Biosafety Considerations Research Articles

Electroencephalography in emerging viral infections: Lessons learned from implementing an EEG unit in a Lassa fever isolation ward in Nigeria.

Electroencephalography (EEG) has been used for almost a century in well-equipped medical centers to facilitate the diagnosis of epilepsy and other brain disorders. Lassa fever (LF) and other emerging viral infections (EVI) are known to cause neurological complications, including meningitis, seizures, and encephalopathy, though to date it remains unclear whether these are secondary to metabolic disturbances caused by the disease or by direct involvement of the central nervous system (CNS). To better characterize how Lassa virus (LASV) affects the CNS, we established an EEG diagnostic unit in the LF isolation ward at Irrua Specialist Teaching Hospital in Edo State, Nigeria. Here, we report on the specific difficulties to successful implementation of EEG in this highly challenging setting. Technical artefacts due to electrical interferences and interrupted power supply, artefacts deriving from a partly improvised EEG setup within a high consequence pathogen isolation ward, and environmental factors, such as heat in the endemic West African setting are among the main difficulties encountered when setting up this diagnostic facility. It takes experienced neurophysiologists to distinguish such artefacts from actual EEG abnormalities as many of them are not commonly encountered to this extent in well-equipped EEG laboratories and can easily be confused with pathologies. The EEG recording process is further complicated by biosafety considerations and the necessity of wearing extensive personal protective equipment. Nevertheless, with the help of experienced neurophysiologists, it is possible to correctly set up the facility and interpret recordings. Taking the above into consideration, EEG is valuable in identifying CNS involvement in emerging infections, particularly regarding assessment of encephalitis, differential diagnosis of impaired consciousness and treatment adjustment in patients with symptomatic seizures. Although highly challenging under these circumstances, EEG can be an important, noninvasive diagnostic tool for neurological complications in EVI where other more advanced imaging modalities are not available.

Influence of flaxseed mucilage on the formation, composition, and properties of exopolysaccharides produced by different strains of lactic acid bacteria

Microorganisms produce a wide variety of polysaccharides. Due to biosafety considerations, lactic acid bacteria (LAB) are popular producers of exopolysaccharides (EPS) for various applications. In this study, we analyzed the composition and properties of EPS produced by L. delbrueckii ssp. bulgaricus and LAB from clover silage (L. fermentum AG8, L. plantarum AG9) after growth on Man, Rogosa, and Sharpe broth (MRS) and with the addition of flaxseed mucilage (FSM) using chromatography, microscopy, and biochemical methods. We found that adding 0.4 % FSM does not drastically alter the medium's rheology but substantially increases EPS yield (by 3.1 to 3.8 times) and modifies the composition and macrostructure of EPS, as well as changes the spatial organization of LAB cells. The presence of FSM led to the production of xylose- and glucose-enriched EPS, which also contained varying proportions of fucose, rhamnose, arabinose, mannose, glycosamines, and uronic acids, depending on the strain. Most EPS had a low molecular weight (up to 32 kDa), except for EPS produced by L. fermentum AG8 in FSM-containing medium, which had molecular weight of 163 kDa. All EPS exhibited a porous microstructure and demonstrated scavenging capacity for OH- and DPPH-radicals, as well as high levels of α-glucosidase and lipase inhibitory activities, even at low concentrations (<1 g·L−1 of EPS). These characteristics make them promising for use in functional food production and medicine.

Nanotechnology-Driven Innovations in Malaria Treatment and Control: Current Challenges and Pharmaceutical Strategies

In the ongoing fight against malaria, an age-old disease responsible for hundreds of thousands of deaths annually, advancements in nanotechnology present new horizons for innovative interventions. This review provides a comprehensive review of the current state of nanotechnology-driven solutions in malaria treatment and control, outlining the ground-breaking opportunities they present and their challenges. Nanoparticle-based drug delivery systems have shown enhanced therapeutic efficacy, targeted delivery, and reduced side effects. Similarly, nanotechnology has paved the way for improved diagnostic tools with higher sensitivity and rapid detection capabilities. Furthermore, nano-enhanced vector control strategies have emerged, aiming to tackle malaria transmission at its source. Despite these advancements, challenges such as scale-up, biosafety, environmental concerns, and cost considerations persist. By bridging the gap between current challenges and pharmaceutical strategies, this review sheds light on the future direction of nanotechnology in malaria eradication, underscoring the potential it holds for revolutionizing the field and bringing us closer to a malaria-free world.

