromosomes). A novel and strong positive correlation between the Johnsen's score and XIST demethylation was identified (r = 0.75, p < 0.0001). CONCLUSIONS: The X chromosome inactivation event and demethylated XIST promoter are promising biomarkers for TGCTs and for assessing spermatogenesis quality.

Parameters of glucose metabolism and the aging brain: a magnetization transfer imaging study of brain macro- and micro-structure in older adults without diabetes.

Given the concurrent, escalating epidemic of diabetes mellitus and neurodegenerative diseases, two age-related disorders, we aimed to understand the relation between parameters of glucose metabolism and indices of pathology in the aging brain. From the Leiden Longevity Study, 132 participants (mean age 66 years) underwent a 2-h oral glucose tolerance test to assess glucose tolerance (fasted and area under the curve (AUC) glucose), insulin sensitivity (fasted and AUC insulin and homeostatic model assessment of insulin sensitivity (HOMA-IS)) and insulin secretion (insulinogenic index). 3-T brain MRI was used to detect macro-structural damage (atrophy, white matter hyper-intensities, infarcts and/or micro-bleeds) and magnetization transfer imaging (MTI) to detect loss of micro-structural homogeneity that remains otherwise invisible on conventional MRI. Macro-structurally, higher fasted glucose was significantly associated with white matter atrophy (P = 0.028). Micro-structurally, decreased magnetization transfer ratio (MTR) peak height in gray matter was associated with higher fasted insulin (P = 0.010), AUCinsulin (P = 0.001), insulinogenic index (P = 0.008) and lower HOMA-IS index (P < 0.001). Similar significant associations were found for white matter. Thus, while higher glucose was associated with macro-structural damage, impaired insulin action was associated more strongly with reduced micro-structural brain parenchymal homogeneity. These findings offer some insight into the association between different parameters of glucose metabolism (impairment of which is characteristic of diabetes mellitus) and brain aging.

Associations between insulin action and integrity of brain microstructure differ with familial longevity and with age.

Impaired glucose metabolism and type 2 diabetes have been associated with cognitive decline, dementia, and with structural and functional brain features. However, it is unclear whether these associations differ in individuals that differ in familial longevity or age. Here, we investigated the association between parameters of glucose metabolism and microstructural brain integrity in offspring of long-lived families ("offspring") and controls; and age categories thereof. From the Leiden Longevity Study (LLS), 132 participants underwent an oral glucose tolerance test (OGTT) to assess glycemia [fasted glucose and glucose area-under-the-curve (AUC)], insulin resistance [fasted insulin, AUCinsulin, and homeostatic model assessment of insulin resistance (HOMA-IR)], and pancreatic Beta cell secretory capacity (insulinogenic index). 3 Tesla MRI and Magnetization Transfer (MT) imaging MT-ratio (MTR) peak-height was used to quantify differences in microstructural brain parenchymal tissue homogeneity that remain invisible on conventional MRI. Analyses were performed in offspring and age-matched controls, with and without stratification for age. In the full offspring group only, reduced MTR peak-height in gray and white matter was inversely associated with AUCinsulin, fasted insulin, HOMA-IR and insulinogenic-index (all p < 0.01). When dichotomized for age (≤65 years and >65 years): in younger controls, significantly stronger inverse associations were observed between MTR peak-height and fasted glucose, AUCglucose, fasted insulin, AUCinsulin and HOMA-IR in gray matter; and for AUCglucose, fasted insulin and HOMA-IR in white matter (all P-interaction < 0.05). Although the strength of the associations tended to attenuate with age in the offspring group, the difference between age groups was not statistically significant. Thus, associations between impaired insulin action and reduced microstructural brain parenchymal tissue homogeneity were stronger in offspring compared to controls, and seemed to diminish with age.

Microstructural brain tissue damage in metabolic syndrome.

