Interactions between human norovirus and intestinal microbiota/microbes: A scoping review.

Human norovirus (HuNoV) is an important foodborne virus, which causes non-bacterial acute gastroenteritis and is associated with a high disease burden. Recently, researchers have focus on the interaction between HuNoV and intestinal microbiota/microbes and engaged in studies investigating the implications of this interaction on HuNoV infection. However, the interaction mechanism and the implication of this interaction on host remain obscure. Current scoping review aimed to systematically investigate the interaction between HuNoV and intestinal microbiota, as well as their implication on HuNoV or HuNoV related symptoms. We found that HuNoV could bind to intestinal microbes and affect the intestinal microbial composition, diversity, and microbial gene expression. In reverse, intestinal microbes could affect HuNoV infectivity, although demonstrating contradictory effects (i.e., promote or inhibit HuNoV replication). These contradictory effects existed among microbes, in part, could be attributed to the differences among microbes (histo-blood group antigens and/or other small molecule substances). Results of current scoping review could assist in the selection and isolation of potential microbial candidates to prevent and/or alleviate HuNoV related symptoms.

Bioaccumulation Pattern of the SARS-CoV-2 Spike Proteins in Pacific Oyster Tissues.

There is mounting evidence of the contamination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the sewage, surface water, and even marine environment. Various studies have confirmed that bivalve mollusks can bioaccumulate SARS-CoV-2 RNA to detectable levels. However, these results do not provide sufficient evidence for the presence of infectious viral particles. To verify whether oysters can bind the viral capsid and bioaccumulate the viral particles, Pacific oysters were artificially contaminated with the recombinant SARS-CoV-2 spike protein S1 subunit (rS1). The bioaccumulation pattern of the rS1 in different tissues was investigated by immunohistological assays. The results revealed that the rS1 was bioaccumulated predominately in the digestive diverticula. The rS1 was also present in the epithelium of the nondigestive tract tissues, including the gills, mantle, and heart. In addition, three potential binding ligands, including angiotensin-converting enzyme 2 (ACE 2)-like substances, A-type histo-blood group antigen (HBGA)-like substances, and oyster heat shock protein 70 (oHSP 70), were confirmed to bind rS1 and were distributed in tissues with various patterns. The colocalization analysis of rS1 and those potential ligands indicated that the distributions of rS1 are highly consistent with those of ACE 2-like substances and oHSP 70. Both ligands are distributed predominantly in the secretory absorptive cells of the digestive diverticula and may serve as the primary ligands to bind rS1. Therefore, oysters are capable of bioaccumulating the SARS-CoV-2 capsid readily by filter-feeding behavior assisted by specific binding ligands, especially in digestive diverticula. IMPORTANCE This is the first article to investigate the SARS-CoV-2 spike protein bioaccumulation pattern and mechanism in Pacific oysters by the histochemical method. Oysters can bioaccumulate SARS-CoV-2 capsid readily by filter-feeding behavior assisted by specific binding ligands. The new possible foodborne transmission route may change the epidemic prevention strategies and reveal some outbreaks that current conventional epidemic transmission routes cannot explain. This original and interdisciplinary paper advances a mechanistic understanding of the bioaccumulation of SARS-CoV-2 in oysters inhabiting contaminated surface water.

Isochoric supercooling cryomicroscopy.

We introduce an isochoric (constant-volume) supercooling cryomicroscope (ISCM), enabling the ice-free study of biological systems and biochemical reactions at subzero temperatures at atmospheric pressure absent ice. This technology draws from thermodynamic findings on the behavior of water in isochoric systems at subfreezing temperatures. A description of the design of the ISCM and a demonstration of the stability of the supercooled solution in the ISCM is followed by an illustration of the possible use of the ISCM in the preservation of biological matter research. A comparison was made between the survival of HeLa cells in the University of Wisconsin (UW) solution in the ISCM at +4 °C under conventional atmospheric conditions and at -5 °C under isochoric supercooled conditions. Continuous real-time monitoring at cryopreservation temperature via fluorescence microscopy showed that after three days of isochoric supercooling storage, the percentage of compromised cells remained similar to fresh controls, while storage at +4 °C yielded approximately three times the mortality rate of cells preserved at -5 °C.

