Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and F?rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.     

Reverse transfection on cell arrays for high content screening microscopy

Here, we describe a robust protocol for the reverse transfection of cells on small interfering (siRNA) arrays, which, in combination with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in intact human cells. The automatic production of 48 'transfection ready' siRNA arrays, each containing 384 samples, takes in total 7 h. Pre-fabricated siRNA arrays can be used without loss of transfection efficiency at least up to 15 months after printing. Different human cell lines that have been successfully transfected using the protocol are presented here. The present protocol has been applied to two genome-wide siRNA screens addressing mitosis and constitutive protein secretion.     

Robust dose-response curve estimation applied to high content screening data analysis

Background and method

Successfully automated sigmoidal curve fitting is highly challenging when applied to large data sets. In this paper, we describe a robust algorithm for fitting sigmoid dose-response curves by estimating four parameters (floor, window, shift, and slope), together with the detection of outliers. We propose two improvements over current methods for curve fitting. The first one is the detection of outliers which is performed during the initialization step with correspondent adjustments of the derivative and error estimation functions. The second aspect is the enhancement of the weighting quality of data points using mean calculation in Tukey’s biweight function.

Results and conclusion

Automatic curve fitting of 19,236 dose-response experiments shows that our proposed method outperforms the current fitting methods provided by MATLAB®;’s nlinfit nlinfit function and GraphPad’s Prism software.

     

Prostate cancer: epidemiology, screening, and biomarkers

Carcinoma of the prostate continues to be a major health problem in the United States. Beginning in 1988, a marked increase in detection of prostate cancer occurred due to the development of a test for prostate-specific antigen (PSA). Controversy exists, however, about the value of PSA as a tumor marker. Although it has prognostic significance both before and after definitive therapy for prostate cancer, it is unclear whether routine PSA screening will translate into a survival advantage for patients. Because of its limitations, PSA may not ultimately be a good enough marker to be used as a screening tool. However, molecular biology has led to a rapid rise in the number of potential new prostate tumor markers, which may eventually overcome the weaknesses of PSA. Considerable progress has occurred in the diagnosis and management of prostate cancer: more is understood about the risk factors for the disease, possible ways to prevent it, and new ways to diagnose and monitor it. These developments have already translated into better patient care, while also identifying where further improvements are needed.     

Post-high-throughput screening analysis: an empirical compound prioritization scheme

An empirical scheme to evaluate and prioritize screening hits from high-throughput screening (HTS) is proposed. Negative scores are given when chemotypes found in the HTS hits are present in annotated databases such as MDDR and WOMBAT or for testing positive in toxicity-related experiments reported in TOXNET. Positive scores were given for higher measured biological activities, for testing negative in toxicity-related literature, and for good overlap when profiled against drug-related properties. Particular emphasis is placed on estimating aqueous solubility to prioritize in vivo experiments. This empirical scheme is given as an illustration to assist the decision-making process in selecting chemotypes and individual compounds for further experimentation, when confronted with multiple hits from high-throughput experiments. The decision-making process is discussed for a set of G-protein coupled receptor antagonists and validated on a literature example for dihydrofolate reductase inhibition.     

Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis

The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase^® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10–15% and an increased glucose release by increasing the enzyme content to 15 U L^?1. The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD₆₀₀) of approximately 40 AU (corresponding to over 60 g L^?1 wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches.     

Lymphocyte mitochondria: toward identification of peripheral biomarkers in the progression of Alzheimer disease

Alzheimer disease (AD) is an age-related neurodegenerative condition. AD is histopathologically characterized by the presence of three main hallmarks: senile plaques (rich in amyloid-β peptide), neuronal fibrillary tangles (rich in phosphorylated tau protein), and synapse loss. However, definitive biomarkers for this devastating disease in living people are still lacking. In this study, we show that levels of oxidative stress markers are significantly increased in the mitochondria isolated from lymphocytes of subjects with mild cognitive impairment (MCI) compared to cognitively normal individuals. Further, an increase in mitochondrial oxidative stress in MCI is associated with MMSE score, vitamin E components, and β-carotene. Further, a proteomics approach showed that alterations in the levels of thioredoxin-dependent peroxide reductase, myosin light polypeptide 6, and ATP synthase subunit β might be important in the progression and pathogenesis of AD. Increased understanding of oxidative stress and protein alterations in easily obtainable peripheral tissues will be helpful in developing biomarkers to combat this devastating disorder.     

