Minimal Sample Requirements Research Articles

From hemolymph to in-vivo: The potential of a 1 mm microlitre flow probe with separate lock chamber for NMR metabolomics in mass limited environmental samples

Metabolomics is one of the leading approaches for understanding the toxic-mode-of-action of environmental contaminants. Nuclear Magnetic Resonance (NMR) spectroscopy has been commonly used in metabolomic studies; however, its main drawback is its relatively low sensitivity, making it challenging to study mass limited but environmentally crucial samples. In this work a 1 mm microlitre probe modified with a separate lock chamber to address this challenge, provided substantial improvements in mass sensitivity relative to conventional 5 mm NMR probes. The 1 mm probe is used to analyze various components of the model organism Daphnia magna, including hemolymph, parthenogenetic eggs, dormant eggs, and neonates. A μL volume flow system is designed for the 1 mm probe to perform an in-vivo exposure of neonates to high salt concentrations. The metabolic investigation of these samples was only achieved due to the minimum sample requirements and high salt tolerance of the probe, demonstrating that the 1 mm microlitre probe modified with a separate lock chamber holds significant potential for future metabolomic studies of mass limited samples.

Time Resolved-Fluorescence Resonance Energy Transfer platform for quantitative nucleosome binding and footprinting.

Quantitative analysis of chromatin protein-nucleosome interactions is essential to understand regulation of genome-templated processes. However, current methods to measure nucleosome interactions are limited by low throughput, low signal-to-noise, and/or the requirement for specialized instrumentation. Here, we report a Lanthanide Chelate Excite Time-Resolved Fluorescence Resonance Energy Transfer (LANCE TR-FRET) assay to efficiently quantify chromatin protein-nucleosome interactions. The system makes use of commercially available reagents, offers robust signal-to-noise with minimal sample requirements, uses a conventional fluorescence microplate reader, and can be adapted for high-throughput workflows. We determined the nucleosome-binding affinities of several chromatin proteins and complexes, which are consistent with measurements obtained through orthogonal biophysical methods. We also developed a TR-FRET competition assay for high-resolution footprinting of chromatin protein-nucleosome interactions. Finally, we set up a TR-FRET competition assay using the LANA peptide to quantitate nucleosome acidic patch binding. We applied this assay to establish a proof-of-principle for regulation of nucleosome acidic patch binding by methylation of chromatin protein arginine anchors. Overall, our TR-FRET assays allow facile, high-throughput quantification of chromatin interactions and are poised to complement mechanistic chromatin biochemistry, structural biology, and drug discovery programs.

An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies.

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.

Prevalence and Determinants of Central Obesity at Urban Slum Dwellers in North Jakarta

Central obesity, widely known as a major cardiovascular disease risk factor, threatens the urban poor more than other socio-economic groups. The study intended to disclose the prevalence and determinants of central obesity in urban slum dwellers in North Jakarta. We conducted a survey study on residents aged 15 and above. The study randomly recruited two hundred forty-eight residents for direct interviews and body measurements according to the minimum sample requirement. Sociodemographic and Noncommunicable Diseases (NCDs) risk factors questionnaires were used and asked after weight, height, and waist circumference measurements. Descriptive and multivariate analyses were conducted to determine the central obesity prevalence and associated determinants. The central obesity prevalence in urban slum dwellers reaches 68.1%, presiding in women (69.4%) than men (65.3%). The multivariate binary logistic regression analysis indicates that central obesity was higher in the 30-59 y.o age group (Prevalence Ratio [PR]= 1.68; 95% Confidence Interval [CI]=1.19-2.37) and ≥ 60 y.o age group (PR = 1.76; 95%CI = 1.20-1.41). Besides, the residents who do not eat fruits and vegetables daily (PR=1.20; 95%CI=1.01-1.41) and practice less physical activity (PR=1.25; 95%CI=1.01-1.56) were more likely to have central obesity. The productive age group and elderly are at a higher risk for central obesity in an urban slum. Although notable as the main risk factors for central obesity, less consumption of fruits, vegetables and lack of physical activity highlight the urgent need to provide affordable, healthier options for urban slum dwellers.