OBJECTIVE: We investigated the association between metabolic syndrome risk factors and brain tissue integrity, as assessed by magnetic resonance imaging. RESEARCH DESIGN AND METHODS: From the Leiden Longevity Study, which is a community-based study of long-lived subjects, their offspring, and partners thereof, 130 subjects (61 men; mean age 66 years) were included. A metabolic syndrome score was computed by summing the individual number of components according to the Adult Treatment Panel III criteria. We performed linear and logistic regression analysis and used standardized ß-values to assess the association between metabolic syndrome and brain macrostructure (brain volume and white matter lesion load, lacunar infarcts, and cerebral microbleeds) and microstructure (mean magnetization transfer ratio [MTR], MTR histogram peak height, fractional anisotropy, and mean diffusivity [MD]). Linear and stepwise regression analysis was performed to identify the individual contribution of one metabolic syndrome parameter adjusting for the four other parameters. Models were adjusted for age, sex, and relation to long-lived family. RESULTS: Brain macrostructure was not associated with metabolic syndrome. In contrast, metabolic syndrome was associated with decreased gray (ß = -0.3 P = 0.001) and white matter peak height (ß = -0.3, P = 0.002) and increased gray matter MD (ß = 0.2, P = 0.01, P = 0.01). Serum HDL cholesterol (ß = 0.22, P = 0.012), triglycerides (ß =-0.25, P = 0.002), BMI (ß =-0.2, P = 0.014), and diastolic blood pressure (ß = -0.17, P = 0.047, and ß = -0.23, P = 0.009, for gray and white matter, respectively) were independent factors in these changes in brain microstructure. CONCLUSIONS: In early manifest metabolic syndrome, brain tissue decline can be detected. Serum HDL cholesterol, triglycerides, BMI, and diastolic blood pressure were independent factors in brain tissue integrity.

Development and characterization of an automated high throughput screening method for optimization of protein refolding processes.

Optimization of protein refolding parameters by automated, miniaturized, and parallelized high throughput screening is a powerful approach to meet the demand for fast process development with low material consumption. In this study, we validated methods applicable on a standard liquid handling robot for screening of refolding process parameters by dilution of denatured lysozyme in refolding buffer systems. Different approaches for the estimation of protein solubility and folding were validated concerning resolution and compatibility with the robotic system and with the complex buffer and protein structure composition. We established an indirect method to assess soluble lysozyme concentration independent of matrix effects and protein structure varieties by automated separation of aggregated protein, resolubilization, and measurement of absorption at 280 nm. Using this nonspecific solubility assays the correlation between favorable parameters for high active and soluble lysozyme yields were evaluated. An overlap of good refolding buffer compositions was found provided that the redox environment was controlled with redox reagents. In addition, the need to control unfolding conditions like time, temperature, lysozyme, and dithiothreitol concentration was pointed out as different feedstocks resulted in different refolding yields.

Examination of a genetic algorithm for the application in high-throughput downstream process development.

Compared to traditional strategies, application of high-throughput experiments combined with optimization methods can potentially speed up downstream process development and increase our understanding of processes. In contrast to the method of Design of Experiments in combination with response surface analysis (RSA), optimization approaches like genetic algorithms (GAs) can be applied to identify optimal parameter settings in multidimensional optimizations tasks. In this article the performance of a GA was investigated applying parameters applicable in high-throughput downstream process development. The influence of population size, the design of the initial generation and selection pressure on the optimization results was studied. To mimic typical experimental data, four mathematical functions were used for an in silico evaluation. The influence of GA parameters was minor on landscapes with only one optimum. On landscapes with several optima, parameters had a significant impact on GA performance and success in finding the global optimum. Premature convergence increased as the number of parameters and noise increased. RSA was shown to be comparable or superior for simple systems and low to moderate noise. For complex systems or high noise levels, RSA failed, while GA optimization represented a robust tool for process optimization. Finally, the effect of different objective functions is shown exemplarily for a refolding optimization of lysozyme.

Effectiveness of percutaneous laser disc decompression versus conventional open discectomy in the treatment of lumbar disc herniation; design of a prospective randomized controlled trial.

