Preparation and benchmarking of novel cellulose nanopaper.

Synthetic polymers and plastics which are currently used as barrier materials in packaging applications are neither renewable nor biodegradable. Nanopaper, which is obtained by breaking down cellulose fibers into nanoscale particles, have unique properties with the potential to replace synthetic packaging materials, but requires very high energy to mechanically process the fibers into nanopaper. This research investigates whether refining alone can be used to produce nanopaper with sufficient quality for packaging applications. Nanopaper was produced from Bleached Eucalyptus Kraft (BEK) refined with a PFI mill and from Northern Bleached Softwood Kraft (NBSK) refined in a pilot disc refiner. Both trials found a plateau for oxygen permeability and water vapour permeability that was reached after 1800 kWh/t and 12,000 kWh/t for refining in the pilot disc refiner and PFI mill, respectively. Refining beyond these optima produced either little or no reduction in permeability, while increasing the drainage time to form a sheet. However, elastic modulus, strain at break and sheet light transmittance did continue to increase. The plateau oxygen permeability of ~ 1.24 (cc µm)/(m2 day kPa) is 1-3 orders of magnitude lower than the oxygen permeability for PET and LDPE, respectively, while the plateau water vapour permeability ~ 3 × 10-11 g/m.s. Pa was 1-2 orders of magnitude higher than for PET and LDPE. The improved strength and barrier properties of nanopaper achieved at lab and pilot scale mechanical refining process promises a sustainable alternative to conventional packaging. Supplementary Information: The online version contains supplementary material available at 10.1007/s10570-022-04563-0.

MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins.

BACKGROUND: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M-protein). Some patients have more than one identifiable M-protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M-protein, (2) an M-protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M-protein with light chain glycosylation, or (4) two distinct biclonal M-proteins. METHODS: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M-proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. RESULTS: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. CONCLUSIONS: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M-proteins is useful for monitoring of patients with PCDs.

Acute Late-Stage Myocarditis in the Crab-Eating Macaque Model of Hemorrhagic Smallpox.

Hemorrhagic smallpox, caused by variola virus (VARV), was a rare but nearly 100% lethal human disease manifestation. Hemorrhagic smallpox is frequently characterized by secondary bacterial infection, coagulopathy, and myocardial and subendocardial hemorrhages. Previous experiments have demonstrated that intravenous (IV) cowpox virus (CPXV) exposure of macaques mimics human hemorrhagic smallpox. The goal of this experiment was to further understand the onset, nature, and severity of cardiac pathology and how it may contribute to disease. The findings support an acute late-stage myocarditis with lymphohistiocytic infiltrates in the CPXV model of hemorrhagic smallpox.

Monitoring Ravulizumab effect on complement assays.

Ravulizumab is a new C5 inhibitor therapeutic monoclonal antibody with a longer half-life than eculizumab. Monitoring complete complement blockade by eculizumab has allowed personalized therapy in specific settings. Similar action is expected with ravulizumab. Ravulizumab has 4 different amino acids from eculizumab, which allow greater affinity for the FcRn immunoglobulin receptor and change the affinity of the molecule for C5. Here we investigate if clinical lab tests traditionally used to monitor complement blockade for eculizumab are appropriate for monitoring complement blockade caused by ravulizumab. De-identified serum samples with known normal complement activity were spiked with increasing amounts of ravulizumab, from zero to 1000 µg/mL. Measurement of classical pathway function (CH50) and C5 function using a liposome method (Wako Diagnostics) showed >50% complement inhibition starting with 50 µg/mL of ravulizumab, but inhibition >95% of complement activity was not achieved, with residual measurements of 11% at 700 µg/mL. In contrast, measurement of alternative pathway function using an ELISA (AH50, Wieslab) showed alternative pathway function inhibition of 80% at 50 µg/mL of ravulizumab and > 95% at 200 µg/mL, which is consistent with expected therapeutic concentrations of ravulizumab >175 µg/mL. If replicated in patient sera, AH50 could be a suitable therapeutic monitoring tool.

Theoretical analysis of LC-refining - pressure screening systems in TMP.

LC refining of mechanical pulps has proven to save energy in the production of TMP pulps. However, the specific role of LC refining as part of a TMP system has not been thoroughly studied since it is difficult to conceive any particular system at industrial-scales and impractical at pilot-scales. In this study, pressure screening and LC refining models that describe fibre length distributions, together with correlations to predict refining power were used to model three basic refining systems. From the simulation results, the impact of important variables such as reject ratio, refiner gap and refining net-power was studied. Performance curves of length-weighed average fibre length were generated from simulation results and were used to assess each system behaviour and also to make comparisons between systems. Data from an industrial scale TMP mill sub-system was gathered and compared to simulation results showing relative errors between 0-18 % on the predicted variables.