Discovering novel chemical inhibitors of human cyclophilin A: virtual screening, synthesis, and bioassay

Cyclophilin A (CypA) is a member of cyclophilins, a family of the highly homologous peptidyl prolyl cis-trans isomerases (PPIases), which can bind to cyclosporin A (CsA). CypA plays critical roles in various biological processes, including protein folding, assembly, transportation, regulation of neuron growth, and HIV replication. The discovery of CypA inhibitor is now of a great special interest in the treatment of immunological disorders. In this study, a series of novel small molecular CypA inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay. The SPECS_1 database containing 85,000 small molecular compounds was searched by virtual screening against the crystal structure of human CypA. After SPR-based binding affinity assay, 15 compounds were found to show binding affinities to CypA at submicro-molar or micro-molar level (compounds 1-15). Seven compounds were selected as the starting point for the further structure modification in considering binding activity, synthesis difficulty, and structure similarity. We thus synthesized 40 new small molecular compounds (1-6, 15, 16a-q, 17a-d, and 18a-l), and four of which (compounds 16b, 16h, 16k, and 18g) showed high CypA PPIase inhibition activities with IC50s of 2.5-6.2 microM. Pharmacological assay indicated that these four compounds demonstrated somewhat inhibition activities against the proliferation of spleen cells.     

Rhesus macaques (Macaca mulatta) are not neophobic toward novel food with a high sugar content

While we know that food neophobia in nonhuman primates is affected by social factors, little is known about how palatability, and specifically sugar content, might ameliorate a monkey's hesitancy to eat a novel food. It is likely that an innate preference for sweet substances would alter an animal's typical neophobic response. To test this, I presented nine male rhesus macaques with novel foods that contained a low, high, or no amount of sugar. As expected, the monkeys exhibited a neophobic response to the no-sugar novel foods but not to the high-sugar novel foods. Previous research on food neophobia may need to be reevaluated in light of the effects of sugar content on the neophobic response.     

Development toward rapid and efficient screening for high performance hydrolysate lots in a recombinant monoclonal antibody manufacturing process

Plant‐derived hydrolysates are widely used in mammalian cell culture media to increase yields of recombinant proteins and monoclonal antibodies (mAbs). However, these chemically varied and undefined raw materials can have negative impact on yield and/or product quality in large‐scale cell culture processes. Traditional methods that rely on fractionation of hydrolysates yielded little success in improving hydrolysate quality. We took a holistic approach to develop an efficient and reliable method to screen intact soy hydrolysate lots for commercial recombinant mAb manufacturing. Combined high‐resolution ¹H nuclear magnetic resonance (NMR) spectroscopy and partial least squares (PLS) analysis led to a prediction model between product titer and NMR fingerprinting of soy hydrolysate with cross‐validated correlation coefficient R² of 0.87 and root‐mean‐squared‐error of cross‐validation RMSECV% of 11.2%. This approach screens for high performance hydrolysate lots, therefore ensuring process consistency and product quality in the mAb manufacturing process. Furthermore, PLS analysis was successful in discerning multiple markers (DL‐lactate, soy saccharides, citrate and succinate) among hydrolysate components that positively and negatively correlate with titer. Interestingly, these markers correlate to the metabolic characteristics of some strains of taxonomically diverse lactic acid bacteria (LAB). Thus our findings indicate that LAB strains may exist during hydrolysate manufacturing steps and their biochemical activities may attribute to the titer enhancement effect of soy hydrolysates. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1061–1068, 2012     