BACKGROUND: The usual surgical treatment of refractory sciatica caused by lumbar disc herniation, is open discectomy. Minimally invasive procedures, including percutaneous therapies under local anesthesia, are increasingly gaining attention. One of these treatments is Percutaneous Laser Disc Decompression (PLDD). This treatment can be carried out in an outpatient setting and swift recovery and return to daily routine are suggested. Thus far, no randomized trial into cost-effectiveness of PLDD versus standard surgical procedure has been performed. We present the design of a randomized controlled trial, studying the cost-effectiveness of PLDD versus conventional open discectomy in patients with sciatica from lumbar disc herniation. METHODS/DESIGN: The study is a randomized prospective multi-center trial, in which two treatment strategies are compared in a parallel group design. Patients (age 18-70 years) visiting the neurosurgery department of the participating hospitals, are considered for inclusion in the trial when sciatica due to lumbar disc herniation has lasted more than 8 weeks. Patients with disc herniation smaller than 1/3 of the spinal canal diameter, without concomitant lateral recess stenosis or sequestration, are eligible for participation, and are randomized into one of two treatment arms; either Percutaneous Laser Disc Decompression or conventional discectomy. The functional outcome of the patient, as assessed by the Roland Disability Questionnaire for Sciatica at 8 weeks and 1 year after treatment, is the primary outcome measure. The secondary outcome parameters are recovery as perceived by the patient, leg and back pain, incidence of re-intervention, complications, quality of life, medical consumption, absence of work and secondary costs. DISCUSSION: Open discectomy is still considered to be the golden standard in the surgical treatment of lumbar disc herniation. Whether Percutaneous Laser Disc Decompression has at least as much efficacy as the standard surgical procedure, and is more cost-effective, will be determined by this trial.

Association of the Tim14.Tim16 subcomplex with the TIM23 translocase is crucial for function of the mitochondrial protein import motor.

Tim14 and Tim16 are essential components of the import motor of the mitochondrial TIM23 preprotein translocase. Tim14 contains a J domain in the matrix space that is anchored in the inner membrane by a transmembrane segment. Tim16 is a J-related protein with a moderately hydrophobic segment at its N terminus. The J and J-like domains function in the regulation of the ATPase activity of the Hsp70 chaperone of the import motor. We report here on the role of the hydrophobic segments of Tim16 and Tim14 in the TIM23 translocase. Yeast cells lacking the hydrophobic N-terminal segment in either Tim16 or Tim14 are viable but show growth defects and decreased import rates of matrix-targeted preproteins into mitochondria. The interaction of the Tim14.Tim16 complex with the core complex of the TIM23 translocase is destabilized in these cells. In particular, the N-terminal domain of Tim16 is crucial for the interaction of the Tim14.Tim16 complex with the TIM23 preprotein translocase. Deletion of hydrophobic segments in both, Tim16 and Tim14, is lethal. We conclude that import into the matrix space of mitochondria requires association of the co-chaperones Tim16 and Tim14 with the TIM23 preprotein translocase.

The import motor of the yeast mitochondrial TIM23 preprotein translocase contains two different J proteins, Tim14 and Mdj2.

The import motor of the mitochondrial (mt)TIM23 complex drives translocation of presequence-containing preproteins across the mitochondrial inner membrane in an ATP-dependent manner. Tim44 is the central component of the motor. It recruits mtHsp70, which binds the incoming preproteins. The J protein Tim14 stimulates the ATPase activity of mtHsp70 and thereby enables efficient binding of mtHsp70 to preproteins. Tim16 is a J-like protein that forms a stable subcomplex with Tim14 and recruits it to the translocase. All subunits of the TIM23 translocase but one are essential for yeast cell viability. Yeast cells contain a close homologue of Tim14, Mdj2. In contrast to Tim14, its deletion leads to no obvious growth defect. In the present study we analyzed Mdj2 and compared it with Tim14. Mdj2 forms a complex with Tim16 and is recruited to the TIM23 translocase. It stimulates the ATPase activity of mtHsp70 to the same extent that Tim14 does. Mdj2 is expressed at a lower level compared with Tim14, and its complex with Tim16 is less stable. However, overexpressed Mdj2 fully restores the growth of cells lacking Tim14. We conclude that Mdj2 is a functional J protein and a component of the mitochondrial import motor.