Cyberbiosecurity in high-containment laboratories.

High-containment laboratories (HCLs) conduct critical research on infectious diseases, provide diagnostic services, and produce vaccines for the world's most dangerous pathogens, often called high-consequence pathogens (HCPs). The modernization of HCLs has led to an increasingly cyber-connected laboratory infrastructure. The unique cyberphysical elements of these laboratories and the critical data they generate pose cybersecurity concerns specific to these laboratories. Cyberbiosecurity, the discipline devoted to the study of cybersecurity risks in conjunction with biological risks, is a relatively new field for which few approaches have been developed to identify, assess, and mitigate cyber risks in biological research and diagnostic environments. This study provides a novel approach for cybersecurity risk assessment and identification of risk mitigation measures by applying an asset-impact analysis to the unique environment of HCLs. First, we identified the common cyber and cyberphysical systems in HCLs, summarizing the typical cyber-workflow. We then analyzed the potential adverse outcomes arising from a compromise of these cyber and cyberphysical systems, broadly categorizing potential consequences as relevant to scientific advancement, public health, worker safety, security, and the financial wellbeing of these laboratories. Finally, we discussed potential risk mitigation strategies, leaning heavily on the cybersecurity materials produced by the Center for Internet Security (CIS), including the CIS Controls®, that can serve as a guide for HCL operators to begin the process of implementing risk mitigation measures to reduce their cyberbiorisk and considering the integration of cyber risk management into existing biorisk management practices. This paper provides a discussion to raise awareness among laboratory decision-makers of these critical risks to safety and security within HCLs. Furthermore, this paper can serve as a guide for evaluating cyberbiorisks specific to a laboratory by identifying cyber-connected assets and the impacts associated with a compromise of those assets.

Host population structure for tolerance determines the evolution of plant-virus interactions.

Heterogeneity for plant defences determines both the capacity of host populations to buffer the effect of infection and the pathogen´s fitness. However, little information is known on how host population structure for tolerance, a major plant defence, impacts the evolution of plant-pathogen interactions. By performing 10 serial passages of Turnip mosaic virus (TuMV) in Arabidopsis thaliana populations with varying proportion of tolerant genotypes simulating different structures for this trait, we analysed how host heterogeneity for this defence shapes the evolution of both virus multiplication, the effect of infection on plant fecundity and mortality, and plant tolerance and resistance. Results indicated that a higher proportion of tolerant genotypes in the host population promotes virus multiplication and reduces the effect of infection on plant mortality, but not on plant fecundity. These changes resulted in more effective plant tolerance to virus infection. Conversely, a lower proportion of tolerant genotypes reduced virus multiplication, boosting plant resistance. Our work for the first time provides evidence of the main role of host population structure for tolerance on pathogen evolution and on the subsequent feedback loops on plant defences.

Exploring Goal Conflicts and How They Are Managed in a Biomedical Laboratory Using Rasmussen's Model of Boundaries.

Introduction: Occupational health and safety management systems are widely used as a systematic approach to managing occupational health and safety. However, sometimes they are restrictive and underspecified to deal with dynamic workplace demands. Rasmussen used a model of boundaries to conceptualize this dynamic model of safety, where the space of possibilities lay within 3 boundaries and workers used various means to stay within the boundaries to remain both productive and safe at work. Methods: This study applied the Rasmussen model of boundaries to understand the factors that formed the boundaries, the gradients, and countergradients in a biomedical laboratory. Results: The most central goal was to be the first to publish, and this formed the boundary to scientific output failure; the boundary to unacceptable workload and boundary to functionally acceptable performance were the other 2 boundaries in line with the Rasmussen model. The workers had developed methods (mental risk assessment, teamwork, and experience and familiarity) of working, which ensured they remained productive and safe. This can be described as resilient performance, where resilience is not something that a system has but something it does to adjust their performance when faced with expected or unexpected changes. Discussion and Conclusion: A customized portfolio of rule-based non negotiable instructions and a risk assessment-based approach would be best suited for a biomedical laboratory. The workers have learned resilient performance on their own and unknowingly are already practicing this. It is now time to formally incorporate such practices into the safety systems of biomedical laboratories.

