Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL (Duffy binding like), CIDR (cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally. METHODS: A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli. RESULTS: Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1α (46.8%) > CIDR2β (42.9%) > DBL2-4γ (31.7%) > DBL2β + C2 (30.6%) > DBL1α (24.9%) > DBL2-7ε (23.1%) > DBL2-5δ (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions. CONCLUSION: The acidic terminal segment (ATS) and CIDR α/β domain types are suitable for recombinant expression in E. coli while all DBL subtypes (α, β, γ, δ, ε) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains

Study of difference in cognitive functions after a single manic episode versus recurrent episodes in euthymic bipolar 1 patients

Background: Neurocognitive deficits have been substantially documented in patients with bipolar disorder in the euthymic state. The assessment of cognitive change from first episode mania is crucial in understanding whether cognitive deficits are progressive or already present from the first diagnostic episode of bipolar I disorder. The objective of the study is to assess and compare the cognitive function in bipolar I patients with one manic episode and recurrent episodes currently in remission.Methods: A cross sectional observational study consisting of 3 groups was carried on eighty cases each of bipolar 1 disorder patients in euthymic phase with one manic, more than 3 manic episodes and controls. These were subjected to the neuropsychological assessment and then compared.Results: The patients with recurrent episodes group shows poor performance upon digit span test, visuospatial memory test, verbal learning and memory test, color stroop test and trail making test than patients with single manic episode and healthy controls upon these cognitive tests.Conclusions: The present finding suggest that there is impairment in various cognitive domains like executive function, attention, memory even in bipolar patients after single manic episode

Identifying Implementation Bugs in Machine Learning based Image Classifiers using Metamorphic Testing

We have recently witnessed tremendous success of Machine Learning (ML) in practical applications. Computer vision, speech recognition and language translation have all seen a near human level performance. We expect, in the near future, most business applications will have some form of ML. However, testing such applications is extremely challenging and would be very expensive if we follow today's methodologies. In this work, we present an articulation of the challenges in testing ML based applications. We then present our solution approach, based on the concept of Metamorphic Testing, which aims to identify implementation bugs in ML based image classifiers. We have developed metamorphic relations for an application based on Support Vector Machine and a Deep Learning based application. Empirical validation showed that our approach was able to catch 71% of the implementation bugs in the ML applications.Comment: Published at 27th ACM SIGSOFT International Symposium on Software Testing and Analysis (ISSTA 2018

Developing a Multi-modal Listing Service for Real Estate Agency Practice in Nigeria

Fraudsters posing as real estate agents threaten the reputation of real estate agencies in Nigeria. These fraudsters have continually defrauded unsuspecting members of the public. The major cause of this lapse is due largely to the fact that there is no known platform provided in the country that allows members of the public to verify a given real estate agent. This paper aims to provide support to real estate agency practice in Nigeria by developing a multi-modal listing service for verifying registered real estate agents and to also provide information on real estates available for sale, lease or rent. The requirements for the system were gathered through observation, literature survey and user survey. These requirements were then modelled using the Unified Modelling Language (UML). The system is developed both as a web and mobile application using an open source content management system (WordPress). This paper essentially presents the: requirements gathering process, design and implementation of the multimodal listing service as well as how it compares with other similar services developed elsewhere. The multi-modal listing service developed in this study is a welcome development due to the availability and widespread adoption of the Internet and Internet-enabled mobile devices in Nigeria. The tool can be of use to the National Association of Estate Agents in Nigeria - a body saddled with the responsibility of rebranding the real estate agency profession in Nigeria

Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. METHODS: Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. RESULTS AND CONCLUSIONS: When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons

Cloning and differential expression of QM like protein homologue from tea [Camellia sinensis (L.) O. Kuntze]

