Rolling circle amplification to screen yam germplasm for badnavirus infections and to amplify and characterise novel badnavirus genomes

Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem et al., 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al., 1999; Seal and Muller, 2007; Bömer et al., 2016 and 2017; Sukal et al., 2017; Umber et al., 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam badnaviruses (Bömer et al., 2016). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of badnavirus diversity as well as the identification of potentially integrated badnavirus sequences in the host genome (Turaki et al., 2017). Here, we describe the step-by-step protocol to screen yam germplasm for badnavirus infections using RCA as an efficient research tool in the amplification and characterization of novel badnavirus genomes

PCR-DGGE analysis: Unravelling complex mixtures of badnavirus sequences present in yam germplasm

Badnaviruses (family Caulimoviridae, genus Badnavirus) have emerged as serious pathogens especially affecting the cultivation of tropical crops. Badnavirus sequences can be integrated in host genomes, complicating the detection of episomal infections and the assessment of viral genetic diversity in samples containing a complex mixture of sequences. Yam (Dioscorea spp.) plants are hosts to a diverse range of badnavirus species, and recent findings have suggested that mixed infections occur frequently in West African yam germplasm. Historically, the determination of the diversity of badnaviruses present in yam breeding lines has been achieved by cloning and sequencing of polymerase chain reaction (PCR) products. In this study, the molecular diversity of partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences from yam badnaviruses was analysed using PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE). This resulted in the identification of complex ‘fingerprints’ composed of multiple sequences of Dioscorea bacilliform viruses (DBVs). Many of these sequences show high nucleotide identities to endogenous DBV (eDBV) sequences deposited in GenBank, and fall into six monophyletic species groups. Our findings highlight PCR-DGGE as a powerful tool in badnavirus diversity studies enabling a rapid indication of sequence diversity as well as potential candidate integrated sequences revealed by their conserved nature across germplasm

Professor Bennie van der Walt: a bridge between white Afrikaners and black Africans

This article honours Professor Bennie van der Walt as a bridge builder between white Afrikaners and black Africans as well as a renowned Christian scholar. Historical Western colonialism in South Africa divided its citizens against each other by means of white racism and apartheid. The whites in general were pitched against the blacks on the basis of white racism and its doctrine of apartheid. This doctrine of separation of races kept the white Afrikaners from the Bantu Africans. However, apartheid as a form of political, social, cultural and religious racism is now history in South Africa. The role which Professor Van der Walt played in bridging the gap between this racial divide is highly commendable and needs to be acknowledged and appreciated, hence the primary objective of this article in honour of his 71st birthday. Furthermore, the article discusses the immense contributions of Professor Bennie van der Walt to Christian scholarship in Africa

A sequence-independent strategy for amplification and characterisation of episomal badnavirus sequences reveals three previously uncharacterised yam badnaviruses

Yam (Dioscorea spp.) plants are potentially hosts to a diverse range of badnavirus species (genus Badnavirus, family Caulimoviridae), but their detection is complicated by the existence of integrated badnavirus sequences in some yam genomes. To date, only two badnavirus genomes have been characterised, namely, Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform SN virus (DBSNV). A further 10 tentative species in yam have been described based on their partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences, generically referred to here as Dioscorea bacilliform viruses (DBVs). Further characterisation of DBV species is necessary to determine which represent episomal viruses and which are only present as integrated badnavirus sequences in some yam genomes. In this study, a sequence-independent multiply-primed rolling circle amplification (RCA) method was evaluated for selective amplification of episomal DBV genomes. This resulted in the identification and characterisation of nine complete genomic sequences (7.4–7.7 kbp) of existing and previously undescribed DBV phylogenetic groups from Dioscorea alata and Dioscorea rotundata accessions. These new yam badnavirus genomes expand our understanding of the diversity and genomic organisation of DBVs, and assist the development of improved diagnostic tools. Our findings also suggest that mixed badnavirus infections occur relatively often in West African yam germplasm

Effects of Parasitic Infections on Erythrocyte Indices of Camels in Nigeria

This study was conducted to determine the prevalence and effect of parasitic infection on erythrocyte indices in trade camels slaughtered in Maiduguri, Nigeria. Two hundred adult one humped camels comprised of 87 (43.5 %) males and 113 (56.5 %) females were examined for helminths and hemoparasites at their slaughter time according to the standard procedures. An overall prevalence of 79 % for single and mixed infections was observed. Examination of faecal samples from camels shows 82 (41 %) were harbouring different nematodes, mostly Strongyle, Strongyloides and Hemonchus species. Buffy coat and thin smear examination of blood samples showed Babesia and Anaplasma species. More females (44.5 %) than males (34.5 %) were positive for various parasitic infections. But the percentage was not statistically significant (P > 0.05). Packed cell volume (PCV), mean haemoglobin concentration (MCH), mean corpuscular haemoglobin concentration (MCHC) and red blood cell counts were significantly (P < 0.01) affected in the infected camels compared to the non-infected ones. Parasite infection in camels leads to macrocytic anaemia