Late-Stage Functionalization and Characterization of Drugs by High-Throughput Desorption Electrospray Ionization Mass Spectrometry.

Late-stage functionalization (LSF) of drug molecules is an approach to generate modified molecules that retain functional groups present in the active drugs. Here, we report a study that seeks to characterize the potential value of high-throughput desorption electrospray ionization mass spectrometry (HT DESI-MS) for small-scale rapid LSF. In conventional route screening, HT-based DESI-MS provides contactless, rapid analysis, reliable and reproducible data, minimal sample requirement, and exceptional tolerance to high salt concentrations. Ezetimibe (E), an established hypertension drug, is targeted for modification by LSF. C-H alkenylation and azo-click reactions are utilized to explore this approach to synthesis and analytical characterization. The effect of choice of reactant, stoichiometry, catalyst, and solvent are studied for both reactions using high throughput DESI-MS experiments. Optimum conditions for the formation of LSF products are established with identification by tandem mass spectrometry (MS/MS).

Transbronchial lung cryobiopsy: prospective safety evaluation and 90-day mortality after a standardized examination protocol.

Background:Transbronchial lung cryobiopsy (TBLC) is a new method of bronchoscopic tissue sampling in patients with unclear diffuse parenchymal lung disease (DPLD). While not the gold standard, TBLC has a good diagnostic correlation with surgical lung biopsy, and retrospective analyses of peri-interventional complications and mortality are promising. However, prospective reports on 90-day mortality are lacking.Objectives:This study addresses morbidity and 30- and 90-day mortality in TBLC after a standardized protocol.Methods:In this prospective study, 75 patients with DPLD requiring tissue sampling were included. A standardized protocol (including prophylactic use of an endobronchial balloon, postinterventional observation, and minimum sampling requirements) was used in all patients. Adverse events (pneumothorax, bronchial bleeding, premature discontinuation, prolonged monitoring at ICU, and fatal outcome) and 30- and 90-day mortality rates were recorded.Results:A total of 308 cryobiopsies were performed in 75 patients. Peri- and postinterventional pneumothorax were observed in 20% (9.3% mild and 10.7% moderate with the necessity of chest drainage), and bronchial bleeding was found in 29.3% (22.7% moderate and 6.7% severe). Total lung capacity below normal value was associated with the risk of pneumothorax (p = 0.009), and diffusion limitation for carbon monoxide below normal value was associated with the risk of bronchial bleeding (p = 0.044). No fatal events were observed within 30 days, and the 90-day mortality rate was 1.3%, but not related to the procedure itself.Conclusion:As it gradually becomes the invasive procedure of choice in unclear DPLD, TBLC is a safe procedure with a low 30- and 90-day mortality.Trial registration ID: DRKS00026746 (German Clinical Trial Register)

Molecular and MALDI-TOF MS identification of swallow bugs Cimex hirundinis (Heteroptera: Cimicidae) and endosymbionts in France

BackgroundThe Cimicidae are obligatory blood-feeding ectoparasites of medical and veterinary importance. We aim in the current study to assess the ability of MALDI-TOF MS to identify Cimex hirundinis swallow bugs collected in house martin nests.MethodsSwallow bugs were picked out from abandoned nests of house martin swallows and identified morphologically to the species level. The bugs were randomly selected, dissected and then subjected to MALDI-TOF MS and molecular analyses.ResultsA total of 65 adults and 50 nymphs were used in the attempt to determine whether this tool could identify the bug species and discriminate their developmental stages. Five adults and four nymphs of C. hirundinis specimens were molecularly identified to update our MS homemade arthropod database. BLAST analysis of COI gene sequences from these C. hirundinis revealed 98.66–99.12% identity with the corresponding sequences of C. hirundinis of the GenBank. The blind test against the database supplemented with MS reference spectra showed 100% (57/57) C. hirundinis adults and 100% (46/46) C. hirundinis nymphs were reliably identified and in agreement with morphological identification with logarithmic score values between 1.922 and 2.665. Ninety-nine percent of C. hirundinis specimens tested were positive for Wolbachia spp. The sequencing results revealed that they were identical to Wolbachia massiliensis, belonging to the new T-supergroup strain and previously isolated from C. hemipterus.ConclusionsWe report for the first time to our knowledge a case of human infestation by swallow bugs (C. hirundinis) in France. We also show the usefulness of MALDI-TOF MS in the rapid identification of C. hirundinis specimens and nymphs with minimal sample requirements. We phylogenetically characterized the novel Wolbachia strain (W. massiliensis) infecting C. hirundinis and compared it to other recognized Wolbachia clades.Graphical

