Molecular Biology Laboratory Research Articles

Effect of Aluminium Phosphide on Some Nutrients and Anti-Nutritional Factors in Arachis hypogaea

Aims: To comparatively study the effect of aluminium phosphide preservative on some nutrientional and anti-nutritional factors in Arachis hypogaea.
 Study Design: Randomized design
 Place and Duration of Study: Department of Biochemistry and Molecular Biology Laboratory, Nasarawa State University, Keffi between May and August, 2019.
 Methodology: Two portions of A. hypogaea weighing 500g each, were obtained, one portion preserved with Aluminium phosphide tablet while the second portion was without any preservative. The preservation lasted for 30 days after which the A. hypogaea was blended using a mechanical blender before analysis for nutritional and anti-nutritional compositions.
 Result: The proximate compositions were significantly (p<0.05) higher in the NAPP than APP except the fiber (03.01±0.06 mg/100 g; 03.80±0.06 mg/100 g) and ash (01.00±0.06 mg/100 g; 02.23±0.06 mg/100 g). Carbohydrates (07.40±0.06 mg/100 g; 03.48±0.06 mg/100 g), protein (19.93±0.06 mg/100 g; 14.94±0.06 mg/100), fat (74.60±0.06 mg/100g; 68.08±2.75 mg/100 g), and moisture content (06.40±0.06 mg/100 g; 3.00±0.06 mg/100 g). Exception of phosphorus (65.00±0.06mg/100g; 63.00±0.06mg/100g), the minerals concentration was significantly(p<0.05) higher in NAPP compared to APP. Iron (76.20±0.06 mg/100 g; 62.00±0.06 mg/100 g), potassium (38.01±0.06mg/100 g; 26.20±0.06mg/100g), manganese (26.02±0.06mg/100g; 15.10±0.06 mg/100 g), magnesium (11.00±0.06mg/100g; 06.00±0.06 mg/100 g), calcium (82.10±0.06 mg/100 g; 72.00±0.06 mg/100 g) and zinc (25.01±0.06 mg/100 g; 14.01±0.06 mg/100 g).The anti-nutritional factors showed significant difference (p<0.05) higher in tannin (9.33±0.69 mg/100 g; 1.90±0.02 mg/100 g), oxalate (32.50±0.60 mg/100 g; 42.50±.60 mg/100 g), phytate (16.72±0.60 mg/100 g; 5.48±0.60 mg/100 g), alkaloid (16.72±0.60 mg/100g; 5.48±0.60 mg/100 g) and saponins (40.74±0.60 mg/100 g; 38.20±0.60 mg/100 g) except cyanide (0.02±0.00 mg/100 g; 0.02±0.00 mg/100 g).
 Conclusion: The study showed that aluminium phosphide negatively affected the nutritional profile of A. hypogaea. Thus, the effect of aluminium phosphide should be further investigated in vivo.

PH-dependent sedimentation of DNA in the presence of divalent, but not monovalent, metal ions

Precipitation of DNA is performed frequently in molecular biology laboratories for the purpose of purification and concentration of samples and also for transfer of DNA into cells. Metal ions are used to facilitate these processes, though their precise functions are not well characterized. In the current study we have investigated the precipitation of double-stranded DNA by group 1 and group 2 metal ions. Double-stranded DNAs were not sedimented efficiently by metals alone, even at high concentrations. Increasing the pH to 11 or higher caused strong DNA precipitation in the presence of the divalent group 2 metals magnesium, calcium, strontium and barium, but not group 1 metals. Group 2 sedimentation profiles were distinctly different from that of the transition metal zinc, which caused precipitation at pH 8. Analysis of DNAs recovered from precipitates formed with calcium revealed that structural integrity was retained and that sedimentation efficiency was largely size-independent above 400 bp. Several tests supported a model whereby single-stranded DNA regions formed by denaturation at high pH became bound by the divalent metal cations. Neutralization of negative surface charges reduced the repulsive forces between molecules, leading to formation of insoluble aggregates that could be further stabilized by cation bridging (ionic crosslinking).

Women of the Nobel factory share their stories

From sequencing to stardom — alumnae of the Laboratory of Molecular Biology reflect on how they succeeded in science. From sequencing to stardom — alumnae of the Laboratory of Molecular Biology reflect on how they succeeded in science.

3D-printed assistive pipetting system for gel electrophoresis for technicians with low acuity vision.