Broad-range and effective detection of human noroviruses by colloidal gold immunochromatographic assay based on the shell domain of the major capsid protein.

BACKGROUND: Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all age groups worldwide. HuNoVs can be detected in vitro using molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced personnel, and extended time to obtain results. Besides, the diversity of viral genotypes is high. Therefore, methods that are rapid, broad-range and effective in the detection of HuNoVs are desiderated for screening the feces or vomit of infected people during outbreaks. RESULTS: In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for effective detection of HuNoVs in clinical samples. Monoclonal antibodies (MAbs) against the shell (S) domain in the major capsid protein of HuNoVs were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2 × 106 genomic copies per gram of stool sample (gc/g) and 4.4 × 105 gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 clinical samples were tested for HuNoVs by ICA and compared against RT-qPCR. The relative sensitivity, specificity and agreement of ICA was 84.2% (95% CI: 83.6-84.8%), 100.0% (95% CI: 98.5-100.0%) and 87.7% (95% CI: 85.6-89.8%), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA detected a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs. CONCLUSIONS: This study demonstrates that ICA targeting the S domain of VP1 is a promising candidate for effectively identifying the different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.

Normal and fibrotic liver parenchyma respond differently to irreversible electroporation.

BACKGROUND: The safety and efficacy of irreversible electroporation (IRE) in treating hepatic, biliary, and pancreatic malignancies are active areas of clinical investigation. In addition, recent studies have shown that IRE may enable regenerative surgery and in vivo tissue engineering. To use IRE effectively in these clinical applications, it is important to understand how different tissue microenvironments impact the response to IRE. In this study, we characterize the electrical and histological properties of non-fibrotic and fibrotic liver parenchyma before and after IRE treatment. METHODS: Electrical resistivity and histology of fibrotic liver from C57BL/6 mice fed a 0.1% 3,5-diethylcarbonyl-1,4-dihydrocollidine (DDC) diet were compared to those of non-fibrotic liver from matched control mice before and after IRE treatment. RESULTS: At baseline, the electrical resistivity of fibrotic liver was lower than that of non-fibrotic liver. Post-IRE, resistivity of non-fibrotic liver declined and then recovered back to baseline with time, correlating with hepatocyte repopulation of the ablated parenchyma without deposition of fibrotic scar. In contrast, resistivity of fibrotic liver remained depressed after IRE treatment, correlating with persistent inflammation. CONCLUSION: Non-fibrotic and fibrotic liver respond to IRE differently. The underlying tissue microenvironment is an important modifying factor to consider when designing IRE protocols for tissue ablation.

Molecular and histological study on the effects of non-thermal irreversible electroporation on the liver.

Non-thermal irreversible electroporation (NTIRE) is a biophysical phenomenon in which certain electric fields delivered across the cell membrane in tissue, cause cell death, without affecting the extracellular matrix. "Minimally invasive regenerative surgery" is a new medical modality for treatment of end-stage organ or tissue failure in which exogenous cells are implanted in a decellularized niche in tissue, formed by the delivery of NTIRE electric fields across a targeted volume of tissue. We anticipate that the success of the procedure will depend on the time of implantation relative to the application of NTIRE. This study was performed to elucidate the histological and molecular events that occur within 24 h after NTIRE, in the context of optimal criteria for the time of implantation. To this end, we examined the histology of NTIRE treated rat liver with H&E, Masson trichrome and TUNEL staining. Western blot was used to examine pro and cleaved caspase-3 (marker for apoptosis), pro and cleaved caspase-1 and gasdermin D (markers for pyroptosis), and RIP3 and MLKL (markers for necroptosis). The key findings are that, complete hepatocytes disintegration within an intact extracellular matrix is seen at 6 h and, new hepatocytes are seen in the treated region at 24 h, after NTIRE. There is no evidence of apoptotic cell death from NTIRE, contrary to commonly made claims in the NTIRE literature. However, molecular pathways of pyroptosis and necroptosis, programed necrosis associated with inflammation, are activated at 6 h after NTIRE and are not evident at 24 h after NTIRE. These are fundamental new findings of basic value to the field of NTIRE in all its applications. Taken together the results suggest the hypothesis that an optimal time for implantation is about 24 h after NTIRE. Future studies in which exogenous cells are implanted at different times after NTIRE are required to examine this hypothesis.

