Optimization of Expression and Purification of Acetoacetyl-CoA Thiolase from Sunflower

The [beta]-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the existence of two different thiolase isoforms, Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9 AACT) and Thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16 OACT). Glyoxysomal AACT is the last enzyme in the p-oxidation of fatty acids in plant glyoxysomes. Glyoxysomal AACT has been successfully cloned, expressed and purified from sunflower cotyledons (Dyer et al., 2006). In this paper we investigate the optimal conditions for both expression and purification. These include the choice of vector, the growth conditions and the purification parameters for the maximum possible yield of AACT protein

Branching into RNAi: Synthesis, Characterization and Biology of Branch and Hyperbranch siRNAs

The cancer epidemic continues to afflict millions of humans world-wide each year and despite a renewed hope with the development of new and improved forms of therapy, a cure for cancer remains an elusive goal. This is partly related to the rise of resilient forms of tumors that have evolved with resistance towards conventional chemotherapy and radiation treatments. Moreover, these non-specific therapeutic regimens are highly toxic, leading to severe immunosuppressive effects which poisons the body and compromises the road towards remission. In an effort to mitigate these limitations, cancer-targeting approaches are currently experiencing a renaissance in the translation of new medicines from pre-clinical to bedside use. Notably, gene therapy has recently gained widespread traction in cancer research in the advent of the first RNA interference (RNAi) application in humans. RNAi solicits the use of a double-stranded RNA substrate, aptly named short-interfering RNA (siRNA), which binds to and triggers the degradation of a targeted complementary mRNA strand within the catalytic site of the RNA-Induced Silencing Complex (RISC). In this manner, malignant mRNA expression is silenced, thereby inhibiting the translation of proteins that can lead to the production of pathological disorders such as cancer. In spite of their utility, several challenges still remain towards the development of a fruitful cancer-targeting gene therapy approach. Here, a new class of siRNA motifs is presented to increase substrate efficacy in the RNAi application. Our biological target is a member of the heat shock family of chaperone proteins, the Glucose Regulated Protein of 78 kilodaltons (GRP78) which signals tumor initiation, proliferation and resistance towards chemotherapy. Moreover, GRP78 is overexpressed and cell surface localized on a wide range of resilient tumor types but not on healthy cells, making it a viable bio-marker for the development of the proposed cancer-targeting gene therapy approach. Significantly, an efficient solid-phase synthesis method is described for the production of linear, V-shape, Y-branch and hyperbranch GRP78-silencing siRNAs. The novel V-shape, Y-branch and hyperbranch motifs were then studied by CD spectroscopy and thermal denaturation experiments. CD spectroscopy was used to characterize the requisite A-type double-stranded RNA helix for RNAi application; whereas thermal denaturation experiments were used to validate siRNA hybrid stabilities. With stable siRNA hybrids in hand, their biological activity was assessed in HepG2 hepatoblastoma cells, which constitutes a morbid form of pediatric liver cancer and a valid tumor model for studying our GRP78-targeting strategy. The GRP78 silencing activity of the putative branch and hyperbranch siRNAs is discussed and related to its underlying mechanisms for inducing apoptosis in cancer. Biological studies confirmed potent suppression of GRP78 expression (50-60%) while compromising cancer cell viability by ~20%. The development of an effective cancer-targeting gene therapy approach is highlighted by preliminary results that showcase the utility of a cancer-targeting peptide (CTP) to condense and deliver siRNA within cancer cells for therapeutic treatment. The latter forms the basis of our cancer-targeting gene therapy approach. Thus, branched and hyperbranched siRNAs may serve as potent siRNA candidates in cancer gene therapy applications

A Model for Estimating Network Infrastructure Costs: A Case for All-Fibre Networks