In molecular biology laboratories, many tasks require fine motor control and high acuity vision. For example, lab technicians with visual impairment experience difficulty loading samples into the small wells of a horizontal agarose gel. We have developed a 3D-printable gel loading system which allows technicians with low-contrast vision to load gels correctly. It includes a casting tray, a bridge, and a modified comb. The system provides a high-contrast visual field to improve visibility, and the bridge allows pipette tips to be inserted at the correct location and only to the correct depth. The necessary computer files for printing this device are freely available to increase the accessibility of molecular biology laboratories to people with visual impairment.

Evaluation of Genetic Diversity of QPM, Provit-A, and Elite Maize Inbreds Resistant to Downy Mildew Disease Using Simple Sequence Repeats

<p>Functional maize tends to be more susceptible to major maize diseases, particularly Downy mildew. Among the functional maize are Quality Protein Maize (QPM) and Provit A maize. The presence of higher amino acid and beta carotene in functional maize might have caused these types of maize more susceptible to Downy mildew disease. The objective of the research was to identify the heterotic pairs among maize inbreds i.e. QPM, Provit A, and local maize varieties resistant to Downy mildew disease using Simple Sequence Repeats (SSR) marker. The research was conducted from April to July 2017 at the Molecular Biology Laboratory of Indonesian Cereals Research Institute. A total of five QPM inbreds, 15 Provit A inbreds, and 11 Downy mildew resistant local varieties of maize were used in the experiment using 34 SSR markers. Results indicated that among 34 SSR locus analysed, variation of allele lengths ranged from 74 bp to 500 bp. A total of 125 alleles ranging from two to nine alleles per locus with an average of 3.68 alleles were generated. The data indicated wide genetic variations among characters. DNA band profile showed that nc130 marker produced the highest PIC (over 0.83) and allele value (8.00). Genetic distance analysis found a total of 21 heterotic genotypes with genetic distance exceeds 0.65. </p>

Student perceptions of an inquiry-based molecular biology lecture and lab following a mid-semester transition to online teaching.

The transition to online learning in spring 2020 was abrupt for both students and instructors. While many instructors moved to asynchronous classes, some institutions relied more heavily on synchronous online courses. Here, we evaluate student perceptions of an inquiry-based molecular biology lecture and lab course following this transition by comparing student survey responses from spring 2019, when the lecture and lab were fully in person, to spring 2020, when the lecture and lab started in person before transitioning to a synchronous online format. Students were asked to identify the main factors that supported their learning in lecture and lab, characterize the main barriers to learning in those courses, and discuss their preference of having an inquiry-based lab or a traditional "cookbook" lab with pre-determined answers. We coded these responses and provide one of the first studies to examine the impact of this online transition on student perceptions of learning in an inquiry-based molecular biology lecture and lab course.

Delay of Detection of COVID-19 Patients in Bangladesh; An Application to Cox Proportional Hazard Model

Background: We depicted the survival rate of the patients with coronavirus disease 2019 (COVID-19). We focused on the patients who were detected lately after symptom development. The aim is to determine the socio-demographic risk factors associated with the delaying detection of COVID-19 patients. Methods: We selected 300 COVID-19 patients from May to July 2020 for this cross-sectional study in the Molecular Biology Laboratory of Chittagong Medical College, Chattogram, Bangladesh. We recorded clinical characteristics from over phone interviews and laboratory diagnosis by Real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR). We applied the Cox proportional hazard model to estimate risk factors affecting the delaying detection of COVID-19 patients. Results: The female mortality rate was 44.9% higher compared to males. The graduates died 32% more than undergraduates, and unmarried peoples’ death rate was 56% more than married. Besides, those who traveled irregularly and in contact with symptomatic patients were 86% more died than non-travelers. Conclusion: Early diagnosis of COVID-19 can save enormous life, and it should emphasize special attention on the significant explanatory variable.

Xây dựng quy trình realtime RT-PCR phát hiện SARS-CoV-2 trong mẫu bệnh phẩm lâm sàng

The verification of SARS-CoV-2 virus detection procedure has been executed in the Laboratory of Molecular Biology- Bio-Medicine Institute. The results showed that the sensitivity, specificity and accuracy of procedure were 100% by using selected samples. Using the external control samples provided by the Center for Medical Testing Quality Control - Hanoi Medical University and the National Institute of Hygiene and Epidemiology Institute provided accurate results. It can be said that process validation improves test quality, thereby contributing to ensuring the quality of laboratory tests. The detection results of SARS-CoV-2 are highly reliable, timely respond to the current epidemic prevention.