Gene Therapy in the Light of Lifestyle Diseases: Budesonide, Acetaminophen and Simvastatin Modulates rAAV Transduction Efficiency.

Recombinant AAV (rAAV) vectors are increasingly favored for gene therapy due to their useful features of vectorology, such as transfection of dividing and nondividing cells, the presence of tissue-specific serotypes, and biosafety considerations. This study investigates the impact of commonly used therapeutic drugs-acetaminophen, budesonide, and simvastatin-on rAAV transduction efficiency in HEK-293 cells. Cells were transduced with an AAV mosaic vector under the control of a cytomegalovirus (CMV) promoter encoding green fluorescent protein (GFP). Transduction efficiency was assessed by qPCR and fluorescent microscopy. Analysis of functional interactions between genes potentially involved in rAAV transduction in drug-exposed cells was also performed. This study showed a clear effect of drugs on rAAV transmission. Notably, acetaminophen enhanced transduction efficiency by 9-fold, while budesonide and simvastatin showed 2-fold and 3-fold increases, respectively. The gene analysis illustrates the possible involvement of genes related to cell membranes in the potentiation of rAAV transduction induced by the drugs under investigation. Attention should be paid to S100A8, which is a common drug-modified gene for drugs showing anti-inflammatory effects (budesonide and simvastatin), demonstrating an interaction with the gene encoding the receptor for AAV (HGFR). This study underscores the significance of assessing rAAV pharmacokinetics/pharmacodynamics (PKs/PDs) and drug-gene therapy interactions in optimizing gene therapy protocols.

Nanogels: Recent Advances in Synthesis and Biomedical Applications.

In the context of advanced nanomaterials research, nanogels (NGs) have recently gained broad attention for their versatility and promising biomedical applications. To date, a significant number of NGs have been developed to meet the growing demands in various fields of biomedical research. Summarizing preparation methods, physicochemical and biological properties, and recent applications of NGs may be useful to help explore new directions for their development. This article presents a comprehensive overview of the latest NG synthesis methodologies, highlighting advances in formulation with different types of hydrophilic or amphiphilic polymers. It also underlines recent biomedical applications of NGs in drug delivery and imaging, with a short section dedicated to biosafety considerations of these innovative nanomaterials. In conclusion, this article summarizes recent innovations in NG synthesis and their numerous applications, highlighting their considerable potential in the biomedical field.

A Survey of Recent Developments in Magnetic Microrobots for Micro-/Nano-Manipulation.

Magnetically actuated microrobots have become a research hotspot in recent years due to their tiny size, untethered control, and rapid response capability. Moreover, an increasing number of researchers are applying them for micro-/nano-manipulation in the biomedical field. This survey provides a comprehensive overview of the recent developments in magnetic microrobots, focusing on materials, propulsion mechanisms, design strategies, fabrication techniques, and diverse micro-/nano-manipulation applications. The exploration of magnetic materials, biosafety considerations, and propulsion methods serves as a foundation for the diverse designs discussed in this review. The paper delves into the design categories, encompassing helical, surface, ciliary, scaffold, and biohybrid microrobots, with each demonstrating unique capabilities. Furthermore, various fabrication techniques, including direct laser writing, glancing angle deposition, biotemplating synthesis, template-assisted electrochemical deposition, and magnetic self-assembly, are examined owing to their contributions to the realization of magnetic microrobots. The potential impact of magnetic microrobots across multidisciplinary domains is presented through various application areas, such as drug delivery, minimally invasive surgery, cell manipulation, and environmental remediation. This review highlights a comprehensive summary of the current challenges, hurdles to overcome, and future directions in magnetic microrobot research across different fields.

Biosafety considerations for laboratory work with SARS-CoV-2

Today, the world is affected by a COVID-19 epidemic. Early diagnosis can play an important role in the control and prevention of this disease, so the study and practice of biosafety among staff of diagnostic laboratories have a key role in the healthiness of health fighters against COVID-19. In this study, we will review laboratory biosafety considerations for the diagnosis process of SARS-CoV-2 in three phases: (1) the preanalytical phase, (2) the analytical phase, and (3) the postanalytical phase. It should be noted that employees with high-risk underlying health conditions, like lung or heart disease or diabetes, should be moved to lower-risk environments. We believe that this review will help to restrict the risk of developing COVID-19, while working in clinical laboratories.

Single-Cell RNA-Sequencing of RVFV Infection.