离体诱变定向培育高油花生新品种宇花9号

笔者前期研究发现经干旱胁迫处理后的花生叶片水势与其籽仁含油率呈显著正相关。文中对羟脯氨酸作为水势(渗透压)调节物质用于离体定向筛选花生高油突变体及培育花生新品种进行了研究。以花生品种花育20号胚小叶作为外植体,平阳霉素作为诱变剂添加于体胚诱导培养基上进行离体诱变培养。形成的体胚转移到添加6 mmol/L羟脯氨酸(培养基水势为-2.079 MPa)的体胚萌发和再生培养基上诱导体胚萌发成苗,同时进行高油突变体定向筛选。再生小苗经嫁接移栽田间,从再生植株后代中获得了132份含油率55%以上的高油突变体,其中27份含油率超过58%,2份超过60%。再生植株后代结合系谱育种法育成了高产高油花生新品种宇花9号,在辽宁省花生新品种备案试验中,籽仁产量比对照品种增产14.0%,并通过了国家非主要农作物品种登记。宇花9号含油率达61.05%,比亲本花育20号高11.55个百分点,是目前国际上含油率最高的花生品种。本研究结果表明,利用离体诱变、培养基低水势定向筛选及其再生植株后代结合常规育种法选择是定向培育高油花生品种的有效方法。     

High throughput ratio imaging to profile caspase activity: potential application in multiparameter high content apoptosis analysis and drug screening

Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non-classical apoptosis inducing agents, the greatest advantage being that the high content information obtained are from individual cells rather than being population based.     

Compound transfer efficiency from polystyrene surfaces: application to microarrayed compound screening

In microarrayed compound screening (microARCS), compounds are spotted and dried onto a polystyrene sheet (ChemCard)ata high density and introduced into the assay by contacting with agarose gels that contain reagents for the assay. The authors have conducted studies to characterize the compound transfer process using 59 compounds of diverse properties. The amount of compounds remaining on the ChemCard was determined by liquid chromatography/mass spectrometry after incubation with agarose gels for predetermined time periods. The results showed good correlation with kinetics of compound transfer to phosphate-buffered saline (PBS) buffer, but only moderate correlation with equilibrium solubility of compounds in PBS buffer. These observations indicate that the major factor determining compound transfer efficiency is the kinetics of dissolution of compounds, rather than equilibrium solubility and diffusion of compounds in the gel. Compounds of lower ClogP showed a higher rate of transfer to agarose gels and vice versa. Other compound properties such as molecular weight, size, acid-base, and H-bonding properties did not significantly affect compound transfer. Importantly, the majority of the compounds studied show greater than 20% transfer after a 10-min incubation with agarose gels, providing sufficient amounts of compounds for screening purposes.     

Dementia screening, biomarkers and protein misfolding: Implications for public health and diagnosis