Escherichia coli viability in an isochoric system at subfreezing temperatures.

In comparison with isobaric (constant pressure) freezing, isochoric (constant volume) freezing reduces potential mechanical damage from ice crystals and exposes stored biological matter to a lower extracellular concentration, at the price of increased hydrostatic pressure. This study evaluates the effects of isochoric freezing to low temperatures and high pressures on Escherichia coli (E. coli) survival. The viability of E. coli was examined after freezing to final temperatures between -5 °C and -20 °C for periods from 0.5 h to 12 h, with recovery periods from 0 h to 24 h. Freezing for up to two hours to -10 °C and -15 °C had little effect on the percentage of viable E. coli, relative to the controls. However, after two hours of exposure at -20 °C, when left to recover for 24 h, a 75% reduction in survival is observed. Furthermore, after 12 h of isochoric freezing at -15 °C and -20 °C, E. coli population is reduced by 2.5 logs while freezing to these temperatures in conventional isobaric atmospheric conditions reduces population by only one log. This suggests that the combination of low temperature and high pressure experienced during isochoric freezing close to the triple point may be more detrimental to biological matter survival than the combination of elevated concentration, low temperature, and ice crystallization experienced during conventional freezing, and that this effect may be related to the time of exposure to these conditions.

Isochoric and isobaric freezing of fish muscle.

We have recently shown that, a living organism, which succumbs to freezing to -4 °C in an isobaric thermodynamic system (constant atmospheric pressure), can survive freezing to -4 °C in an isochoric thermodynamic system (constant volume). It is known that the mechanism of cell damage in an isobaric system is the freezing caused increase in extracellular osmolality, and, the consequent cell dehydration. An explanation for the observed survival during isochoric freezing is the thermodynamic modeling supported hypothesis that, in the isochoric frozen solution the extracellular osmolality is comparable to the cell intracellular osmolality. Therefore, cells in the isochoric frozen organism do not dehydrate, and the tissue maintains its morphological integrity. Comparing the histology of: a) fresh fish white muscle, b) fresh muscle frozen to -5 °C in an isobaric system and c) fresh muscle frozen to -5 °C I in an isochoric system, we find convincing evidence of the mechanism of cell dehydration during isobaric freezing. In contrast, the muscle tissue frozen to -5 °C in an isochoric system appears morphologically identical to fresh tissue, with no evidence of dehydration. This is the first experimental evidence in support of the hypothesis that in isochoric freezing there is no cellular dehydration and therefore the morphology of the frozen tissue remains intact.

Non-electrolytic microelectroporation.

Micro and nano technologies are of increasing importance in microfluidics devices used for electroporation (electroporation - the permeabilization of the cell membrane with brief high electric field pulses). Electrochemical reactions of electrolysis occur whenever an electric current flows between an electrode and an ionic solution. It can have substantial detrimental effects, both on the cells and solutions during the electroporation. As electrolysis is a surface phenomenon, between electrodes and solution, the extent of electrolysis is increased in micro and nano electroporation over macro-electroporation, because the surface area of the electrodes in micro and nano electroporation is much larger. A possible way to eliminate the electrolytic effect is to develop non-electrolytic microelectroporation by coating the microelectroporation devices with a dielectric insulating layer. In this study, we examine the effect of a dielectric insulating layer on the performance of a singularity microelectroporation device that we have recently designed. Using numerical analysis, we study the effects of various design parameters including, input sinusoidal voltage amplitude and frequency, geometrical configuration and material electrical properties on the electroporation performance of the non-electrolytic microelectroporation device. In the simulation, we used properties of four real dielectric materials and four solutions of interest for microelectroporation. We characterized the effect of various design parameters of relevance to singularity based microelectroporation, on non-electrolytic microelectroporation. Interestingly, we found that the system behaves in some aspects as a filter and in many circumstances saturation of performance is reached. After saturation is reached, changes in parameters will not affect the performance of the device.

