Biological Research Laboratories Research Articles

Preparation empty peptide-receptive MHC class I complex for large-scale detection through photolabile peptide ligands

Peptide-major histocompatibility complex (pMHC) multimers are wide recognized as the premier technique for detecting, characterizing, and isolating antigen-specific CD8+ T-cell subsets. These multimers are specifically useful in studying infections, autoimmune conditions, and cancer through single-cell analysis techniques such as flow cytometry and fluorescence microscopy. However, the development of high-throughput assays with commercially available pMHC tetramers can be expensive, while in-house production may pose challenges for most biology research laboratories. In this context, we introduce a cost-friendly and uncomplicated protocol to prepare empty MHC class I tetramers using disulfide-stabilized molecules and photolabile peptide ligands. Our method relies on disulfide bond-stabilized MHC-I molecules, which demonstrated stability when folded into stable monomers in the presence of a photolabile epitope. These monomers, upon ultraviolet irradiation and streptavidin binding, efficiently assemble into tetramers devoid of any peptide. Following a short incubation with the peptide of interest under gentle conditions, the resulting pMHC tetramer effectively detects patient-sourced, neoantigen-specific T cells. Our unique approach streamlines large-scale pMHC generation, thus paving the way for advancements in T cell-based diagnostics and personalized therapies.

The Effect of Giving Trichoderma asperellum on Rice Plant Height in Organic and Inorganic Cultivation

The agricultural sector cannot be separated from the role of inorganic fertilizers to support plant growth, as is the case with rice cultivation. Farmers' dependence on using inorganic fertilizers has led to decreased soil fertility and damage to natural resources. One way to overcome this is to implement organic farming. Organic cultivation in this research uses the SRI (System of Rice Intensification) method, while inorganic cultivation follows conventional methods from local communities. The aim of the research was to determine the effect of giving Trichoderma asperellum on rice plant height in organic and inorganic cultivation. The research was carried out in September-December 2022 in Jorong Galo gandang, Nagari III Koto, Tanah Datar Regency, Biological Research Laboratory, and Plant Physiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Padang State University. This type of research is experimental. The research was structured using a Randomized Block Design, with 4 treatments, namely; O (Organic) OT (Organic + Trichoderma asperellum), A (Inorganic), AT (Inorganic + Trichoderma asperellum) and 5 repetitions. Observational data was analyzed by analysis of variance (ANOVA) using the SPSS application. If there are differences, continue with the DNMRT test (Duncan's New Multiple Range Test). The results showed that giving Trichoderma asperellum had no significant effect on rice plant height in organic and inorganic cultivation. Because the high rainfall at the research location caused the nutrients from each experimental plot to be washed away by the water.

Diverse Durham collection phages demonstrate complex BREX defense responses.

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.

A collaborative approach to promote use of 3D printing in a biology research laboratory.

Three dimensional (3D) design and printing are customizable and cost-effective approaches to developing small equipment and other items for use in various interdisciplinary applications. However, many pedagogical approaches to 3D printing focus more on the generation of artifacts than on the involvement of students as creators. Moreover, library makerspaces offer 3D printing services but cannot always engage the students with practical applications of their designs. We sought to determine if promoted use of 3D printing could be developed in biology laboratory trainees, ranging from undergraduate students to postdoctoral fellows. We combined two instructional workshops in the San Diego State University Library build IT makerspace, with two individual assignments to build items for the research laboratory. Evaluation of the course revealed that participants had expected the design and print processes to be of high complexity, but learned that the necessary skills could be acquired and applied in a relatively short period of time. Also, we found that trainees became proficient in 3D design and printing, and that a majority of individuals used 3D printing for subsequent applications. This effective translation of 3D printing to the research laboratory can be a paradigm for how 3D fabrication is taught. Moreover, this approach required the collaboration of library makerspace and research faculty, underlining the value of embedded librarianship in enhancing training and knowledge.

Circular RNA Expression Profiling by Microarray-A Technical and Practical Perspective.

Circular RNAs, as covalently circularized RNA loops, have many unique biochemical properties. Many circRNA biological functions and clinical indications are being continually discovered. Increasingly, circRNAs are being used as a new class of biomarkers, which are potentially superior to linear RNAs due to the unusual cell/tissue/disease specificities and the exonuclease-resistant stabilized circular form in the biofluids. Profiling circRNA expression has been a common step in circRNA research to provide much needed insight into circRNA biology and to facilitate rapid advances in the circRNA field. We will review circRNA microarrays as a practical and effective circRNA profiling technology for regularly equipped biological or clinical research labs, share valuable experiences, and highlight the significant findings from the profiling studies.