Trade-offs between host tolerances to different pathogens in plant-virus interactions.

Although accumulating evidence indicates that tolerance is a plant defence strategy against pathogens as widespread as resistance, how plants evolve tolerance is poorly understood. Theory predicts that hosts will evolve to maximize tolerance or resistance, but not both. Remarkably, most experimental works failed in finding this trade-off. We tested the hypothesis that the evolution of tolerance to one virus is traded-off against tolerance to others, rather than against resistance and identified the associated mechanisms. To do so, we challenged eighteen Arabidopsis thaliana genotypes with Turnip mosaic virus (TuMV) and Cucumber mosaic virus (CMV). We characterized plant life-history trait modifications associated with reduced effects of TuMV and CMV on plant seed production (fecundity tolerance) and life period (mortality tolerance), both measured as a norm of reaction across viral loads (range tolerance). Also, we analysed resistance-tolerance and tolerance-tolerance trade-offs. Results indicate that tolerance to TuMV is associated with changes in the length of the pre-reproductive and reproductive periods, and tolerance to CMV with resource reallocation from growth to reproduction; and that tolerance to TuMV is traded-off against tolerance to CMV in a virulence-dependent manner. Thus, this work provides novel insights on the mechanisms of plant tolerance and highlights the importance of considering the combined effect of different pathogens to understand how plant defences evolve.

COVID-19 and Beyond: Safety and Design Considerations for the Development of a Mobile Biocontainment Laboratory.

Introduction: An infectious disease outbreak like the current COVID-19 pandemic can lead to particularly high infection rates in areas where diagnostic laboratory support is limited. The deployment of mobile laboratories can help to detect pathogens, monitor the presence in a population, and inform public health authorities to take measures aimed at reducing pathogen spread. Materials and Methods: Available layouts and operational descriptions of mobile laboratories were analyzed for their suitability for the envisioned purpose and to assure high standards of biosafety and biosecurity. Recent media coverage on creative solutions for the diagnostics of SARS-CoV-2 (drive-through test centers, self-swab, inverse gloveboxes to protect health care workers) from various countries were considered. Results: A minimalistic and optimized design to construct a multifunctional laboratory on the chassis of a regular-sized box truck is proposed and can serve as a blueprint to rapidly develop additional diagnostic capacities. Discussion: For acute health threats including the current COVID-19 outbreak, rapid diagnosis of infection is key to recommend measures aimed at preventing the spread of the pathogen. Laboratory layouts that are similar to the one proposed here are used in stationary setups, and mobile laboratories have been built on varying platforms (trailers, shipping containers, etc).

Chloroquine: A Potential Drug in the COVID-19 Scenario.

Today, the whole world is fighting a public health emergency called 'COVID-19' caused by a new infectious virus called SARS-CoV2. Any person can catch COVID-19 from an infected person via aerosol droplets when the person coughs, sneezes, or speaks. To limit such a transmission, World Health Organization (WHO) has recommended people to wear masks and physically distance themselves by staying at least 1 m (3 feet) away from others. As aerosol droplets (by cough or sneeze) land on objects and surfaces around the person such as tables, doorknobs and handrails, and remain active on these surfaces for hours to days, people are advised to use soaps for at least 20 s. and alcohol-based sanitizers as well. As the public made efforts, clinicians and researchers investigated and found that drugs which were initially used to treat other diseases may work as a treatment option for COVID-19. One of those drugs was Chloroquine and its related derivative called hydroxychloroquine. In this review article, we have systematically searched for details of COVID-19 pandemic till May 2020 and assembled few data pertaining to (i) Corona viruses; (ii) SARS-CoV2, the virus that causes COVID-19' and (iii) How chloroquine and hydroxychloroquine mediates anti-viral effect in both prophylactic and therapeutic setting. These data have been acquired mostly from PubMed and websites of WHO and Indian Council for Medical Research (ICMR). We did a systematic search and found that the properties of chloroquine are very much essential for the COVID-19 scenario. We also bring to you some evidence that the anti-lysosomal activity of chloroquine may be increased by botanicals like betulinic acid.