Lab hands and knowing toxic substances in Uganda

How does one know a toxic substance? Perhaps paradoxically, through contact with it, as I show ethnographically from a molecular biology laboratory in Uganda, a site where people routinely handle a range of chemicals, including potentially hazardous toxicants. I analyse an everyday situation in which researchers worked with ethidium bromide, a substance regularly used to stain DNA bands in gels at this Ugandan lab but also in many comparable labs elsewhere. Ethidium bromide is one of the most fear‐inspiring chemicals at this lab; it is considered a potent mutagen and thus counts as highly carcinogenic. While the lab has clear safety procedures concerning how to handle this substance correctly, these are often not implemented in a hard‐to‐control lab setting marked by both material deprivation and a steady flow of unremunerated volunteers who seek to gain practical experience. I show how experienced Ugandan scientists have learned to mitigate their own risk of exposure to ethidium bromide by developing choreographed routines that prevent them from inadvertently touching it. I examine this as a way of ‘handling’ a dangerous substance, developing and habituating skilled movements of hands and bodies that then are passed on to other researchers. It is not an abstract knowledge of a substance's chemical properties that counts here; instead, knowing a toxic substance implies practical routines of handling.

Hepatitis B genotyping in chronic hepatitis B patients not on treatment in a tertiary medical centre in Kuala Lumpur, Malaysia

Background: Distribution, clinical characteristics and clinical response to antiviral treatment vary among hepatitis B viral (HBV) genotypes. This study aimed to describe the prevalence, demography and clinical characteristics of the HBV genotypes among chronic hepatitis B patients who were not on treatment in Universiti Kebangsaan Malaysia Medical Centre (UKMMC). Methods & Materials: A total of 1200 plasma samples received by the Molecular Biology Laboratory from 2015 to 2017 for HBV viral load were screened for volume adequacy and viral load of at least 1000 IU/mL. Records were traced to include cases not on any antiviral therapy, cases without co-infection with other hepatitis virus or HIV, and cases without other causes of liver disease. Real-time polymerase chain reaction with 3 steps melting curve analysis was done. Sequencing was done in a private laboratory for 7 samples with undifferentiated genotypes using the platform of ABI 3730 XL (Applied Biosystems). The association among genotypes, demographic and clinical characteristic data (viral load, HBeAg, AntiHBe, ALT, AFP, cirrhosis/HCC) were analysed. Results: Eighty samples (n=80) were successfully genotyped. Of these, 73% (n=59) were genotype B, 20% genotype C (n=16) and 2.5% genotype A (n=2). One sample which constituted 1.3% (n=1) each for genotype D, mixed genotype A+B and mixed genotype B+C. Sixty percent were female (48/80) and mean age was 49 years. Chinese ethnic group recorded the highest cases (78.8%), followed by Malays (17.5%). Majority of the study population (85%) had no cirrhosis by ultrasonography. Normal level of AFP and ALT were predominant (median AFP: 2.46 μg/mL; median ALT: 26 U/L). Anti-Hbe reactivity was significantly higher in those with genotype B than C with normal ultrasound findings (p = 0.035). Individuals infected by genotype A and mixed genotype B+C showed more benign clinical characteristics while genotype B, C, and mixed genotype A+B were more aggressive although not statistically significant. Genotype B was also the most prevalent among the subjects with advanced liver disease in 4 out of 6 subjects (66.7%). Conclusion: Genotypes B and C were most prevalent among chronic hepatitis B patients who were not on treatment in UKMMC. Genotype B was also most prevalent among subjects with advanced liver disease.

Gallic Acid Content and Antioxidant Activity of Pomegranate Peel Ethanol Extract

Ethanol extract of Pomegranate peel is proven to have the ability to inhibit the proliferation of cervical cancer HeLa cells. It was reported that this extract contains ellagic acid, gallic acid, and punicalagin which are identified as major effectors of antitumor activity. Gallic acid has been demonstrated to have an antioxidant activity that can influence cancer cell proliferation. The aim of the study was to identify the gallic acid content in pomegranate peel ethanol extract and to measure its antioxidant. This study was an experimental study performed at the Biochemistry and Molecular Biology Laboratory and Central Laboratory of Universitas Padjadjaran Bandung, Indonesia during September-December 2019. Gallic acid concentration was measured by using a high-performance liquid chromatography method (HPLC) and the antioxidant activity was measured by using 1,1-diphenyl-2-picrylhydrazyl (DPPH). The extract contained 616.97 mg/kg of gallic acid with an antioxidant activity of IC50 6.1 µg/mL. In conclusion, gallic acid has cytotoxic and antitumor effects from its ability to modulate antioxidant/pro-oxidant balance and inhibit reactive oxygen species (ROS). Therefore, both gallic acid content and antioxidant activity parameters can be used as markers in pomegranate peel extract which shows activities that inhibit HeLa cell proliferation.