The Role of Compensation as a Determinant of Employee Performance and Employee Job Satisfaction: A Study in PT Bank Rakyat Indonesia (Persero) Tbk.

This paper conducts a more in-depth study of employee performance to connect with compensation and job satisfaction and the work environment of employees in PT Bank Rakyat Indonesia (Persero) Tbk. (BRI). This research was conducted at PT Bank Rakyat Indonesia (Persero) Tbk. operating in Indonesia. The research population was all employees of the Branch Offices of PT Bank Rakyat Indonesia (Persero) Tbk. spread over in Sumatera, Jawa, Kalimantan, Sulawesi, Bali and Jayapura. The sample was collected using Proportional Random Sampling Stratified from six locations spread throughout Indonesia. Considering the wide area, the sampling technique is adjusted to the area, so sampling is based on the island as a sub-population. The sample of this study was 212 employees. This amount has met the minimum sampling requirements if using the Generalised Structural Component Analysis (GSCA) method. Compensation does not directly affect employee performance. Working environment directly influences the employee performance in PT Bank Rakyat Indonesia (Persero) Tbk. There is an influence of compensation and working environment on job satisfaction and employee performance in PT Bank Rakyat Indonesia (Persero) Tbk. The leader of PT Bank Rakyat Indonesia (Persero) Tbk. should improve employees’ performance by taking into account the improvement of compensation which includes the suitability of wages and salaries, incentives, benefits and facilities with their performance.

Minimum training sample size requirements for achieving high prediction accuracy with the BN model: A case study regarding seismic liquefaction

The complexity of a Bayesian network (BN) model and the number of training samples used have significant impacts on the prediction accuracy of the model regarding seismic liquefaction. The required training sample size for ensuring that a BN model has high generalization ability is a critical issue in parameter learning. To address this issue, this study analyses the relationship between the predictive performance of the BN model and the complexity of the model, training sample size, and average discrete intervals. Taking seismic liquefaction prediction as an example, 4536 statistical experiments are designed to investigate the training and testing performances of 21 different BN models under the conditions of nine different training sample size ratios (5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80% of all data) and testing samples using 20% of all data. The results reveal that the learning performance of a BN is not sensitive to the training sample size but is related to the complexity of the model. The larger the sample size is, the stronger the generalization ability of the model. The minimum training sample requirements are related to the maximum in-degrees and the average discrete intervals, not the numbers of nodes and edges and the maximum out-degree of the BN structure. In addition, a modified structural entropy can characterize the complexity of a BN structure better than the existing structural entropy, but it has a worse relationship with the minimum training sample requirements than that of the maximum in-degree. To quickly determine the minimum training sample size requirements of a BN model with a predictive accuracy of 80%, a fitting function that considers the effects of the maximum in-degree and the average discrete intervals is presented, and its effectiveness is validated by two examples.

Optimizing extracellular vesicles' isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant.

In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.