Misfolded proteins are at the core of many neurodegenerative diseases, nearly all of them associated with cognitive impairment. For example, Creutzfeldt-Jacob disease is associated with aggregation of prion protein,^1,2 Lewy body dementia and Parkinson disease with α-synuclein^3,4 and forms of frontotemporal dementia with tau, TDP43 and a host of other proteins.^5,6 Alzheimer disease (AD), the most common cause of dementia,⁷ and its prodromal syndrome mild cognitive impairment (MCI)⁸ are an increasing public health problem and a diagnostic challenge to many clinicians. AD is characterized pathologically by the accumulation of amyloid β-protein (Aβ)^9,10 as senile plaques and in the walls of blood vessels as amyloid angiopathy.^11,12 Additionally, there are accumulations of tau-protein as neurofibrillary tangles and dystrophic neurites.^11,12 Biological markers of AD and MCI can serve as in vivo diagnostic indicators of underlying pathology, particularly when clinical symptoms are mild^13–15 and are likely present years before the onset of clinical symptoms.^16–19 Research to discover and refine fluid and imaging biomarkers of protein aggregation has undergone a rapid evolution^20–22 and combined analysis of different modalities may further increase diagnostic sensitivity and specificity.^23–26 Multi-center trials are now investigating whether imaging and/or cerebrospinal fluid (CSF) biomarker candidates can be used as outcome measures for use in phase III clinical trials for AD.^27–29Key words: dementia, screening, biomarkers, amyloid, tau, Alzheimer disease, preclinical, presymptomaticCurrently, the diagnosis of AD is based on exclusion of other forms of impairment with definitive diagnosis requiring autopsy confirmation.³⁰ Thus, there is a strong need to find easily measurable in vivo AD biomarkers that could facilitate early and accurate diagnosis³¹ as well as prognostic data to assist in monitoring therapeutic efficacy.³² Although biological markers such as MRI, PET scans and CSF increase the diagnostic likelihood that AD is present,^{9,18–20,33,34} biomarkers are invasive, uncomfortable, expensive and may not be readily available to rural areas, underserved communities, underinsured individuals or developing countries, making them impractical for broad use. However, the lessons learned from biomarkers can be applied to increase the likelihood that clinicians will be able to detect disease at earlier stages in the form of dementia screening.Public health may be best defined as the organized efforts of society to improve health, often framed in terms of primary, secondary and tertiary prevention. Prevention encompasses an understanding of causation, alteration of natural history of disease and understanding of pathophysiological mechanisms.³⁵ The clearest application of this from a public health perspective is in the setting of secondary prevention (i.e., screening)—early detection as a core element, coupled with treatments or preventative actions to reduce the burden of disease.³⁵ In this instance we seek to identify individuals in whom a disease has already begun and who may be experiencing very mild clinical symptoms but have not yet sought out medical care. The objective of effective screening is to detect the disease earlier than it would have been detected with usual care. Recent healthcare reform (Accountable Care Act)³⁶ proposes a Personalized Prevention Plan including screening for cognitive disorders, reimbursable through Medicare. Thus tying knowledge about dementia screening with underlying biology of protein misfolding associated with neurodegenerative disease can have enormous implications.A review of the natural history of dementia illustrates this point (Fig. 1). The timeline of disease from presumptive start to the patient demise is plotted. Stage I marks the biologic onset of disease; however this point often cannot be identified and may begin years to decades before any evidence is apparent (represented by dashed lines). As this stage is subclinical, it is difficult to study in humans but lends itself nicely to animal models. At some point in the progression of the biology, stage II begins heralding the first pathologic evidence of disease could be obtained—in the case of AD this could include CSF measurements of amyloid and tau^22,26,27 or PET imaging with amyloid ligands.^18,37 Subsequently, the first signs and symptoms of disease develop (stage III). Till this point, the disease process has been entirely presymptomatic. Beginning with the onset of symptoms, the patient may seek medical care (stage IV) and eventually be diagnosed (stage V). From stage III onwards, the patient enters the symptomatic phase of disease. From this point, the patient is typically treated with various pharmacologic and nonpharmacologic approaches towards some outcome. Another way to envision the disease spectrum is from the biological onset to the seeking of medical attention as the preclinical phase of disease with the clinical phase beginning with the initial clinical investigations into the cause of the patients'' symptoms.Open in a separate windowFigure 1Model of the natural history of AD. Timeline from presumptive start of AD through patient diagnosis is plotted. The initiation of biological changes (stage I) marks the onset of disease and begins years to decades before any evidence is apparent (represented by dashed lines). At some point the first pathologic evidence of disease (stage II) begins and in theory can be detected with biomarkers such as CSF measurements of amyloid and tau or PET imaging with amyloid ligands. Subsequently, the first signs and symptoms of disease develop (stage III) followed by the patient seeking medical attention (stage IV) and finally a diagnosis is established (stage V). This timeline can be clustered into a presymptomatic phase (stages I–III) and a symptomatic phase (stages III–V). An alternative way to envision the disease spectrum is from the biological onset to the seeking of medical attention (stages I–IV) as the preclinical phase of disease with the clinical phase beginning with the initial clinical investigations into the cause of the patients'' symptoms (stages IV and V). Stage III is the ideal time for dementia screening.What is the value of thinking about disease in this fashion? Such models allow researchers and clinicians to model the approach to finding and applying new diagnostics and offering new interventions. From stage I to stage III, the patient is the presymptomatic, preclinical phase of disease. The only means of detection would be with a biological marker that reflected protein misfolding or some proxy marker of these events. Although longitudinal evidence of cognitive change exist from 1–3 years before clinical diagnosis, raw scores on neuropsychological testing during this time remains in the normal range.