Discovery of Bioactive Compounds from Traditionally used Medicinal Plants: A Profile of Nilufar Z. Mamadalieva

Discovery of Bioactive Compounds from Traditionally used Medicinal Plants: A Profile of Nilufar Z. Mamadalieva

Evaluation of the Amplex eazyplex Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Pneumocystis jirovecii Pneumonia.

Quantitative PCR (qPCR) assays are the gold standard for diagnosis of Pneumocystis jirovecii pneumonia (PCP). However, they are laborious and require skilled personnel. Therefore, execution outside regular working hours of the molecular biology laboratory is limited. The eazyplex P. jirovecii assay (PJA) uses loop-mediated isothermal amplification for detection of P. jirovecii It is performed directly with respiratory specimens, without the need for special skills, and delivers a result within 3 to 25 min. The goal of our study was to compare the performance of the eazyplex PJA with that of established P. jirovecii qPCR assays. All archived bronchoalveolar lavage fluid (BALF) samples that had previously tested positive for P. jirovecii by qPCR assay and 50 control samples (retrospective part), as well as all BALF samples received for P. jirovecii analysis over a period of 4 months (prospective part), were tested. Forty-nine patients with proven PCP and 126 patients without PCP were included. The sensitivity and specificity of the eazyplex PJA (95.7% and 96.5%, respectively) were comparable to those for three different P. jirovecii qPCR assays. The detection limit of the eazyplex PJA was analogous to 103 copies of the major surface glycoprotein gene per 25 μl of BALF, corresponding to 10 to 20 P. jirovecii cells. The eazyplex PJA reliably discriminated patients with PCP from patients with P. jirovecii colonization. It delivered a positive result within a mean of 9 min 38 s and required a hands-on time of 2 min 45 s. In summary, the eazyplex PJA showed identical performance for the diagnosis of PCP, compared to qPCR assays. However, in terms of time to result, practicability, and robustness, the eazyplex PJA is clearly superior and allows for around-the-clock molecular testing.

Cell scientist to watch – Hayley Sharpe

ABSTRACT Hayley Sharpe graduated from the University of Bath, UK, with a degree in biochemistry. She then joined the laboratory of Sean Munro for her PhD in cell and molecular biology at the Medical Research Council Laboratory of Molecular Biology (MRC-LMB) in Cambridge, UK. There, she investigated the properties of protein transmembrane domains embedded within the organelle membranes of the secretory pathway. She next moved to San Francisco, USA, for postdoctoral work at Genentech, where she studied clinical resistance mechanisms to a Hedgehog pathway inhibitor in skin cancer. In 2016, Hayley returned to the UK to establish her own research group at the Cambridge Institute for Medical Research, and in 2019, she moved to the Babraham Institute, also in Cambridge. Her laboratory works on understanding how protein tyrosine phosphatases function in signalling and cell–cell communication, with a particular focus on a family of cell surface receptors. Hayley became part of the EMBO Young Investigator Programme in 2019 and was awarded the Lister Institute Research Prize in 2020.

Polymorphism of p53 Codon 72 Gene on Cervical Cancer Incidence in Malay Population

In Indonesia, the cases of cervical cancer are estimated at around 50 per 100.000 people. It was estimatedthattherearemore than1 millionwomenworldwidewho have cervical cancer,andmostofthemhave not been diagnosed yet or do not have access to screening and medical treatment. P53 codon 72 polymorphism can affect the risk of cervical cancerthrough the regulationofproliferationandcellapoptosis.The purpose of this research was to investigate the association between p53 codon 72 polymorphism and cases of cervical cancer. This research was observational analytic research. The research was done by examining in the laboratory of Molecular Biology, Faculty of Medicine, Universitas Sriwijaya by using a case-control study approach.Thesampleoftheresearchconsistedof 70subjects,andtheyweredividedintotwogroups:35in case group and 35 in the control group. The determination of genotype and allotype was done by using PCR-RFLP technics. The results of the research showed there was a significant difference between p53 codon 72 polymorphism between the case group and control group. The results of genotypes of p53 codon 72 polymorphism in the case group were seven respondents (20.0%) with Pro/Pro genotype, five respondents (14.3%) with Arg/Arg genotype, and 23 respondents (65.7%) with Pro/Arg. While in control group, there were 28 respondents (80.0%) with Pro/Pro genotype, 0 respondent (0.0%) with Arg/Arg genotype, and 7 respondents (20%) with Pro/Arg. The frequency of the Prolin allele in case group was 37 (52.9%), and the Arginin allele was 33 (47.1%), while the frequency of the Prolin allele in the control group was 63 (90%), and the Arginin allele was 7 (10%).TheChi-Squareofgenotypewas valued0.000,OR1,304andCI95%1,071-5,891,whiletheallelewas valued 0.000,OR8.027 andCI95% 3.228-19.962. Therewas an associationbetweengenotypeand allele of p53 codon72 polymorphismandcasesofcervicalcancer