Interaction Between SARS-CoV-2 Spike Protein S1 Subunit and Oyster Heat Shock Protein 70.

There is growing evidence that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contaminates the marine environment and is bioaccumulated in filter-feeding shellfish. Previous study shows the Pacific oyster tissues can bioaccumulate the SARS-CoV-2, and the oyster heat shock protein 70 (oHSP70) may play as the primary attachment receptor to bind SARS-CoV-2's recombinant spike protein S1 subunit (rS1). However, detailed information about the interaction between rS1 and oHSP70 is still unknown. In this study, we confirmed that the affinity of recombinant oHSP70 (roHSP70) for rS1 (KD = 20.4 nM) is comparable to the receptor-binding affinity of rACE2 for rS1 (KD = 16.7 nM) by surface plasmon resonance (SPR)-based Biacore and further validated by enzyme-linked immunosorbent assay (ELISA). Three truncated proteins (roHSP70-N/C/M) and five mutated proteins (p.I229del, p.D457del, p.V491_K495del, p.K556I, and p.ΣroHSP70) were constructed according to the molecular docking results. All three truncated proteins have significantly lower affinity for rS1 than the full-length roHSP70, indicating that all three segments of roHSP70 are involved in binding to rS1. Further, the results of SPR and ELISA showed that all five mutant proteins had significantly lower affinity for rS1 than roHSP70, suggesting that amino acids at these sites are involved in binding to rS1. This study provides a preliminary theoretical basis for the bioaccumulation of SARS-CoV-2 in oyster tissues or using roHSP70 as the capture unit to selectively enrich virus particles for detection.

Inulin mitigated antibiotic-induced intestinal microbiota dysbiosis - a comparison of different supplementation stages.

Antibiotics are unavoidable to be prescribed to subjects due to different reasons, and they decrease the relative abundance of beneficial microbes. Inulin, a fructan type of polysaccharide carbohydrate, on the contrary, could promote the growth of beneficial microbes. In this study, we investigated the effect of inulin on antibiotic-induced intestinal microbiota dysbiosis and compared their overall impact at different supplementation stages, i.e., post-antibiotic, at the time of antibiotic administration or prior to antibiotic treatment, in the C57BL/6 mice model. Although supplementation of inulin after antibiotic treatment could aid in the reconstruction of the intestinal microbial community its overall impact was limited and no remarkable differences were identified as compared to the spontaneous restoration. On the contrary, the effect of simultaneous and pre-supplementation was more remarkable. Simultaneous inulin supplementation significantly mitigated the antibiotic-induced dysbiosis based on alterations as evaluated using weighted and unweighted UniFrac distance between baseline and after treatment. Moreover, comparing the effect of simultaneous supplementation, pre-supplemented inulin further mitigated the antibiotic-induced dysbiosis, especially on the relative abundance of dominant microbes. Collectively, the current study found that the use of inulin could alleviate antibiotic-induced microbiota dysbiosis, and the best supplementation stage (overall effect as evaluated by beta diversity distance changes) was before the antibiotic treatment, then simultaneous supplementation and supplementation after the antibiotic treatment.

Fully Integrated Microfluidic Platform for Multiplexed Detection of Hunov by a Dynamic Confined-Space-Implemented One-Pot Rpa-Lamp System.

Human norovirus (HuNoV) is the leading cause of nonbacterial acute gastroenteritis, which is highly infectious, rapidly evolving, and easily transmitted through feces. The accurate and early detection of HuNoV subtypes is essential for effective treatment, early surveillance, risk assessment, and disease prevention. In this study, a portable multiplex HuNoV detection platform that combines integrated microfluidics and cascade isothermal amplification, using a streamlined protocol for clinical fecal-based diagnosis is presented. To overcome the problems of carryover contamination and the incompatibility between recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), a Dynamic confined-space-implemented One-pot RPA-LAMP colorimetric detection system (DORLA) is developed by creating a hydrogen bond network. The DORLA system exhibits excellent sensitivity, with detection limits of 10 copies µL-1 and 1 copy µL-1 for HuNoV GI and GII, respectively. In addition, a portable diagnostic platform consisting of a thermostatic control module and an integrated 3D-printed microfluidic chip for specific HuNoV capture, nucleic acid pretreatment, and DORLA detection, which enables simultaneous diagnosis of HuNoV GI and GII is developed. A DORLA-based microfluidic platform exhibits satisfactory performance with high sensitivity and portability, and has high potential for the rapid point-of-care detection of HuNoV in clinical fecal samples, particularly in resource-limited settings.

Identification of Potential Proteinaceous Ligands of GI.1 Norovirus in Pacific Oyster Tissues.

Human norovirus (HuNoV) is the leading foodborne pathogen causing nonbacterial gastroenteritis worldwide. The oyster is an important vehicle for HuNoV transmission, especially the GI.1 HuNoV. In our previous study, oyster heat shock protein 70 (oHSP 70) was identified as the first proteinaceous ligand of GII.4 HuNoV in Pacific oysters besides the commonly accepted carbohydrate ligands, a histo-blood group antigens (HBGAs)-like substance. However the mismatch of the distribution pattern between discovered ligands and GI.1 HuNoV suggests that other ligands may exist. In our study, proteinaceous ligands for the specific binding of GI.1 HuNoV were mined from oyster tissues using a bacterial cell surface display system. Fifty-five candidate ligands were identified and selected through mass spectrometry identification and bioinformatics analysis. Among them, the oyster tumor necrosis factor (oTNF) and oyster intraflagellar transport protein (oIFT) showed strong binding abilities with the P protein of GI.1 HuNoV. In addition, the highest mRNA level of these two proteins was found in the digestive glands, which is consistent with GI.1 HuNoV distribution. Overall the findings suggested that oTNF and oIFT may play important roles in the bioaccumulation of GI.1 HuNoV.

Individual Microparticle Manipulation Using Combined Electroosmosis and Dielectrophoresis through a Si3N4 Film with a Single Micropore.

Porous dielectric membranes that perform insulator-based dielectrophoresis or electroosmotic pumping are commonly used in microchip technologies. However, there are few fundamental studies on the electrokinetic flow patterns of single microparticles around a single micropore in a thin dielectric film. Such a study would provide fundamental insights into the electrokinetic phenomena around a micropore, with practical applications regarding the manipulation of single cells and microparticles by focused electric fields. We have fabricated a device around a silicon nitride film with a single micropore (2-4 µm in diameter) which has the ability to locally focus electric fields on the micropore. Single microscale polystyrene beads were used to study the electrokinetic flow patterns. A mathematical model was developed to support the experimental study and evaluate the electric field distribution, fluid motion, and bead trajectories. Good agreement was found between the mathematic model and the experimental data. We show that the combination of electroosmotic flow and dielectrophoretic force induced by direct current through a single micropore can be used to trap, agglomerate, and repel microparticles around a single micropore without an external pump. The scale of our system is practically relevant for the manipulation of single mammalian cells, and we anticipate that our single-micropore approach will be directly employable in applications ranging from fundamental single cell analyses to high-precision single cell electroporation or cell fusion.

Preservation of grape tomato by isochoric freezing.

This study investigated the potential of isochoric freezing to preserve tomatoes. Isochoric freezing is an emerging technology that preserves biological matter at subfreezing temperatures without any ice damage. Isochoric freezing was compared with freezing under isobaric conditions and with preservation techniques used in the food industry: cold storage at 10 °C and individual quick freezing (IQF). Physicochemical and nutritional properties were evaluated weekly for four weeks. Preservation under isochoric conditions maintained the mass, color, nutrient content (ascorbic acid, lycopene and phenolics) and antioxidant activity of the fresh tomatoes. Also, isochoric preservation led to minimal texture damage. In comparison, mass loss of tomatoes stored at 10 °C for 3 weeks contributed to changes in overall visual quality and firmness as well as significant losses in nutrient content. The greatest mass, texture, and nutrients losses were obtained for tomatoes subjected to IQF and isobaric freezing. The results show that isochoric freezing has the potential to preserve tomatoes while maintaining physicochemical and nutritional properties similar to those of fresh tomatoes which might find application in the commercial preservation of tomatoes.