On the RNA level, viral infections are characterized by perturbations in the host cell transcriptome as well as the development of viral genetic information. Investigating the abundance and dynamic of RNA molecules can provide ample information to understand many aspects ofthe infection, from viral replication to pathogenesis. A key aspect therein is the resolution of the data, as infections are generally highly heterogeneous. Even in simple model systems such as cell lines, viral infections happen in a very asynchronous way. Quantifying RNAs at single-cell resolution can therefore substantially increase our understanding of these processes.Whereas measuring the RNA in bulk, that is, in samples containing thousands to hundreds of thousands of cells, is established and widely used since many years, methods for studying not only just a few different RNAs in individual cells became widely available only recently. Here, I outline and compare current concepts and methodologies for using single-cell RNA-sequencing to study virus infections. This covers sample preparation, cell preservation, biosafety considerations, and various experimental methods, with a special focus on the aspects that are important for studying virus infections. Since there is not "the one" method for doing single-cell RNA-sequencing, I will not provide a detailed protocol. Rather, this chapter should serve as a primer for getting started with single-cell RNA-sequencing experiments of virus infections and discusses the criteria that allow readers to choose the best procedures for their specific research question.

A rapid and stable spontaneous reprogramming system of Spermatogonial stem cells to Pluripotent State

BackgroundThe scarcity of pluripotent stem cells poses a major challenge to the clinical application, given ethical and biosafety considerations. While germline stem cells commit to gamete differentiation throughout life, studies demonstrated the spontaneous acquisition of pluripotency by spermatogonial stem cells (SSCs) from neonatal testes at a low frequency (1 in 1.5 × 107). Notably, this process occurs without exogenous oncogenes or chemical supplementation. However, while knockout of the p53 gene accelerates the transformation of SSCs, it also increases risk and hampers their clinical use.ResultsWe report a transformation system that efficiently and stably convert SSCs into pluripotent stem cells around 10 passages with the morphology similar to that of epiblast stem cells, which convert to embryonic stem (ES) cell-like colonies after change with ES medium. Epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and fresh mouse embryonic fibroblast feeder (MEF) are essential for transformation, and addition of 2i (CHIR99021 and PD0325901) further enhanced the pluripotency. Transcriptome analysis revealed that EGF activated the RAS signaling pathway and inhibited p38 to initiate transformation, and synergically cooperated with LIF to promote the transformation.ConclusionThis system established an efficient and safe resource of pluripotent cells from autologous germline, and provide new avenues for regenerative medicine and animal cloning.

Biosafety and biohazard considerations of HSV-1-based oncolytic viral immunotherapy.

Oncolytic viral immunotherapies are agents which can directly kill tumor cells and activate an immune response. Oncolytic viruses (OVs) range from native/unmodified viruses to genetically modified, attenuated viruses with the capacity to preferentially replicate in and kill tumors, leaving normal tissue unharmed. Talimogene laherparepvec (T-VEC) is the only OV approved for patient use in the United States; however, during the last 20years, there have been a substantial number of clinical trials using OV immunotherapies across a broad range of cancers. Like T-VEC, many OV immunotherapies in clinical development are based on the herpes simplex virus type 1 (HSV-1), with genetic modifications for tumor selectivity, safety, and immunogenicity. Despite these modifications, HSV-1 OV immunotherapies are often treated with the same biosafety guidelines as the wild-type virus, potentially leading to reduced patient access and logistical hurdles for treatment centers, including community treatment centers and small group or private practices, and healthcare workers. Despite the lack of real-world evidence documenting possible transmission to close contacts, and in the setting of shedding and biodistribution analyses for T-VEC demonstrating limited infectivity and low risk of spread to healthcare workers, barriers to treatment with OV immunotherapies remain. With comprehensive information and educational programs, our hope is that updated biosafety guidance on OV immunotherapies will reduce logistical hurdles to ensure that patients have access to these innovative and potentially life-saving medicines across treatment settings. This work reviews a comprehensive collection of data in conjunction with the opinions of the authors based on their clinical experience to provide the suggested framework and key considerations for implementing biosafety protocols for OV immunotherapies, namely T-VEC, the only approved agent to date.

Implantable microfluidics: methods and applications.