³⁸ After stage IV, the patient is in the symptomatic, clinical phase of disease. Testing here is centered on confirming the suspected diagnosis, correctly staging the disease and initiating the appropriate therapies. Basic scientific approaches focusing on the presymptomatic, preclinical phase and clinical care approaches focusing on the symptomatic, clinical phase are well established and will continue to benefit from additional research.However, if we focus only on these two phases, an opportunity will be missed to make a decidedly important impact in the patient''s well-being. From stage III to stage IV, the patient enters symptomatic, preclinical phase of disease; symptomatic because the patient or family is beginning to detect some aspect of change, but preclinical because these signs and symptoms have not yet been brought to medical attention. In the case of AD (and the other forms of dementia) this period may go for an extended length of time as patients, families and clinicians dismiss early cognitive symptoms as part of the normal aging process. Thus, the rationale for screening is that if we can identify disease earlier in its natural history than would ordinarily occur, intervention measures (those currently available and those that are being developed) would be more effective. Dementia screening therefore would be best suited to detect cognitive impairment at the beginning of disease signs (stage III), particularly if these screening measures reflect what is known about the symptomatic, clinical phase of disease and correlate with the pathologic changes occurring in the brain during the pre-symptomatic, preclinical phase of disease.In a recent paper, we evaluated the relationship between several dementia screening tests and biomarkers of AD.⁴⁰ We tested whether a reliable and validated informant-based dementia screening test (the AD8)^41,42 correlates with changes in AD biomarkers and, if positive, screening with the AD8 clinically supports an AD clinical phenotype, superior to a commonly used performance-based screening tests including the Mini Mental State Exam (MMSE)⁴³ and the Short Blessed Test (SBT).⁴⁴ A total of 257 participants were evaluated, administered a comprehensive clinical and cognitive evaluation with the Clinical Dementia Rating scale (CDR)⁴⁵ used as the gold standard. Participants consented to and completed a variety of biomarker studies including MRI, amyloid imaging using the Pittsburgh Compound B (PiB)^37,46 and CSF studies of Aβ₄₂, tau and phosphorylated tau at Serine 181 (p-tau₁₈₁).^23,24 The sample had a mean age of 75.4 ± 7.3 years with 15.1 ± 3.2 years of education. The sample was 88.7% Caucasian and 45.5% male with a mean MMSE score of 27.2 ± 3.6. The formal diagnoses of the sample was 156 CDR 0 cognitively normal, 23 CDR 0.5 MCI, 53 CDR 0.5 very mild AD and 25 CDR 1 mild AD. Participants with positive AD8 scores (graded as a score of 2 or greater) exhibited the typical AD fluid biomarker phenotype characterized by significantly lower mean levels of CSF Aβ₄₂, greater CSF tau, p-tau₁₈₁ and the tau(s)/Aβ₄₂ ratios.^26,27 They also exhibited smaller temporal lobe volumes and increased mean cortical binding potential (MCBP) for PiB imaging similar to studies of individuals with AD.^18,19 These findings support that informant-based assessments may be superior to performance-based screening measures such as the MMSE or SBT in corresponding to underlying AD pathology, particularly at the earliest stages of decline. The use of a brief test such as the AD8 may improve strategies for detecting dementia in community settings where biomarkers may not be readily available and also may enrich clinical trial recruitment by increasing the likelihood that participants have underlying biomarker abnormalities.⁴⁰To gain a better understanding of changes in biomarkers in the symptomatic, preclinical phase, a post hoc evaluation of the 156 individuals who were rated as CDR 0 no dementia at the time of their Gold Standard assessment was completed. Some of these nondemented individuals have abnormal AD biomarkers, but in the absence of performing lumbar punctures or PET scans, is it possible to detect evidence of change? AD8 scores for 132 individuals were less than 2; thus their screening test suggests no impairment (mean AD8 score = 0.30 ± 0.46). However 25 of these individuals had AD8 scores (≥2) suggesting impairment (mean AD8 score = 2.4 ± 0.91). Applying the model described in Figure 1, some of these individuals are hypothesized to be in the symptomatic, preclinical phase of disease. No difference in age, education, gender or brief performance tests (MMSE or SBT) were detected between groups (45 is increased in the individuals with higher AD8 scores supporting that informants were noticing and reporting changes in the participants cognitive function. A review of the individual AD8 questions that were first reported to change suggest that informants endorsement of subtle changes in memory (repeats questions, forgets appointments) and executive ability (trouble with judgment, appliances, finances) are valuable early signs. This is consistent with previous reports that changes in memory and judgment/problem solving CDR boxscores in nondemented individuals correlate with findings of AD pathology at autopsy.¹⁷ Although biomarkers do not reach significance in this small sample, the direction of change in favor of “Alzheimerization” of this group suggests that some of these individuals may be in the symptomatic, preclinical phase of disease. More research with larger sample sizes and longitudinal follow-up is needed to confirm this hypothesis. It should be also noted that not all individuals with an AD8 score of 2 or greater have AD. The AD8 was designed to detect cognitive impairment from all causes, and as such, these mildly affected individuals may have other causes for their cognitive change such as depression, Lewy body dementia or vascular cognitive impairment.^41,42