Biological research and self-driving labs in deep space supported by artificial intelligence

Biological research and self-driving labs in deep space supported by artificial intelligence

Facile methods for reusing laboratory plastic in developmental biology experiments

Plastic pollution negatively affects ecosystems and human health globally, with single-use plastic representing the majority of marine litter in some areas. Life science laboratories prefer pristine conditions for experimental reliability and therefore make use of factory standardized single-use plastic products. This contributes to overall plastic waste in the United States and globally. Here, we investigate the potential of reusing plastic culture dishes and subsequently propose methods to mitigate single-use plastic waste in developmental biology research laboratories. We tested the efficacy of bleach and ethyl alcohol in sterilizing used dishes. We then tested the feasibility of washing and reusing plastic to culture Xenopus laevis embryos subjected to various manipulations. Cleaning and reusing laboratory plastic did not affect the development or survival of X. laevis, indicating that these cleaning methods do not adversely affect experimental outcome and can be used to sterilize plastic before reuse or recycling. Lastly, we performed a survey of various life science laboratories to estimate both waste reduction and savings associated with recycling single-use plastics. Standardization of these procedures would allow research laboratories to benefit economically while practicing environmentally conscious consumption.

BIOINSECTICIDE ACTIVITY OF SILVER NANOPARTICLES PRODUCT OF BIOSYNTHESIS USING ENTOMOPATHOGEN AGAINST THE BEET CATERPILLAR SPODOPTERA EXIGUA

Spodoptera exigua (Hubner) is a polyphagous insect pest that is detrimental to farmers. Nanotechnology exists as a renewable technology that can increase effectiveness due to the small size of particles it produces. This study aimed to determine the effectiveness of biosynthesis of silver nanoparticles from the fungi Metarhizium, Trichoderma, and The Bacillus bacteria which are biocontrol agents. The research started in February and ended in May 2020, at the Biological Research Laboratory, Faculty of Mathematics and Natural Sciences Education, University of Pendidikan Indonesia. The experiment was based on a completely randomized design method (CRD). Onion armyworm (Spodoptera exigua), two isolates from the fungi Metarhizium and Trichoderma were obtained from the Indonesia Vegetable Research Institute (IVERRI). Each extract of Metarhizium and Trichoderma fungi and 50 mL of extract from Bacillus bacteria were added to 50 mL of Ag + 60 ppm solution in a 250 mL glass tube. The mixture of Metarhizium mushroom extract with 60 ppm Ag + solution or Trichoderma mushroom with Ag + 60 ppm solution was incubated at 400 C with a rotation speed of 200 rpm for five days under a dark condition. For the mixture of bacterial extracts with Ag + 60 ppm solution, incubation was at room temperature with a rotation speed of 150 rpm for three days. Analysis of the effect of NPP on the mortality of S. exiguawas done using the treatments, Metarhizum, Trichoderma, Bacillus, NPP-Metarhizium, NPP-Trichoderma, NPP-Bacillus, NPP, and distilled water as controls. Third instar larvae of S. exigua were immersed for 30 seconds. After immersion, the larvae are placed in sterile Petri dishes that have been lined with sterile filter paper and provided with caterpillar feed. Observation of caterpillar mortality was recorded at 2, 4, 6, 9, 24, 48, 64, 72, 96 and 108 hours after treatment. There were three replicates the results showed that all the treatments caused death of the insect, both entomopathogens and entomopathogenic nanoparticles. We found the three treatments of silver nanoparticles highly lethal to S. exigua. Two treatments, NPP-Metarhizium and NPP-Bacillus even caused 100% mortality after 120 hrs treatment. The possible reason for the pathogenicity of these products is considered to be biosynthesis of nanoparticles and enzymatic substances

Maternal survival costs in an asocial mammal.