Implantable microfluidics involves integrating microfluidic functionalities into implantable devices, such as medical implants or bioelectronic devices, revolutionizing healthcare by enabling personalized and precise diagnostics, targeted drug delivery, and regeneration of targeted tissues or organs. The impact of implantable microfluidics depends heavily on advancements in both methods and applications. Despite significant progress in the past two decades, continuous advancements are still required in fluidic control and manipulation, device miniaturization and integration, biosafety considerations, as well as the development of various application scenarios to address a wide range of healthcare issues. In this review, we discuss advancements in implantable microfluidics, focusing on methods and applications. Regarding methods, we discuss progress made in fluid manipulation, device fabrication, and biosafety considerations in implantable microfluidics. In terms of applications, we review advancements in using implantable microfluidics for drug delivery, diagnostics, tissue engineering, and energy harvesting. The purpose of this review is to expand research ideas for the development of novel implantable microfluidic devices for various healthcare applications.

CONTROVERSY OVER HEALTH IMPLICATIONS OF GENETICALLY MODIFIED ORGANISMS: Which way for developing countries?

The Ghana Public Health Association organized a scientific seminar in April 2014 to examine the introduction of genetically modified organisms (GMOs) into public use, and their potential health consequences. The seminar was driven by current public debate on the subject. Concerns have been raised about use of GMOs, especially in developing countries, for a variety of reasons including ethical and biosafety considerations. The seminar identified some of their advantages such as introduction of pest resistant, disease resistant, and drought tolerant crops; potential for reduced use of chemicals and pesticides; improving the nutritional properties of crops, and huge potential for production of pharmaceuticals and vaccines. Potential health concerns raised included current efforts focusing primarily on a few crop/trait combinations of high commercial value; most trial designs are laboratory design and of short duration; increasing observation of trends in organ disease with use of GMOs, and lack of labeling of GMO products that hinder post-marketing surveillance. There is the need to enhance local capacity to research the introduction and use of GMOs; to put in place appropriate regulatory mechanisms including, particularly, the labeling of their products, and long-term post-marketing surveillance for possible negative health consequences. Furthermore, the appropriate state agency should put in place advocacy and communication strategies to keep the public informed about the health and other consequences of GMOs, for informed public choices.

Chemical tunability of advanced materials used in the fabrication of micro/nanobots.

Micro and nanobots (MNBs) are unprecedented in their ability to be chemically tuned for autonomous tasks with enhanced targeting and functionality while maintaining their mobility. A myriad of chemical modifications involving a large variety of advanced materials have been demonstrated to be effective in the design of MNBs. Furthermore, they can be controlled for their autonomous motion, and their ability to carry chemical or biological payloads. In addition, MNBs can be modified to achieve targetability with specificity for biological implications. MNBs by virtue of their chemical compositions may be limited by their biocompatibility, tissue accumulation, poor biodegradability and toxicity. This review presents a note on artificial intelligence materials (AIMs), their importance, and the dimensional scales at which intrinsic autonomy can be achieved for diverse utility. We briefly discuss the evolution of such systems with a focus on their advancements in nanomedicine. We highlight MNBs covering their contemporary traits and the emergence of a few start-ups in specific areas. Furthermore, we showcase various examples, demonstrating that chemical tunability is an attractive primary approach for designing MNBs with immense capabilities both in biology and chemistry. Finally, we cover biosafety and ethical considerations in designing MNBs in the era of artificial intelligence for varied applications.

Sustainability Considerations for Consumables Used in Microbiological and Biomedical Laboratories.

The 6th edition of the Biosafety in Microbiological and Biomedical Laboratories includes Appendix L on sustainability that describes a series of considerations for biocontainment facilities. But many biosafety practitioners may not be familiar with sustainability options that are available, feasible, and safe for laboratory settings as training in this realm is not common. With a particular focus on consumable products used in containment laboratory operations, a comparative assessment was made regarding sustainability activities employed in the healthcare setting, where significant advances have been achieved. Table 1 has been created that describes various consumables that result in waste as part of normal laboratory operations, and the biosafety and infection prevention considerations have been highlighted along with options regarding waste elimination or minimization that have been successfully employed. Even if a containment laboratory has already been designed, constructed, and is in operation, sustainability opportunities exist for the reduction of environmental impacts without compromising safety.

Discontinuation of DNA Probes for Identification of Dimorphic Fungi Growing in Culture: What's a Lab To Do?