Development and characterization of supramolecular calcitonin assembly and assessment of its interactions with the bone remodelling process.

Osteoporosis is the most common metabolic bone disease, which poses an immense socio-economic burden on the society. Human calcitonin, though safe, is not considered as a therapeutic option because of its high tendency to self-associate to form amyloid fibrils thereby affecting its potency. To circumvent this issue we harnessed the inherent capacity of aggregation and developed an assemblage of human calcitonin monomers, [Supramolecular Calcitonin Assembly (SCAI)], which releases biologically active calcitonin monomers in a sustained manner for a period of at least three weeks. AFM and FT-IR analysis showed that SCA-I is amorphous aggregates of calcitonin monomers. Both SCA-I and monomer released from it demonstrated superior anti-osteoclast activity and proteolytic stability in-vitro. SCA-I upon single injection significantly improved bone formation markers and reduced bone resorption markers in ovariectomized (OVX) rat model of postmenopausal osteoporosis. Micro-CT analysis revealed that calcitonin released from SCA-I exhibits its beneficial effect on cortical bone more profoundly compared to trabecular bone. This study demonstrates that SCA-I is more effective compared to the human calcitonin monomers on osteoclasts and has site-specific effect on bone in a model of post-menopausal osteoporosis. This approach opens up an innovative way to use and study the function of human calcitonin.

Modified Efficient and Simple Method for Biological Spill Cleanup.

Introduction: Spill response procedures are an important aspect of laboratories where infectious materials are handled. The decades-old conventional method of spill cleanup uses paper towels to cover the spill. It requires 2 staff and involves a considerable amount of bending and squatting and being able to balance in those positions while wearing personal protective equipment (PPE). In this article, we describe a method that simplifies spill cleanup and compares it to the conventional method. The simplified method can be easily conducted by 1 person, takes about half the time, generates less waste, and reduces the amount of time spent in contaminated areas. Objective: The objective is to describe a modified, simple method of spill cleanup. Methods: A mock spill was created and a spill response initiated per the institution's procedure. The simplified procedure uses a pail filled with decontaminant and a household mop dripping with the decontaminant. Mopping is done from the noncontaminated area toward the contaminated area so the spill does not spread. Mopping is done more than once, and all the materials used, including the mop(s), are disposed as biohazardous material. Results: The simplified spill cleanup process described here can be performed by just one person and does not require bending and squatting while wearing PPE. The kit is very simple, consisting of a pail and a mop, which are common household tools familiar to most people. Conclusion: The mop-and-pail methodology is simple, requires only one staff member, generates less biological waste, and requires less training and practice while effectively cleaning the spill.

Virulence evolution of a sterilizing plant virus: Tuning multiplication and resource exploitation.

Virulence evolution may have far-reaching consequences for virus epidemiology and emergence, and virologists have devoted increasing effort to understand the modulators of this process. However, still little is known on the mechanisms and determinants of virulence evolution in sterilizing viruses that, as they prevent host reproduction, may have devastating effects on host populations. Theory predicts that sterilizing parasites, including viruses, would evolve towards lower virulence and absolute host sterilization to optimize the exploitation of host resources and maximize fitness. However, this hypothesis has seldom been analyzed experimentally. We investigated the evolution of virulence of the sterilizing plant virus Turnip mosaic virus (TuMV) in its natural host Arabidopsis thaliana by serial passage experiments. After passaging, we quantified virus accumulation and infectivity, the effect of infection on plant growth and development, and virulence of the ancestral and passaged viral genotypes in A. thaliana. Results indicated that serial passaging increased the proportion of infected plants showing absolute sterility, reduced TuMV virulence, and increased virus multiplication and infectivity. Genomic comparison of the ancestral and passaged TuMV genotypes identified significant mutation clustering in the P1, P3, and 6K2 proteins, suggesting a role of these viral proteins in the observed phenotypic changes. Our results support theoretical predictions on the evolution of virulence of sterilizing parasites and contribute to better understand the phenotypic and genetic changes associated with this process.