High-throughput screening and spatial profiling of low-mass pesticides using a novel Ti3C2 MXene nanowire (TMN) as MALDI MS matrix

Pesticides play critical roles in agricultural fields; however, pesticide residues can cause serious damage to human health and the ecological environment; therefore, developing a rapid and sensitive method for pesticide detection is urgently needed. Nanostructure-assisted matrix laser desorption/ionization (MALDI) mass spectrometry (MS) has great potential for the detection of low-mass pesticides. In this study, a novel Ti3C2 MXene nanowire (TMN) was prepared by a facile sol-gel method and served as a matrix to enhance MALDI MS performance in the analysis of pesticides in positive ion mode. The TMN showed superior performance in the high-throughput detection of six kinds of pesticides (organophosphorus, organochlorine, carbamate, neonicotinoids, triazole, and oxadiazines), with ultrahigh sensitivity (detection limits at sub-ppt levels), remarkable repeatability, excellent salt tolerance, and extremely low background compared to traditional organic matrices due to the specific polyaromatic structure and the doping of nitrogen. Furthermore, this matrix was successfully employed for the analysis of residual pesticides in traditional Chinese herbs, and the level of diniconazole was quantified with a linear range of 0–50 ng/mL (R2 > 0.99). More importantly, the spatial distribution of various endogenous compounds (e.g., amino acids and saccharides, fatty acids, alkaloids, and plant hormones) and xenobiotic pesticides from the intact root of the medicinal plant P. quinquefolium was clearly visualized using the TMN self-assembly film as a matrix for MALDI imaging mass spectrometry (IMS). With superior advantages such as sensitivity, simplicity, rapidness, and minimal sample requirement, TMN as a matrix-assisted MALDI MS shows great promise for various applications.

Shock waves exposed α-Fe2O3 nanoparticles for electrochemical sensing of riboflavin, uric acid and folic acid

Multi-analyte detection has become an increasingly important need for on-line and in-situ monitoring in food process, biomedical and environmental monitoring industries due to its advantages like minimum sample and time requirement for analysis, improved functionality, and portability of the devices. Here, we report a novel electrochemical sensor based on shock wave treated Iron (ΙΙΙ) oxide (α-Fe2O3) nanoparticles for simultaneous detection of Riboflavin (RF), Uric acid (UA), and Folic acid (FA). The α-Fe2O3 NPs were synthesized by the sol–gel method and modified by exposing shock waves (50,100,150 pulses) with a time interval of 3 s. Both the pristine and modified nanoparticles were characterized by powder XRD, HRTEM, XPS, Raman, and Photoluminescence spectroscopy. Glassy carbon electrodes (GCE) have been modified with the α-Fe2O3NPs and their electrocatalytic activity towards the oxidation of RF, UA, and FA has been investigated in phosphate buffer saline (PBS) at pH 7.4. Among various electrodes, the α-Fe2O3 NPs exposed to 150 shock waves exhibited sharp oxidation peaks at −0.45 V, 0.4 V, and 0.8 V corresponding to oxidation of RF, UA, and FA respectively. The fabricated sensor responded selectively over wider dynamic ranges of 0.11 to 624 μM for RF, 0.11 to 722 μM for UA, and 0.11 to 691 μM for FA with the corresponding detection limits of 26, 18, and 25 nM respectively. For simultaneous determination, the SWV oxidation peak currents increased proportionally with the increase in the concentration over the range of 0.111 to 397 µM, with the relatively better LODs of 11, 14, and 16 nM for RF, UA, and FA. The influence of 100-fold excess of ascorbic acid, sucrose, thiamine (Vit.B1), urea, glucose, NaCl, KCl, histamine, ammonia, acetylcholine, ascorbic acid, and L-tyrosine were tested on the SWV response in the presence of riboflavin, uric acid, and folic acid. Applicability of the fabricated electrode has been tested using vitamin tablets and human urine as real samples.