The 21st century is an era that has been characterised by phenomenal growth in data rates at the local area network (intranet), extranet and the Internet, a trend pushed by deployment of “bandwidth hungry” applications such VoIP, security surveillance systems, video conferencing and streaming of online multimedia content. Due to demand placed on network resources by these applications physical layer cabling solutions have had to evolve to support faster, improved LAN technologies such as Gigabit Ethernet. Although new network architectures (such as Centralised Fibre networks) address current and long term demands of the modern networking environment, concerns have been raised about its cost viability. The key problem identified in this study was an inadequacy of suitable tools that aid decision making when estimating the cost of a network infrastructure project. Factors of importance in this regard were collected in a survey and used in development of a cost model. A network was designed based on two architectures – centralised fibre (all-fibre network) and hierarchical star (UTP for horizontal cabling and optical fibre for backbone cabling). Thereafter, cost of implementing these two architectures was calculated using the model. Based on the results computed from the cost model, the all-fibre network (centralised fibre architecture) was found to be more cost effective than the hierarchical star network. Keywords: centralised fibre architecture, hierarchical star architecture, structured cabling, multimode optical fibre, singlemode optical fibre, backbon

Deep transfer learning for detecting Covid-19, Pneumonia and Tuberculosis using CXR images -- A Review

Chest X-rays remains to be the most common imaging modality used to diagnose lung diseases. However, they necessitate the interpretation of experts (radiologists and pulmonologists), who are few. This review paper investigates the use of deep transfer learning techniques to detect COVID-19, pneumonia, and tuberculosis in chest X-ray (CXR) images. It provides an overview of current state-of-the-art CXR image classification techniques and discusses the challenges and opportunities in applying transfer learning to this domain. The paper provides a thorough examination of recent research studies that used deep transfer learning algorithms for COVID-19, pneumonia, and tuberculosis detection, highlighting the advantages and disadvantages of these approaches. Finally, the review paper discusses future research directions in the field of deep transfer learning for CXR image classification, as well as the potential for these techniques to aid in the diagnosis and treatment of lung diseases

An integrated approach to risk management during competitive tendering for local authorities by emerging building contractors in Botswana

The construction industry in Botswana has in the recent years come under severe pressure due to increased competition and a critical shortage of construction work. The competition has been as a result of a dramatic influx of foreign firms that have set up base in Botswana in recent years due to Botswana’s good economic performance. The abundant diamond resources coupled with the government’s sound macroeconomic policies have largely driven this economic growth. As a result of the shortage of construction work, locally owned emerging construction companies have had to change their bidding strategies in order to be competitive and secure construction work. This in turn has led these companies tendering at very low prices and sometimes submitting sub-economic bids. In doing so, these construction companies do not price for all the risks they are expected to carry if their bids mature into a construction contract. This paper investigates the sources of risk due to the client (Botswana local authorities) and due to statutory requirements and whether these sources are perceived by the emerging building contractors to have an influence in their (contractors) pricing strategies. The paper also investigates the risk management strategies, which are employed by these contractors in order to minimize risks during competitive tendering. The study concentrated on those building projects tendered in the public sector within the local authorities in Botswana. This was further narrowed down to specifically those projects tendered for the district councils located in the southern region of Botswana. The study concentrated on building projects and excluded civil engineering projects and projects that are overwhelmingly of specialized electrical or mechanical installations.Thesis (M Sc) (Project Management))--University of Pretoria, 2011.ai201

Demonstrating PM2.5 and road-side dust pollution by heavy metals along Thika superhighway in Kenya, sub-Saharan Africa