Bone morphogenetic protein-5, a key molecule that mediates differentiation in MC3T3E1 osteoblast cell line.

Bone morphogenetic protein-5 (BMP-5) is a member of the TGF receptor-ß family with osteoinductive property. However, its physiological role in osteoblast differentiation is not defined. This study highlights the importance of BMP-5 in MC3T3E1 osteoblast differentiation. Pre-osteoblasts exposed to osteogenic media (ascorbic acid, 50 µg/ml and ß-glycerophosphate, 10 mM) showed high protein expression of BMP-5 in cell lysates and cell culture supernatants, which peaked during early time-points of differentiation and declined with onset of mineralization. Attenuation of endogenous BMP-5 protein expression by RNA interference downregulated the expression of type I collagen (COLIA1), an early osteoblast differentiation marker but not osteocalcin, a late osteoblast differentiation marker. Further experiments to analyze the cell signaling components revealed that BMP-5 modulates COLIA1 expression via p38-Runx2 axis involving Runx2 (Ser19) phosphorylation. These effects were also observed when recombinant BMP-5 was added to pre-osteoblast cultures reinforcing the fact that BMP-5 is a modulator of COLIA1 expression. We conclude that BMP-5 has stage-specific role to play during MC3T3E1 osteoblast differentiation in part by autocrine p38/Runx2/COLIA1 signaling. © 2017 BioFactors, 43(4):558-566, 2017.

Role of osteocytes in mediating bone mineralization during hyperhomocysteinemia.

Hyperhomocysteinemia (HHCY) is a risk factor for osteoporosis but whether HHCY affects bone mineralization or not is still ambiguous. Herein we evaluated whether homocysteine affects tissue mineral density (TMD) of cortical bone and if so the role of osteocytes. CD1 mice administered with homocysteine (5 mg/100 g body weight, i.p.) for 7, 15 and 30 days showed temporal changes in TMD and osteocyte lacunar density in femoral cortices. Short-term administration of homocysteine (day 7) increased osteocyte lacunar density and reduced TMD evidenced by microCT50 while prolonged administration of homocysteine (day 30) reinstated TMD and lacunar density to baseline values. Major differences were decreased number of nucleated osteocyte lacunae, increased number of empty lacunae and cleaved caspase 3-positive osteocyte lacunae in day 30 HHCY bone evidenced by H&E staining and immunohistochemistry. Other differences were induction in mineralization genes like Dmp1, Phex and Sost in cortical bone by real-time PCR and increased number of Dmp1- and Sost-positive osteocyte lacunae in day 30 HHCY bone evidenced by immunohistochemistry. Both HHCY day 7 and day 30 samples showed reduced Young's modulus demonstrating that biomechanical property of bone was lost during early HHCY itself, which did not improve with recovery of TMD. Our results thus demonstrate occurrence of two phases in cortical bone upon HHCY: the early phase that involved loss of TMD and increase in osteocyte numbers and a late phase that involved osteocyte reprogramming, apoptosis and mineralization, which reinstated TMD but compromised biomechanical property. To conclude, osteocytes have a potential role in arbitrating bone pathogenesis during HHCY.

Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1.

In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1α, IL-1ß, G-CSF, GM-CSF, MIP-1α, IFN-γ, IL-17, and TNF-α. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality.

Anti-inflammatory potential of an ethyl acetate fraction isolated from Justicia gendarussa roots through inhibition of iNOS and COX-2 expression via NF-κB pathway.