The Performance Improvement and the Competitiveness of Private Universities (PTS) in North Sumatera through the Strategy of Building Institutional Competence

According to data released by DIKTI 2015 concerning the classification and ranking of the best 200 of higher education in Indonesia is only 5 universities from North Sumatera that are included in the list, both public and private universities. The purpose in this study is to examine whether there in an effect of learning orientation, internal environment and reputation on institutional competence. As well as to test whether there is an effect of competence on the achievement of institutional performance. The unit of analysis in this research is private universities. Respondents of this research were divided into 2 stages: Stage 1 is focused on the leadership of PTS (rector, vice chancellor I, II, or III; director, deputy director I, II, or III; chairman or vice chairman I, II, or III), where one PTS will be represented by one respondent. This study will describe the perceptions of PTS leader regarding organisational learning, the influence of internal environment, reputation, competence strategies and PTS performance. The second stage of respondent was extended to student suppliers, companies using graduate students, students, lectures, administration staff to describe the perception of the institutional competitiveness. The population in this study were all private universities in North Sumatera amounting 267 universities. This study uses 16 indicator so that the minimum sample requirement is 80. The data was collected using questionnaire, while data measured by Likert scale. Data were analysed use Structural Equation Modelling (SEM) through Analysis of Moment Structure (AMOS) version 16.0 program.

Microfluidic Point-of-Care Testing: Commercial Landscape and Future Directions.

Point-of-care testing (POCT) allows physicians to detect and diagnose diseases at or near the patient site, faster than conventional lab-based testing. The importance of POCT is considerably amplified in the trying times of the COVID-19 pandemic. Numerous point-of-care tests and diagnostic devices are available in the market including, but not limited to, glucose monitoring, pregnancy and infertility testing, infectious disease testing, cholesterol testing and cardiac markers. Integrating microfluidics in POCT allows fluid manipulation and detection in a singular device with minimal sample requirements. This review presents an overview of two technologies - (a.) Lateral Flow Assay (LFA) and (b.) Nucleic Acid Amplification - upon which a large chunk of microfluidic POCT diagnostics is based, some of their applications, and commercially available products. Apart from this, we also delve into other microfluidic-based diagnostics that currently dominate the in-vitro diagnostic (IVD) market, current testing landscape for COVID-19 and prospects of microfluidics in next generation diagnostics.

Complex Nucleic Acid Hybridization Reactions inside Capillary-Driven Microfluidic Chips.

Nucleic acid hybridization reactions play an important role in many (bio)chemical fields, for example, for the development of portable point-of-care diagnostics, and often such applications require nucleic acid-based reaction systems that ideally run without enzymes under isothermal conditions. The use of novel capillary-driven microfluidic chips to perform two isothermal nucleic acid hybridization reactions, the simple opening of molecular beacon structures and the complex reaction cascade of a clamped-hybridization chain reaction (C-HCR), is reported here. For this purpose, reagents are arranged in a self-coalescence module (SCM) of a passive silicon microfluidic chip using inkjet spotting. The SCM occupies a footprint of ≈7 mm2 of a ≈0.4×2cm2 microfluidic chip. By means of fluorophore-labeled DNA probes, the hybridization reactions can be analyzed in just ≈2min and using only ≈3µL of the sample. Furthermore, the SCM chip offers a variety of reagent delivery options, allowing, for example, the influence of the initiator concentration on the kinetics of C-HCR to be investigated systematically with minimal sample and time requirements. These results suggest that self-powered microfluidic chips equipped with a SCM provide a powerful platform for performing and investigating complex reaction systems.

Encapsulated Nanodroplet Crystallization of Organic-Soluble Small Molecules

SummarySingle-crystal X-ray diffraction analysis (SCXRD) constitutes a universal approach for the elucidation of molecular structure and the study of crystalline forms. However, the discovery of viable crystallization conditions remains both experimentally challenging and resource intensive in both time and the quantity of analyte(s). We report a robot-assisted, high-throughput method for the crystallization of organic-soluble small molecules in which we employ only micrograms of analyte per experiment. This allows hundreds of crystallization conditions to be screened in parallel with minimal overall sample requirements. Crystals suitable for SCXRD are grown from nanoliter droplets of a solution of analyte in organic solvent(s), each of which is encapsulated within an inert oil to control the rate of solvent loss. This encapsulated nanodroplet crystallization methodology can also be used to search for new crystal forms, as exemplified through both our discovery of a new (13th) polymorph of the olanzapine precursor ROY and SCXRD analysis of the “uncrystallizable” agrochemical dithianon.