This study assessed the level of heavy metal in roadside dust and PM2.5 mass concentrations along Thika superhighway in Kenya. Thika superhighway is one of the busiest roads in Kenya, linking Thika town with Nairobi. Triplicate road dust samples collected from 12 locations were analysed for lead (Pb), chromium (Cr), cadmium (Cd), nickel (Ni), zinc (Zn), and copper (Cu) using atomic absorption spectrophotometry (AAS). PM2.5 samples were collected on pre-weighed Teflon filters using a BGI personal sampler and the filters were then reweighed. The ranges of metal concentrations were 39–101 μg/g for Cu, 95–262 μg/g for Zn, 9–28 μg/g for Cd, 14–24 μg/g for Ni, 13–30 μg/g for Cr, and 20–80 μg/g for Pb. The concentrations of heavy metals were generally highly correlated, indicating a common anthropogenic source of the pollutants. The results showed that the majority of the measured heavy metals were above the background concentration, and in particular, Cd, Pb, and Zn levels indicated moderate to high contamination. Though not directly comparable due to different sampling timeframes (8 h in this study and 24 h for guideline values), PM2.5 for all sites exceeds the daily WHO PM2.5 guidelines of 25 μg/m3. This poses a health risk to people using and working close to Thika superhighway, for example, local residents, traffic police, street vendors, and people operating small businesses. PM2.5 levels were higher for sites closer to Nairobi which could be attributed to increased vehicular traffic towards Nairobi from Thika. This study provides some evidence of the air pollution problem arising from vehicular traffic in developing parts of the world and gives an indication of the potential health impacts. It also highlights the need for source apportionment studies to determine contributions of anthropogenic emissions to air pollution, as well as long-term sampling studies that can be used to fully understand spatiotemporal patterns in air pollution within developing regions

La exclusión de los agentes locales del liderazgo en la coordinación de medidas de protección para la infancia

A pesar de los múltiples compromisos y de las numerosas directrices sobre la conveniencia de que sean los agentes locales quienes lideren la coordinación a nivel nacional, la realidad es que siguen siendo excluidos

Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified <i>arabidopsis</i> RNA-dependent RNA polymerases RDR2 and RDR6

Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants

Malignant Small Round Cell Tumor: A Rare Aggressive Malignancy-Case Report

Background: Malignant Small round Cell Tumor (MSRCT) is very rare group of tumors. Malignant cells have similar histologic appearance making it difficult to distinguish one from another, and poses a diagnostic challenge with needs for a multimodal approach. Immunohistochemistry and immunophenotyping by flow cytometry, reverse transcriptase polymer chain reaction (RT-PCR) and fluorescence in situ hybridization are needed to confirm. The appropriate and best treatment in early stages of MSRCT is early surgical excision with chemoradiotherapy based on patient specification. Inspite of all these, tumors have poor prognosis and chance of recurrence are high. Method: Case study of a 27-year-old male who presented with a non-productive cough for 4 weeks, with feverish feeling with weight loss and shortness of breath and a new onset precordial chest pain non radiating and worse on exertion. On examination his vitals were: BP 121/91 mm of hg, pulse rate 114bpm, temperature 36.3 degrees Celsius, respiratory rate 24/per min, SPO2 96%.On examination, Elevated Jugular Venous Pressure, hyperactive precordium, displaced apex beat to 6th intercostal space anterior axillary line, muffled heart sounds and bilateral lower limb edema. Respiratory exam was unremarkable. Sputum gene expert was negative for mycobacterium tuberculosis and covid 19 rapid test was negative. Chest X Ray showed cardiomegaly and a transthoracic echocardiography revealed moderate-large pericardial effusion, with mild collapse of right atrium in diastole. Pericardiocentesis done showed hemorrhagic effusion with lymphocytes cell component, patient was initiated on treatment for TB pericarditis empirically. Three months later a chest CT scan done revealed extensive mediastinal/pericardial mass with lung nodules. An ultrasound guided mediastinal/Pericardial biopsy was done, revealed a poorly differentiated small round cell carcinoma. Results: Patient expired 14 weeks from the initial onset of symptoms while awaiting immunohistochemistry studies for specific diagnosis of the tumor and oncology review. Discussion: This demonstrates the complexity of establishing differential diagnosis of MSRCT in a resource limited setting with subsequent delay in treatment initiation, heralds poor prognosis. This also confirms difficulties in establishing differential diagnosis in poorly differentiated tumor

Cloning, Expression and Purification of an Acetoacetyl CoA Thiolase from Sunflower Cotyledon

Thiolase I and II coexist as part of the glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT