Biologically Active Micropatterns of Biomolecules and Living Matter Using Microbubble Lithography.

In situ patterning of biomolecules and living organisms while retaining their biological activity is extremely challenging, primarily because such patterning typically involves thermal stresses that could be substantially higher than the physiological thermal or stress tolerance level. Top-down patterning approaches are especially prone to these issues, while bottom-up approaches suffer from a lack of control in developing defined structures and the time required for patterning. A microbubble generated and manipulated by optical tweezers (microbubble lithography) is used to self-assemble and pattern living organisms in continuous microscopic structures in real-time, where the material thus patterned remains biologically active due to their ability to withstand elevated temperatures for short exposures. Successful patterns of microorganisms (Escherichia coli, Lactococcus. lactis and the Type A influenza virus) are demonstrated, as well as reporter proteins such as green fluorescent protein (GFP) on functionalized substrates with high signal-to-noise ratio and selectivity. Together, the data presented herein may open up fascinating possibilities in rapid in situ parallelized diagnostics of multiple pathogens and bioelectronics.

The immune-adjunctive potential of recombinant LAB vector expressing murine IFNλ3 (MuIFNλ3) against Type A Influenza Virus (IAV) infection.

BACKGROUND: The conventional means of controlling the recurring pandemics of Type A Influenza Virus (IAV) infections remain challenging primarily because of its high mutability and increasing drug resistance. As an alternative to control IAV infections, the prophylactic use of cytokines to drive immune activation of multiple antiviral host factors has been progressively recognized. Among them, Type III Interferons (IFNs) exhibit a pivotal role in inducing potent antiviral host responses by upregulating the expression of several antiviral genes, including the Interferon-Stimulated Genes (ISGs) that specifically target the virus replication machinery. To harness the immuno-adjunctive potential, we examined whether pre-treatment of IFNλ3, a Type III IFN, can activate antiviral host responses against IAV infections. METHODS: In the present study, we bioengineered a food-grade lactic acid-producing bacteria (LAB), Lactococcus lactis (L. lactis), to express and secrete functional murine IFNλ3 (MuIFNλ3) protein in the extracellular milieu. To test the immune-protective potential of MuIFNλ3 secreted by recombinant L. lactis (rL. lactis), we used murine B16F10 cells as an in vitro model while mice (BALB/c) were used for in vivo studies. RESULTS: Our study demonstrated that priming with MuIFNλ3 secreted by rL. lactis could upregulate the expression of several antiviral genes, including Interferon Regulatory Factors (IRFs) and ISGs, without exacerbated pulmonary or intestinal inflammatory responses. Moreover, we also showed that pre-treatment of B16F10 cells with MuIFNλ3 can confer marked immune protection against mice-adapted influenza virus, A/PR/8/1934 (H1N1) infection. CONCLUSION: Since the primary target for IAV infections is the upper respiratory and gastrointestinal tract, immune activation without affecting the tissue homeostasis suggests the immune-adjunctive potential of IFNλ3 against IAV infections.

Pre-treatment with chicken IL-17A secreted by bioengineered LAB vector protects chicken embryo fibroblasts against Influenza Type A Virus (IAV) infection.

The recent advances in our understanding of the host factors in orchestrating qualitatively different immune responses against influenza Type A virus (IAV) have changed the perception of conventional approaches for controlling avian influenza virus (AIV) infection in chickens. Given that infection-induced pathogenicity and replication of influenza virus largely rely on regulating host immune responses, immunoregulatory cytokine profiles often determine the disease outcomes. However, in contrast to the function of other inflammatory cytokines, interleukin-17A (IL-17A) has been described as a 'double-edged sword', indicating that in addition to antiviral host responses, IL-17A has a distinct role in promoting viral infection. Therefore, in the present study, we investigated the chicken IL-17A mediated antiviral immune effects on IAVs infection in primary chicken embryo fibroblasts cells (CEFs). To this end, we first bioengineered a food-grade Lactic Acid Producing Bacteria (LAB), Lactococcus lactis (L. lactis), secreting bioactive recombinant chicken IL-17A (sChIL-17A). Next, the functionality of sChIL-17A was confirmed by transcriptional upregulation of several genes associated with antiviral host responses, including granulocyte-monocyte colony-stimulating factor (GM-CSF) (CSF3 in the chickens), interleukin-6 (IL-6), interferon-α (IFN-α), -ß and -γ genes in primary CEFs cells. Consistent with our hypothesis that such a pro-inflammatory state may translate to immunoprotection against IAVs infection, we observed that sChIL-17A pre-treatment could significantly limit the viral replication and protect the primary CEFs cells against two heterotypic IAVs such as A/turkey/Wisconsin/1/1966(H9N2) and A/PR/8/1934(H1N1). Together, the data presented in this work suggest that exogenous application of sChIL-17A secreted by modified LAB vector may represent an alternative strategy for improving antiviral immunity against avian influenza virus infection in chickens.

One-time Electromagnetic Irradiation Modifies Stress-sensitive Gene Expressions in Rice Plant.

Electromagnetic energy is utilized over multiple frequency bands to provide seamless wireless communication services. Plants can well perceive electromagnetic energy present in open environment due to reasonably high permittivity and electrical conductivity of constituent tissues. Moreover, higher surface-to-volume ratio of plant structure facilitates increased interaction with the incident electromagnetic waves. To date, a few well-designed studies have been conducted inside controlled electromagnetic reverberation chambers to investigate either short duration-low amplitude or long duration-periodic electromagnetic irradiation-induced molecular responses in plants. However, as far as is known, studies investigating molecular responses particularly at the mid-vegetative stage in plants following one-time (hours-long) electromagnetic irradiation have not been reported earlier. Hence, the present study aimed at investigating molecular responses in 40-day-old Swarnaprabha rice plants following one-time 1837.50 MHz, 2.75 mW/m2 electromagnetic irradiation of 2 h 30 min duration. Controlled electromagnetic irradiation inside a simple reverberation chamber was ensured to achieve pure electromagnetic environment at 1837.50 MHz with deterministic electromagnetic power density at selected position. Swarnaprabha rice plant was chosen for this investigation since the rice variety is widely cultivated and consumed in the Indian subcontinent. Subsequent alterations in some selected stress-sensitive gene expressions were assayed using real-time quantitative polymerase chain reaction technique-significant upregulation in calmodulin and phytochrome B gene expressions were noted. This investigation was purposefully focused on subsequent molecular responses immediately following electromagnetic irradiation so that the possible effects of secondary stimulations could be avoided. Observed molecular responses strongly suggested that plants perceive 1837.50 MHz, 2.75 mW/m2 electromagnetic irradiation similar to other injurious stimuli. © 2021 Bioelectromagnetics Society.