Abstract B8: Multiplex quantification of immunomodulatory proteins in tissue and plasma using next-gen targeted MRM mass spectrometry

Abstract Quantifying the many immunomodulatory proteins in the “cancer-immunity cycle” is critical for both patient selection and development of novel immunotherapies. Conventional technologies to measure proteins (IHC, ELISA, Western blot, flow cytometry) are often semiquantitative, not multiplexable, feature poor specificity, and are difficult to develop. Due to these limitations, investigators have turned to mRNA-based measurements to infer protein expression of immunomodulatory factors. However, as has been unequivocally demonstrated for human breast, colorectal, and ovarian cancers (Pubmed IDs: 27251275, 25043054, 27372738), RNA levels can be poor predictors of protein levels or activity. Thus, direct quantification of proteins is desirable. As part of the National Cancer Institute’s Cancer Moonshot, we are developing >300 multiplex assays for quantifying immunomodulatory proteins in tissue and plasma using a next-gen technology for protein quantification, based on targeted multiple reaction monitoring mass spectrometry (MRM-MS). MRM-MS complements traditional immunoassays by enabling highly specific, precise, harmonizable quantification of proteins in biospecimens, even at high multiplex levels. As proof of concept, we will present a novel multiplex assay based on next-gen MRM-MS for quantification of 47 immunomodulatory proteins in plasma and/or tumor tissue. The technology uses immunoaffinity enrichment coupled with MRM-MS (immuno-MRM assay). Assay bioanalytical validation was conducted in accordance with industry standards for use in FFPE tissue and plasma matrices and included determination of limits of detection, linear range of response, intraday and interday repeatability, stability of analytes in sample processing, and characterization of reproducibility of endogenous analyte measurement. The mean linear range was over three orders of magnitude with median intra- and interday CVs less than 10%. The characterized assays are being applied to a panel of tumor specimens from a variety of cancer types to document detection and determine minimum sample requirements. Additional assay panels are under development to provide measurements of up to 350 immunomodulatory proteins. Citation Format: Jeffrey R. Whiteaker, Lei Zhao, Richard G. Ivey, Julia Voytovich, Regine M. Schoenherr, Dongqing Huang, Jacob J. Kennedy, Amanda G. Paulovich. Multiplex quantification of immunomodulatory proteins in tissue and plasma using next-gen targeted MRM mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B8.

High-Throughput Oil-Encapsulated Nanodroplet Crystallisation for Organic-Soluble Small Molecule Structure Elucidation and Polymorph Screening

Single crystal X-ray diffraction analysis (SCXRD) constitutes a universal approach for the elucidation of molecular structure and for the study of crystalline forms. However, the discovery of viable crystallisation conditions remains both experimentally challenging and resource intensive, in time and quantity of analyte(s). We report a robot-assisted, high-throughput method for the crystallisation of organic-soluble small molecules, employing only micrograms of analyte per experiment. This allows hundreds of crystallisation conditions to be screened in parallel, with minimal overall sample requirements. Crystals suitable for SCXRD analysis are grown from nanolitre droplets of a solution of analyte in organic solvent(s), each of which is encapsulated within an inert oil to control the rate of solvent loss. This encapsulated nanodroplet crystallisation methodology can also be used in the search for new crystal forms, as exemplified through both our discovery of a new (thirteenth) polymorph of the olanzapine precursor ROY and the SCXRD analysis of the “uncrystallisable” agrochemical dithianon.