Changes in Some Biochemical Parameters and Body Weights of Healthy Albino Rats Following Palm-wine Administration

Aims: To evaluate the effect of palm-wine administration on liver function, antioxidant parameters and body weights in albino rats.
 Study Design: Randomized block design.
 Place and Duration of Study: Department of Biochemistry and Molecular Biology Laboratory, Nasarawa State University, Keffi, Nasarawa State, Nigeria between June and August, 2019.
 Methodology: Twenty adult male albino rats were divided in to five groups of four rats each, group 1 was the control. The experimental groups were administered fresh and prolonged-fermented palm-wine thus; Group 2: 15ml/ of fresh palm wine, Group 3; 15ml/ kg of prolonged fermented palm wine, Group 4; 30ml/ kg of fresh palm wine, Group 5; 30 ml/ kg of prolonged fermented palm wine, for 28 days.
 Results: AST activity decreased significantly (P<0.05) in groups 3 (9.615±0.225), 4 (9.860±0.162) and 5 (9.390±0.624) when compared to the control (10.505±0.121). ALT activity was observed to decrease significantly (P<0.05) in group 5 (7.415±0.548) when compared to control. ALP decreased significantly (P<0.05) in group 3 (7.395±0.710) when compared to control (8.250±0.577). Total Bilirubin, was significantly (P<0.05) lower in groups 3 (0.120±0.041) and 5 (0.170±0.012) compared to control (0.650±0.012). MDA decreased significantly (P< 0.05) in groups 2, 3 and 5 compared to control. SOD decreased significantly (P<0.05) in group 2 (2.735±0.110) compared to control (2.990±0.012). Groups 4 (2.700±0.046) and 5 (2.710±0.346) showed significant (P< 0.05) decrease in GPX when compared to control. GSH was significantly (P< 0.05) higher in groups 4 (5.950±0.058) and 5 (5.925±0.064) compared to control (3.110±0.035). By the third week, the mean body weights of the rats significantly (P<0.05) decreased in all the groups compared to week 2.
 Conclusion: Palm-wine did not exert remarkable toxic effect on the liver biomarkers and may not have induced oxidative stress but reduced body weights of the rats in the third week of administration.

The European Nucleotide Archive in 2020.

The European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena), provided by the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI), has for almost forty years continued in its mission to freely archive and present the world's public sequencing data for the benefit of the entire scientific community and for the acceleration of the global research effort. Here we highlight the major developments to ENA services and content in 2020, focussing in particular on the recently released updated ENA browser, modernisation of our release process and our data coordination collaborations with specific research communities.

The international nucleotide sequence database collaboration.

The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org/) has been the core infrastructure for collecting and providing nucleotide sequence data and metadata for >30 years. Three partner organizations, the DNA Data Bank of Japan (DDBJ) at the National Institute of Genetics in Mishima, Japan; the European Nucleotide Archive (ENA) at the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI) in Hinxton, UK; and GenBank at National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health in Bethesda, Maryland, USA have been collaboratively maintaining the INSDC for the benefit of not only science but all types of community worldwide.

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

We describe the updated size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) set-up used at the P12 bioSAXS beam line of the European Molecular Biology Laboratory (EMBL) at the PETRAIII synchrotron, DESY Hamburg (Germany). The addition of size exclusion chromatography (SEC) directly on-line to the SAXS capillary has become a well-established approach to reduce the effects of the sample heterogeneity on the SAXS measurements. The additional use of multi-angle laser light scattering (MALLS), UV absorption spectroscopy, refractive index (RI), and quasi-elastic light scattering (QELS) in parallel to the SAXS measurements enables independent molecular weight validation and hydrodynamic radius estimates. This allows one to address sample monodispersity as well as conformational heterogeneity. The benefits of the current SEC-SAXS set-up are demonstrated on a set of selected standard proteins. The processed SEC-SAXS data and models are provided in the Small Angle Scattering Biological Data Bank (SASBDB) and are labeled as “bench-marked” datasets that include the unsubtracted data frames spanning the respective SEC elution profiles and corresponding MALLS-UV-RI-QELS data. These entries provide method developers with datasets suitable for testing purposes, in addition to an educational resource for SAS data analysis and modeling.