Sickle cell identification using image processing and red blood cell morphological characteristics

Blood is the most vital liquid that helps sustain the healthiness and life of the human body. Biologically, normal red blood cells have circular shapes and play a key role in transporting oxygen and nutrient to tissues. Red blood cells are bendable, which allows them to pass through the veins and arteries smoothly. Sadly, there are exceptional individuals with abnormal blood cells call sickle cell disease. The physical shape of the abnormal blood cells is in sickle/crescent form. Sickle cell disease is hereditary, and a person becomes affected if at least one of the parents has the abnormal haemoglobin S gene. The danger of sickle cells is that they inflict many severe health conditions such as pain, tiredness, jaundice, kidney problem, and other critical illnesses. For many years, managing and diagnosing sickle patients is performed by collecting blood samples to manually observe the irregular shapes of the red blood cells using a microscope. This process is time-consuming and results in errors for large samples of blood. In this thesis, a compelling image processing method is proposed to optimize the detection of abnormality in human blood cells with the deep learning technique. Ten images of red blood cells were randomly collected from the online source using the Google search engine. Each image was analyzed using MATLAB codes for image processing using the blood cell area, eccentricity, diameter, extension, and form factor as input parameters. The study results show that the proposed technique has 71 – 100 percent accuracy, far higher than what is obtainable in the manual method. This technique can serve and enhance the current manual method of sickle cell segmentation because it is faster and more accurate

Development of Quality Standards of Prosopis africana (Fabaceae) Stem-Bark

Prosopis africana (Guill. &amp; Perr.) Taub. (Fabaceae) is the only known species of its genus found in Africa. Almost all parts of the tree are used in medicine namely as remedies for skin diseases, caries, fevers, gonorrhoea, tooth and stomach-ache, dysentery and bronchitis. It is therefore considered worthwhile to establish quality standard for the stem bark. Pharmacognostic standardization was carried out on the pulverized stem-bark and its anatomical section, by using standard experimental procedures to determine the macro and micro morphological characters, as well as other quantitative and qualitative parametres. The results of this study produced vital data that could be useful in setting some diagnostic indices that could be included in the preparation of a monograph of the plant stem bark. Keywords: Quality standards, development, stem-bark, Mimosoida

Estimation of Serum Osteocalcin Levels in Osteoporotic Postmenopausal Iraqi Women with Type 2 Diabetes Mellitus

Background: Osteoporosis (OP) is a systemic disease characterized by low bone mass and micro architectural deterioration of bone tissue, resulting in an increased risk of fractures and has touched rampant proportions. Osteocalcin, one of the osteoblast-specific proteins, showed that its functions as a hormone improves glucose metabolism and reduces fat mass ratio. This study is aimed to estimate the osteocalcin and glucose level in blood serum of osteoporotic postmenopausal Women with and without Type 2 Diabetes.Materials and methods: 60 postmenopausal women with osteoporosis divided into two groups depending on with or without T2DM, 30 patients for each. Serum samples of 30 healthy postmenopausal women were collected as control group. Osteocalcin was measured by ELISA method using a kit of (CUSABIO. China). Glucose was determined by spectrophotometric method. Results: Mean serum osteocalcin in postmenopausal osteoporotic women without Type II Diabetes is higher than control group and the group with T2DM (p ? 0.001). Conclusion: Bone formation marker increases at postmenopausal osteoporosis women; Hyperglycemia also induces osteoblast function and reduces of production osteocalcin at T2DM

Rapid detection of potyviruses from crude plant extracts

Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA). The developed method, named ‘Direct RT-RPA’, detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation. The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide

Functional properties and storage stability of optimized cereal-based complementary foods

The functional properties and good quality of food materials are important factors that determine the suitability for complementary foods for children. This study evaluated the functional properties and storage stability of cereal-based and legume- based complementary food (CF). Nutri-survey (2007) was used to optimize and generate six composite blends designated F1, F2, F3, F4, F5, and F6 from yellow maize (Zea mays), wheat (Triticum aestivum), millet (Pennisetumglaucum), groundnut (Arachis hypogea), soyabeans (Glycine max), and Moringa oleifera. Freshly prepared samples were subjected to analysis of functional properties. During storage, the composite blends were subjected to sensory evaluation, microbial count, moisture content, peroxide value and free fatty acid determination at 15-day intervals for a period of 60 days. Data obtained were analyzed by ANOVA and results expressed as mean and standard error of mean. Results of functional properties revealed that bulk density ranged from (0.63-0.81 g/cm3), water absorption capacity (86-90%), swelling index (0.33-1.34 cm3/g), reconstitution index (2.20-3.20) and pH (6.52-6.69). The organoleptic properties and keeping quality of the formulated complementary foods were not significantly different (P> 0.05) at baseline and end line. Therefore, this study provides a basis for the development of acceptable complementary foods with optimal functional properties and storage stability