Maternal characteristics, social dynamics, and environmental factors can all influence reproduction and survival and shape trade‐offs that might arise between these components of fitness. Short‐lived mammals like the golden‐mantled ground squirrel (GMGS; Callospermophilus lateralis) tend to maximize effort toward current reproduction at the expense of survival but may be complicated by other aspects of the species’ life history and environment. Here, we use 25 years of data (1995–2020) collected from a population of GMGS at the Rocky Mountain Biological Research Laboratory in Gothic, Colorado, to test the effect of several maternal characteristics (e.g., age, experience, and timing of litter emergence), social context (e.g., litter sex ratio and kin density), and environmental context (e.g., date of bare ground and length of vegetative growing season) on survival of reproductive female GMGS using Cox proportional hazard models. Our results indicated that social dynamics (i.e., density) and environmental conditions (i.e., standardized first day of permanent snow cover and length of growing season) explained significant variation in annual maternal survival, while maternal characteristics did not. A higher density of related breeding females and the total number of females (both related and unrelated to the focal mother) were associated with an increase in the mortality hazard. A later standardized date of the first day of permanent snow cover and a shorter growing season both reduced the maternal mortality hazard. Together, our results suggest that factors extrinsic to the squirrels affect maternal survival and thus may also influence local population growth and dynamics in GMGS and other short‐lived, territorial mammal species.

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish. Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish. Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

First-line Evaluation of Sperm Parameters in Mice ( Mus musculus ).

Infertility has become a major public health problem, with a male factor involved in about half the cases. Mice are the most widely used animal model in reproductive biology research laboratories, but changes in sperm parameters in mice can be subtle and, in the absence of official guidelines, it is important that analyses are carried out in a strict and reproductive manner. This protocol successively details the different steps required to obtain spermatozoa under good conditions, the measurement of sperm motility using a Computer Assisted Sperm Analysis System (CASA) device, the calculation of sperm concentration in the epididymides using a sperm counting cell, and the examination of sperm morphology. The combination of these assays provides an overview of the basic sperm parameters in mice. This is both a diagnostic and a decision-making tool for researchers to orient their scientific strategy according to the observed abnormalities.

Potent mouse monoclonal antibodies that block SARS-CoV-2 infection

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that not only exhibit robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies.

Professor Daniel I.C. Wang: A Legacy of Education, Innovation, Publication, and Leadership.

Professor Daniel I.C. Wang: A Legacy of Education, Innovation, Publication, and Leadership.

Imaging the Electrochemical Impedance of Single Cells via Conductive Polymer Thin Film.

Many fundamentally important biological phenomena involve the cells to establish and break down the adhesive interactions with the substrate. Here, we report a novel optical method that could directly image the electrochemical impedance of cell-substrate interactions at the single cell level with conventional microscopes and cameras. A thin conductive polymer layer on top of the ITO substrate (poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate), PEDOT:PSS) is used as the impedance imaging and sensing layer. A sinusoidal electrochemical potential is applied to the conductive polymer film, and the ion intercalation and transportation in the PEDOT:PSS layer will change the absorption spectrum of the polymer film. The attachment of the cells to the substrate will block and affect the ion doping and dedoping process, and therefore change the color of the polymer film. This process can be captured by any upright or inverted microscope with a simple camera. Utilizing this method, we have successfully imaged the impedance of single-cell attachment, observed the variations of cell-substrate interactions, and measured the impedance changes at different stages of the attachment process. This paper has proposed and successfully demonstrated a new strategy that translates the electrochemical impedance information to an optical signal that could be imaged and used to quantify the local responses. In addition, this method does not need any specially designed optical setup, which may lead to its broad applications in the clinics and biological research laboratories.

Pengaruh Penambahan Gula Terhadap Waktu Fermentasi Sauerkraut Dari Kol (Brassica Oleracea L.)

Fermentasi merupakan suatu proses pengawetan makanan, yang berperan dalam perbaikan dari kandungan nutrisi, memperpanjang masa simpan dari makanan. Sauerkraut merupakan produk hasil bioteknologi konvensional berbahan dasar irisan kol dengan penambahan garam. Sauerkraut dibuat tidak mengunakan tambahan starter kaerna memanfaatkan bakteri asam laktat yang sudah ada pada kol. Penelitian ini dilakukan pada bulan desember 2019 sampai januari 2020 di Laboratorium Penelitian Biologi FMIPA, Universitas Negeri Padang. Penelitian ini merupakan penelitian eksperimen yang disusun dalam Rancangan Acak Lengkap (RAL) dengan empat perlakuan dan lima ulangan. Perlakuannya adalah konsentrasi gula yaitu 2,5%, 5%, 7,5% dan 10%.. Parameter pengamatan utama adalah waktu fermentasi. Hasil penelitian menunjukkan bahwa penambahan gula pada proses fermentasi Sauerkraut berpengaruh terhadap waktu fermentasi.

Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities

Ionizing radiation is widely used to inactivate pathogens. It mainly acts by destroying nucleic acids but causes less damage to structural components like proteins. It is therefore highly suited for the sterilization of biological samples or the generation of inactivated vaccines. However, inactivation of viruses or bacteria requires relatively high doses and substantial amounts of radiation energy. Consequently, irradiation is restricted to shielded facilities—protecting personnel and the environment. We have previously shown that low energy electron irradiation (LEEI) has the same capacity to inactivate pathogens in liquids as current irradiation methods, but generates much less secondary X-ray radiation, which enables the use in normal laboratories by self-shielded irradiation equipment. Here, we present concepts for automated LEEI of liquids, in disposable bags or as a continuous process. As the electrons have a limited penetration depth, the liquid is transformed into a thin film. High concentrations of viruses (Influenza, Zika virus and Respiratory Syncytial Virus), bacteria (E. coli, B. cereus) and eukaryotic cells (NK-92 cell line) are efficiently inactivated by LEEI in a throughput suitable for various applications such as sterilization, vaccine manufacturing or cell therapy. Our results validate the premise that for pathogen and cell inactivation in liquids, LEEI represents a suitable and versatile irradiation method for standard biological research and production laboratories.

Polymerase chain reaction: a creative review

In molecular biology, a scientific technique PCR (polymerase chain reaction) is used to generate thousands to millions of copies of a single particular DNA sequences to amplify a single or few copies of a piece of DNA across several orders of magnitude. For multiple applications, PCR is an ordinary and often vital practice used in medicinal and biological research labs and is used for diagnosis and investigation of multiple diseases. In PCR mainly three major steps are involved. Denaturation, annealing, and extension. PCR can be used to detect not only the human genome but also the genome of viruses and bacteria. PCR is especially useful in forensic laborites because a very small amount of original DNA is required. In the development of cancer, genes have been implicated through PCR

ANTIBACTERIAL EFFECT OF 0.2% CHLORHEXIDINE AND 1% CHITOSAN MOUTHWASH ON BACTERIA DURING ORTHODONTIC MINISCREW USE

Objective: Inflammation is one of the most common complications observed when using orthodontic miniscrews. Chlorhexidine mouthwash can beused to prevent and reduce the degree of inflammation, but long-term use of this solution may lead to some side effects. This study sought to evaluatethe peri-miniscrew antibacterial effect of 1% chitosan, a biomaterial with antibacterial properties, relative to 0.2% chlorhexidine mouthwash.Methods: A randomized, double-blind clinical trial was conducted at the Dental Teaching Hospital and Oral Biology Research Laboratory at theUniversity of Indonesia from February to June 2019. Thirty subjects (25 females and five males) were randomly assigned to rinse with 1% chitosan(n=10), 0.2% chlorhexidine digluconate (n=10), and Aquadest (n=10) in addition to their usual oral hygiene procedure for 4 days. Peri-miniscrewclinical inflammation signs were recorded and peri-miniscrew plaque collected before and after 4 days of rinsing. The total bacterial and red-complexbacteria count in plaque samples were evaluated by a real-time polymerase chain reaction.Results: Chitosan and chlorhexidine showed antibacterial activity, reducing total bacterial count around orthodontic miniscrews (p<0.05). Theantibacterial activity of chitosan on total bacteria was not significantly different from that of chlorhexidine (p≥0.05). Regarding the antibacterialactivity of chitosan on red-complex bacteria, the best result seen was a 58% bacteria count reduction in Tannerella denticola.Conclusion: Chitosan has potential antibacterial activity and could be used in mouthwash to maintain peri-miniscrew hygiene.

Luciferase Enzyme and its Application

Aim: The aim of this review is to know about luciferase enzyme and its application Background : Luciferase is a generic term for the class of oxidsative enzymes that produce bioluminescence and is distinct from a photoprotein. “Firefly luciferase” as a laboratory reagent often refers to P. pyralis luciferase although recombinant luciferases from several other species of fireflies are also commercially available. Firefly luciferase is a euglobulin protein that catalyses the oxygenation of luciferin using ATP and molecular oxygen to yield oxyluciferin, a highly unstable, singlet-excited compound that emits light upon relaxation to its ground state. Luciferase can act as an ATP sensor protein through biotinylation. Bioluminescence assay systems have become increasingly used in biology and medical research laboratories in addition to (or as alternatives to) fluorescence and chemiluminescence detection strategies. Luciferase enzymes isolated from different animal species have inherent variability in light emission, allowing two or more luciferase enzymes to be used in combination for multiplex analyses, including in vivo imaging, cell viability and single and dual-spectral luciferase reporter assays. Reason: This review is made to know in detail about the Luciferase enzyme uses and its application in diagnosis