Table 1

Characteristics of nondemented CDR 0 individuals stratified by AD8 scores

Object-oriented image analysis for high content screening: detailed quantification of cells and sub cellular structures with the Cellenger software.

BACKGROUND: Detailed image analysis still is a considerable bottleneck for many cellular assays, and automated solutions to the problem are desirable. However, dealing with the complexity and variability of structures in cellular images makes detailed and reliable analysis a nontrivial task. METHODS: Therefore, based on the object-oriented image analysis approach, a novel image analysis technology, a flexible and reliable system for image analysis in cellular assays was developed. It contains a library of predefined, adaptable modules, each of them developed for a specific analysis task. The system can be configured easily by combining appropriate modules and adapting them interactively to the specific image data, if necessary. By representing cells and sub cellular structures within a network of interlinked image objects, a large number of parameters can be derived that describe shape, intensity, and relevant structural and relational aspects of any chosen class of structures. RESULTS: Thus, multi-parameter analysis and multiplexing are supported. A sample application based on this approach demonstrates that GFP signals can be distinguished based on their properties and the relative location within the cell.     

Using extracellular biomarkers for monitoring efficacy of therapeutics in cancer patients: an update

Rapidly detectable and easily accessible markers of tumor cell death are needed for evaluating early therapeutic efficacy for immunotherapy and chemotherapy so that patients and their physicians can decide whether to remain with a given therapeutic strategy. Currently, image-based tests such as computed tomography scans and magnetic resonance imaging are used to visualize the response of a patient’s tumor, but often these evaluations are not conducted for weeks to months after treatment begins. While serum levels of secreted proteins such as carcinoembryonic antigen and prostate specific antigen are commonly monitored to gauge tumor status during therapy and between image evaluations, the levels of these proteins do not always correlate well with the actual tumor response. In laboratory studies, it has been shown that tumor cells undergoing apoptosis can release cellular components into cell culture media such as cytochrome c, nucleosomes, cleaved cytokeratin-18 and E-cadherin. Studies of patient sera have found that these and other macromolecules can be found in circulation during cancer therapy, providing a potential source of material for monitoring treatment efficacy. In the future, analysis of biofluids from severe combined immunodeficiency mice bearing patient tumor specimens treated with a targeted therapy such as Apo2L/tumor necrosis factor-related apoptosis-inducing ligand will be useful in the preclinical identification of therapy response markers. In this review, the current status of the identification of serum markers of tumor cell apoptosis is provided, as well as a discussion of critical research questions that must be addressed and the considerations necessary when identifying a marker that reflects true clinical outcome.     

Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening

ContextSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SARS-CoV-2.MethodsVero E6 cells were cultured with or without copper gluconate 18−24 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were incubated in fresh medium containing varying concentration of copper gluconate (supplemented with bovine serum albumin or not) for an additional 48 -h period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT and propidium iodide assays.ResultsThe viability of Vero E6 cells exposed to copper gluconate up to 200 μM was found to be similar to that of unexposed cells, but it dropped below 70 % with 400 μM of this agent after 72 h of continuous exposure. The infection rate was 23.8 %, 18.9 %, 20.6 %, 6.9 %, 5.3 % and 5.2 % in cells treated prior infection with 0, 2, 10, 25, 50 and 100 μM of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71 %, 77 %, and 78 % with 25, 50, and 100 μM of copper gluconate respectively (p < 0.05). In cells treated only post-infection, the rate of infection dropped by 73 % with 100 μM of copper gluconate (p < 0.05). However, the antiviral activity of copper gluconate was abolished by the addition of bovine serum albumin.ConclusionCopper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells but this effect was abolished by albumin, which suggests that copper will not retain its activity in serum. Furthers studies are needed to investigate whether copper gluconate could be of benefit in mucosal administration such as mouthwash, nasal spray or aerosols.