Simultaneous electroporation and dielectrophoresis in non-electrolytic micro/nano-electroporation.

It was recently shown that electrolysis may play a substantial detrimental role in microfluidic electroporation. To overcome this problem, we have developed a non-electrolytic micro/nano electroporation (NEME) electrode surface, in which the metal electrodes are coated with a dielectric. A COMSOL based numerical scheme was used to simultaneously calculate the excitation frequency and dielectric material properties dependent electric field delivered across the dielectric, fluid flow, electroporation field and Clausius-Mossotti factor for yeast and E. coli cells flowing in a channel flow across a NEME surface. A two-layer model for yeast and a three-layer model for E. coli was used. The numerical analysis shows that in NEME electroporation, the electric fields could induce electroporation and dielectrophoresis simultaneously. The simultaneous occurrence of electroporation and dielectrophoresis gives rise to several interesting phenomena. For example, we found that a certain frequency exists for which an intact yeast cell is drawn to the NEME electrode, and once electroporated, the yeast cell is pushed back in the bulk fluid. The results suggest that developing electroporation technologies that combine, simultaneously, electroporation and dielectrophoresis could lead to new applications. Obviously, this is an early stage numerical study and much more theoretical and experimental research is needed.

A comparison of freezing-damage during isochoric and isobaric freezing of the potato.

BACKGROUND: Freezing is commonly used for food preservation. It is usually done under constant atmospheric pressure (isobaric). While extending the life of the produce, isobaric freezing has detrimental effects. It causes loss of food weight and changes in food quality. Using thermodynamic analysis, we have developed a theoretical model of the process of freezing in a constant volume system (isochoric). The mathematical model suggests that the detrimental effects associated with isobaric freezing may be reduced in an isochoric freezing system. To explore this hypothesis, we performed a preliminary study on the isochoric freezing of a produce with which our group has experience, the potato. METHOD: Experiments were performed in an isochoric freezing device we designed. The device is robust and has no moving parts. For comparison, we used a geometrically identical isobaric freezing device. Following freezing and thawing, the samples were weighed, examined with colorimetry, and examined with microscopy. RESULTS: It was found that potatoes frozen to -5 °C in an isochoric system experienced no weight loss and limited enzymatic browning. In contrast the -5 °C isobaric frozen potato experienced substantial weight loss and substantial enzymatic browning. Microscopic analysis shows that the structural integrity of the potato is maintained after freezing in the isochoric system and impaired after freezing in the isobaric system. DISCUSSION: Tissue damage during isobaric freezing is caused by the increase in extracellular osmolality and the mechanical damage by ice crystals. Our thermodynamic analysis predicts that during isochoric freezing the intracellular osmolality remains comparable to the extracellular osmolality and that isochoric systems can be designed to eliminate the mechanical damage by ice. The results of this preliminary study seem to confirm the theoretical predictions. CONCLUSION: This is a preliminary exploratory study on isochoric freezing of food. We have shown that the quality of a food product preserved by isochoric freezing is better than the quality of food preserved to the same temperature in isobaric conditions. Obviously, more extensive research remains to be done to extend this study to lower freezing temperatures and other food items.

Inactivation of Yeast on Grapes by Plasma-Activated Water and Its Effects on Quality Attributes.

Plasma-activated water (PAW) is a promising nonthermal technology in food preservation and food safety. The inactivation efficacy of PAW was investigated against Saccharomyces cerevisiae CICC 1374 inoculated on grape berries. PAW30 and PAW60 were obtained by activating water with plasma for 30 and 60 min, respectively. Grapes were directly treated with PAW, and a 0.38- to 0.53-log CFU/ml reduction of S. cerevisiae was achieved in a time-dependent manner (P < 0.05). The oxidation-reduction potential and pH values of PAW30 and PAW60 were also in a time-dependent manner (P < 0.05). Grape quality assessment demonstrated no significant change in surface color and total anthocyanin content after 30 min of PAW60 treatment (P > 0.05). Although grape quality was unaffected by PAW in this study, this technology should be optimized to enhance inactivation efficiency.