The QM like protein gene encodes for ribosomal protein L10, which is implicated in tumor suppression, transcription factor regulation, and ribosome stability in yeast and mammals. Present study describes cloning of a full-length QM cDNA (CsQM) from tea leaves using differential display of mRNA followed by rapid amplification of cDNA ends. Expression of CsQM was studied in leaves of different stages of development and under various external cues. CsQM contained an open reading frame of 651 bases, encoding 216 amino acids. CsQM shared 71–87% and 85–91% identity at nucleotide and amino acid sequences, respectively with QM genes isolated from other plant species. During active-growth period of tea, higher expression was observed in apical buds that decreased gradually with increasing age of the leaf. During dormancy season, the expression of CsQM gene was severely down-regulated in all the leaves studied. CsQM transcript was found to be down regulated in response to drought stress and abscisic acid treatment but up-regulated by gibberellic acid treatment. A positive association of CsQM transcript abundance with active cellular growth suggested its role in plant growth and developmen

De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments

<p>Abstract</p> <p>Background</p> <p><it>Picrorhiza kurrooa </it>Royle ex Benth. is an endangered plant species of medicinal importance. The medicinal property is attributed to monoterpenoids picroside I and II, which are modulated by temperature. The transcriptome information of this species is limited with the availability of few hundreds of expressed sequence tags (ESTs) in the public databases. In order to gain insight into temperature mediated molecular changes, high throughput <it>de novo </it>transcriptome sequencing and analyses were carried out at 15°C and 25°C, the temperatures known to modulate picrosides content.</p> <p>Results</p> <p>Using paired-end (PE) Illumina sequencing technology, a total of 20,593,412 and 44,229,272 PE reads were obtained after quality filtering for 15°C and 25°C, respectively. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 74,336 assembled transcript sequences were obtained, with an average coverage of 76.6 and average length of 439.5. Guanine-cytosine (GC) content was observed to be 44.6%, while the transcriptome exhibited abundance of trinucleotide simple sequence repeat (SSR; 45.63%) markers.</p> <p>Large scale expression profiling through "read per exon kilobase per million (RPKM)", showed changes in several biological processes and metabolic pathways including <it>cytochrome P450s </it>(<it>CYPs</it>), <it>UDP-glycosyltransferases </it>(<it>UGTs</it>) and those associated with picrosides biosynthesis. RPKM data were validated by reverse transcriptase-polymerase chain reaction using a set of 19 genes, wherein 11 genes behaved in accordance with the two expression methods.</p> <p>Conclusions</p> <p>Study generated transcriptome of <it>P. kurrooa </it>at two different temperatures. Large scale expression profiling through RPKM showed major transcriptome changes in response to temperature reflecting alterations in major biological processes and metabolic pathways, and provided insight of GC content and SSR markers. Analysis also identified putative <it>CYPs </it>and <it>UGTs </it>that could help in discovering the hitherto unknown genes associated with picrosides biosynthesis.</p

Characterization of dihydroflavonol 4-reductase cDNA in tea [Camellia sinensis (L.) O. Kuntze]

Tea leaves are major source of catechins-antioxidant flavonoids. Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is one of the important enzymes that catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of catechins. This manuscript reports characterization of DFR from tea (CsDFR) that comprised 1,413 bp full-length cDNA with ORF of 1,044 bp (115-1,158) and encoding a protein of 347 amino acids. Sequence comparison of CsDFR with earlier reported DFR sequences in a database indicated conservation of 69-87% among amino acid residues. In silico analysis revealed CsDFR to be a membrane-localized protein with a domain (between 16 and 218 amino acids) resembling the NAD-dependent epimerase/dehydratase family. The theoretical molecular weight and isoelectric point of the deduced amino sequence of CsDFR were 38.67 kDa and 6.22, respectively. Upon expression of CsDFR in E. coli, recombinant protein was found to be functional and showed specific activity of 42.85 nmol min(-1) mg protein(-1). Expression of CsDFR was maximum in younger rather than older leaves. Expression was down-regulated in response to drought stress and abscisic acid, unaffected by gibberellic acid treatment, but up-regulated in response to wounding, with concomitant modulation of catechins content. This is the first report of functionality of recombinant CsDFR and its expression in tea