Justicia gendarussa Burm.f. (J. gendarussa) is a plant used as traditional medicine in different parts of India and China to treat inflammatory disorders like rheumatoid arthritis. But its mechanism of anti-inflammatory action is still unclear. Hence in this context, the objective of our study is to reveal the mechanism of anti-inflammatory activity of J. gendarussa which would form an additional proof to the traditional knowledge of this plant. The anti-inflammatory function and mechanism(s) of action was studied in an ethyl acetate fraction isolated from methanolic extract of J. gendarussa roots (EJG). Anti-inflammatory studies were conducted on rats using partitioned fractions isolated from methanolic extract of J. gendarussa roots. In carrageenan-induced rat paw edema, ethyl acetate fraction brought about 80% and 93% edema inhibition at 3rd and 5th hour at a dose of 50 mg/kg, when compared to other extracts and Voveran. We investigated whether EJG inhibits the release of cycloxygenase (COX), 5-lipoxygenase (5-LOX), interleukin-6 (IL-6) and nuclear factor kappa B (NF-κB) in LPS stimulated human peripheral blood mononuclear cells (hPBMCs). Results shows that EJG dose dependently inhibited LPS-activated COX, 5-LOX, IL-6, and NF-κB in hPBMCs. EJG also reduced LPS induced levels of iNOS and COX-2 mRNA expression in hPBMCs. This study provides an insight into the probable mechanism(s) underlying the anti-inflammatory activity of EJG and therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.

Efficacy of Bacopa monniera (L.) Wettst in alleviating lysosomal instability in adjuvant-induced arthritis in rats.

The present study was aimed to assess the effect of Bacopa monniera extract against lysosomal instability during adjuvant-induced arthritis in rats. B. monniera extract was administered orally at a dose of 100 mg/kg body weight for 30 days to the experimental rats after the induction of adjuvant arthritis. The ability of B. monniera extract to stabilize lysosomal enzyme activities in the cartilage of control and experimental rats was done by monitoring the activities of pathophysiological enzymes such as ß-glucuronidase, ß-glucosaminidase, cathepsin D, hyaluronidase, collagenase and the level of protein bound carbohydrates and glycosaminoglycans (GAGs) in arthritic rats. B. monniera extract supplementation significantly inhibited lysosomal instability in different tissues studies and improved the level of glycoproteins in synovial effusate and GAG in the cartilage. To evaluate whether anti-inflammatory property of B. monniera extract was due to lysosomal stability and GAG protection, purified chloroform fraction of B. monniera (CF) was assayed for its role to modulate interleukin-6 (IL-6) expression and prostaglandin E2 (PGE2) production. Administration of CF (50 mg/kg) to arthritic rats significantly downregulated the expression of IL-6 in blood mononuclear cells and PGE2 levels in cartilage tissue. The possible mechanism of action of the B. monniera extract may be through its stabilizing action on lysosomal membranes and hence the decrease in spread of inflammation.

Protective activity of Bacopa monniera Linn. on nicotine-induced toxicity in mice.

Nicotine, a pharmacologically active component of cigarettes smoke causes devastating effects in important biomolecules of the cell through generation of free radicals leading to genomic instability. Bacopa monniera is a reputed drug in Ayurveda known for its hepatoprotective and DNA protective effects. In this study, an aqueous extract of Bacopa monniera (BAE, 50 mg/kg i.p.) was investigated for its ability to reduce nicotine-induced lipid peroxidation (LPO) and confer genoprotection in Swiss mice. Genoprotective effect was assayed using micronucleus (MN) assay. LPO status was studied by evaluating MDA levels and antioxidant status. Nicotine altered hepatic function as evident by increased ALP and GST levels and decreased SOD, catalase and GPx activities. BAE treatment restored antioxidant enzymes such as SOD, catalase and GPx in liver. BAE treatment also significantly reduced the frequency of micronuclei induced by nicotine by decreasing the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Hepatic GSH, ALP and GST levels were brought to normal values indicating protection. The results of the present study suggest that BAE exerts protective effects by modulating the extent of lipid peroxidation and enhancing the antioxidant status.