Abstract 822: PureQuant Methylation Assay for the accurate quantification of immune cell populations

Abstract A key challenge during the development and manufacturing of cell based immunotherapies is the reliable estimation of cell purity. Current flow cytometric methods, while effective for the detection of surface antigens such as CD8 that positively identifies Cytotoxic T lymphocytes, are not ideal for intracellular markers used to positively identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Additionally, characterization methods that utilize live cells pose a challenge in large scale GMP manufacturing environments due to complicated logistics, limited throughput and difficulty in standardization. Therefore, there is an emerging need for alternative assay methods that address some of these challenges. Here, we introduce our newly developed PureQuant™ Assays that specifically measure the unique DNA methylation status of specific immune cell types. Assays were developed specifically for Cytotoxic T Cells, Regulatory T cells and T helper 17 cells by detecting methylation status of CD8, FoxP3 and IL17A, respectively, via qPCR of bisulfite converted genomic DNA. These assays are robust with minimal sample requirement, utilizing fresh/frozen cells or isolated gDNA. Results are represented as a percentage of the total cell population similar to flow cytometry based methods. Comparison of the two methods for surface markers such as CD8 show comparable results. Our results demonstrate that this method which combines accuracy, low sample requirement and flexibility, is an ideal measurement system for confirmation of T cell identity and purity that is critical for cell therapy research and development. Citation Format: Suman Pradhan, Carl Dargitz, Jerry Guzman, Uma Lakshmipathy. PureQuant Methylation Assay for the accurate quantification of immune cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 822.

The development of good governance model for performance improvement

Purpose The purpose of this paper is to re-test the determinant factors of the quality of financial statements and performance of the government by adding contextual factors, such as the personal factor, system/administrative factor and political factor, that may affect the quality of financial statement information and performance of the government. The personal factor is proxied to the competencies that affect the quality of financial statements and performance. The social administrative factor is proxied on the regulations and presentation of quality financial statements. Design/methodology/approach The analysis unit in this study was conducted at the organizational level. The research object was in the South Sulawesi Province. This was a descriptive and verificative research with a survey technique. Based on the objectives of the research, this is an explanatory study. The research method used was an explanatory survey with a quantitative approach. The population of this research was proxied to the Regional Unit Organization (Organisasi Perangkat Desa/OPD) which compiled the financial statements in the South Sulawesi Provincial Government and consisted of 803 units of local government agencies (Satuan Kerja Perangkat Daerah or SKPD). The purposive sampling technique was chosen under the following criteria: the regional government whose financial statement has been audited by the BPK, the regional government whose financial accountability report has been evaluated by Indonesia’s Agency for Financial, and Development Supervision (Badan Pengawasan Keuangan dan Pembangunan or BPKP). In line with the criteria mentioned above, the minimum samples required for 26 observations/indicators are 5 × 26 = 130 respondents. The sample size met the minimum sample requirement of five for each group (cell) (Hair et al., 2006, p. 112). Findings The personal factor “competence” affects the financial statements’ quality. The high personal factor “competence” will affect the high financial statements’ quality. The system/administration factor “regulation” affects the financial statement quality. The high system/administration factor “regulation” will affect the high financial statements’ quality. Political factors affect the financial statements’ quality. The high political factors will affect the high financial statements’ quality. The personal factor “competence” has no direct effect on the performance. The high personal factor “competence” will not affect the high or low of the performance. However, there is a significant indirect effect between the personal factor “competence” on performance through the financial statements’ quality, which means that the higher personal factor “competence” will lead to higher performance through financial statements’ quality. The system/administration factor “regulation” does not directly affect the performance. The high system/administration factor “regulation” will not affect the high or low of the performance. However there is a significant indirect effect between the system/administration factor “regulation” on performance through the financial statements’ quality which means that higher system/administration factor “regulation” will lead to higher performance through financial statements’ quality. The political factor does not directly affect the performance. The high political factors will not affect the high or low of the performance. However there is a significant indirect effect between political factors on performance through the financial statements’ quality which means that the higher political factor will lead to higher performance through the financial statements’ quality. Financial statements’ quality affects the performance. The high financial statements will affect the performance. Originality/value The research issues raised are the increasing public demands for the government services and accountability, while on the other hand, the government is faced with the report and financial quality that are below the expectation. This issue is a national strategic issue, leading this research to aim at providing guidelines that can help the regional government to formulate operational policies and strategies for the quality improvement of financial statement and performance of the regional government.