The most common dimorphic fungi isolated from clinical specimens in North America are Coccidioides immitis, Coccidioides posadasii, Blastomyces dermatitidis species complex, and Histoplasma capsulatum. These organisms are typically definitively identified at reference or public health laboratories, as they are risk group 3 (RG3) pathogens requiring additional biosafety considerations compared to risk group 2 (RG2) pathogens. Reference and public health laboratories have been using organism-specific DNA probes since the early 1990s as the primary method of confirming the identification of morphologically suspect dimorphic fungi growing in culture. At the end of November 2021, manufacturing of these probes was discontinued, leaving clinical laboratories responsible for dimorphic fungus identification with the task of validating and implementing a new identification method for these pathogens. Here, we discuss alternatives to DNA probes for identification of Coccidioides spp., B. dermatitidis species complex, and H. capsulatum growing in culture, including the strengths and limitations of each method.

Potassium Sodium Niobate-Based Lead-Free High-Frequency Ultrasonic Transducers for Multifunctional Acoustic Tweezers

Ultrasonic transducers may need to operate in direct contact with the human body, especially with the skin or closer to blood vessels. Eco-friendly lead-free materials and devices are therefore being vigorously developed for biosafety considerations. This work presents high-performance potassium sodium niobate [(K,Na)NbO3, KNN]-based lead-free ceramics with composition-driven multiphase coexistence and their application on high-frequency ultrasonic transducers for multifunctional acoustic tweezers. A high piezoelectric constant d33 value of 332 pC/N, a good Curie temperature TC value of 348 °C, and improved in situ temperature stability were obtained in the piezoceramics via the construction multiple phases near room temperature and domain engineering. One to three piezocomposites were further fabricated based on the synthesized ceramics for higher electromechanical coupling properties. Lead-free high-frequency transducers as multifunctional acoustic tweezers for precise and selective manipulation of microparticles were designed and manufactured with a high center frequency of 23.4 MHz and a broad -6 dB bandwidth of 75.4%. Additionally, a stable transducer performance was obtained over a test temperature range of 23-60 °C, indicating good thermal stability in environments with fluctuating temperatures. Research on lead-free high-frequency transducers for ultrasound imaging and precise and selective manipulation of microparticles demonstrates their broad potential in fields such as medical therapy and diagnosis.

Biosafety Considerations for Viral Vector Gene Therapy: An Explanation and Guide for the Average Everyday-Hero Pharmacist

Purpose: An overview of the multi-faceted biosafety points that must be taken into consideration by pharmacists and pharmacies in order to provide viral vector gene therapy to their practice site. Summary: As science and medicine evolves, pharmacists and other healthcare workers are continually faced with unique challenges in the workplace. They are expected to be informed and proficient on new therapies and standards of practice, and be able to apply this knowledge appropriately for their patients. One such advancement that seems to be picking up speed in recent years is gene therapy, which is often achieved with the assistance of a viral vector. As these viral vector doses move closer to mainstream medicine, a host of issues and concerns for the pharmacists, nurses, and caregivers that are involved in the process begin to rise to the surface, often rooted in the critical concern: “How do we dispense, utilize, and administer these doses safely?” Unfortunately, there is no singular, concise source of information for addressing biosafety with viral vector products, and guidance must be gathered from a variety of resources in order to mesh together a reasonable working process. Conclusion: While this may seem to be a daunting task, facilities that already meet USP 797 and USP 800 guidelines are well on their way to being ready to provide viral vector doses. By incorporating additional steps and reviewing biosafety specific resources, these sites can easily adapt to provide these new and novel therapies for their patient population.

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as ‘direct to PCR’ assays that do not involve RNA extraction or chemical neutralisation methods.

Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins.

Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.

On-Demand Patient-Specific Phenotype-to-Genotype Ebola Virus Characterization

Biosafety, biosecurity, logistical, political, and technical considerations can delay or prevent the wide dissemination of source material containing viable virus from the geographic origin of an outbreak to laboratories involved in developing medical countermeasures (MCMs). However, once virus genome sequence information is available from clinical samples, reverse-genetics systems can be used to generate virus stocks de novo to initiate MCM development. In this study, we developed a reverse-genetics system for natural isolates of Ebola virus (EBOV) variants Makona, Tumba, and Ituri, which have been challenging to obtain. These systems were generated starting solely with in silico genome sequence information and have been used successfully to produce recombinant stocks of each of the viruses for use in MCM testing. The antiviral activity of MCMs targeting viral entry varied depending on the recombinant virus isolate used. Collectively, selecting and synthetically engineering emerging EBOV variants and demonstrating their efficacy against available MCMs will be crucial for answering pressing public health and biosecurity concerns during Ebola disease (EBOD) outbreaks.