Power-gap relationships in low consistency refining.

Distance between stationary and rotating refining plates, gap, has a direct and significant impact on refining power. Gap is almost universally used to control power in low consistency refining operations. The relationship between power and gap are affected by refiner size, pulp type, plate pattern and refining conditions. In this study, a correlation was developed to describe the power-gap relationships at a wide range of refining conditions and furnish. The correlation was developed using pilot-scale refining data of mechanical pulps. Results showed that a properly defined dimensionless power number is suitable to describe refining power as well as to compare different refiners under the same grounds. The developed correlation was also used to predict mill-scale refining data showing good agreement with between predicted and measured values. Finally, experimental data from force sensor measurements supports the correlation's theoretical assumptions.

iOS Appstore-Based Phone Apps for Diabetes Management: Potential for Use in Medication Adherence.

BACKGROUND: Currently, various phone apps have been developed to assist patients. Many of these apps are developed to assist patients in the self-management of chronic diseases such as diabetes. It is essential to analyze these various apps to understand the key features that would potentially be instrumental in helping patients successfully achieve goals in disease self-management. OBJECTIVE: The objective of this study was to conduct a review of all the available diabetes-related apps in the iOS App Store to evaluate which diabetic app is more interactive and offers a wide variety of operations such as monitoring glucose, water, carbohydrate intake, weight, body mass index (BMI), medication, blood pressure (BP) levels, reminders or push notifications, food database, charts, exercise management, email, sync between devices, syncing data directly to the prescribers, and other miscellaneous functions such as (Twitter integration, password protection, retina display, barcode scanner, apple watch functionality, and cloud syncing). METHODS: Data was gathered using the iOS App Store on an iPad. The search term "diabetes" resulted in 1209 results. Many of the results obtained were remotely related to diabetes and focused mainly on diet, exercise, emergency services, refill reminders, providing general diabetes information, and other nontherapeutic options. We reviewed each app description and only included apps that were meant for tracking blood glucose levels. All data were obtained in one sitting by one person on the same device, as we found that carrying out the search at different times or on different devices (iPhones) resulted in varying results. Apps that did not have a feature for tracking glucose levels were excluded from the study. RESULTS: The search resulted in 1209 results; 85 apps were retained based on the inclusion criteria mentioned above. All the apps were reviewed for average customer ratings, number of reviews, price, and functions. Of all the apps surveyed, 18 apps with the highest number of user ratings were used for in-depth analysis. Of these 18 apps, 50% (9/18) also had a medication adherence function. Our analysis revealed that the Diabetes logbook used by the mySugr app was one of the best; it differentiated itself by introducing fun as a method of increasing adherence. CONCLUSIONS: A large variation was seen in patient ratings of app features. Many patient reviewers desired simplicity of app functions. Glucose level tracking and email features potentially helped patients and health care providers manage the disease more efficiently. However, none of the apps could sync data directly to the prescribers. Additional features such as graph customization, availability of data backup, and recording previous entries were also requested by many users. Thus, the use of apps in disease management and patient and health-care provider involvement in future app refinement and development should be encouraged.

4-Ferrocenylpyridine- and 4-ferrocenyl-3-ferrocenylmethyl-3,4-dihydropyridine-3,5-dicarbonitriles: multi-component synthesis, structures and electrochemistry.

The reactions of 2-cyano-3-ferrocenylacrylonitrile (1) with malononitrile (2) in a MeOH/H2O or 2-PrOH/H2O medium in the presence of Na2CO3 afforded 6-alkoxy-2-amino-4-ferrocenylpyridine-3,5-dicarbonitriles 3a,b (multi-component condensation) and 6-alkoxy-2-amino-4-ferrocenyl-3-ferrocenylmethyl-3,4-dihydropyridine-3,5-dicarbonitriles 4a,b (multi-component cyclodimerization). Analogous reactions of 1 with 2 in an MeOH/H2O medium in the presence of NaOH, piperidine, or morpholine gave compounds 3a, 4a and 2-amino-4-ferrocenyl-6-hydroxy-, 6-piperidino- and 6-morpholinopyridine-3,5-dicarbonitriles 3c-e, respectively. The structures of the compounds 3b, 4a and 4b were established by the spectroscopic data and X-ray diffraction analysis. The electrochemical behaviour of compounds 3b, 3d and 4b was investigated by means of cyclic voltammetry.

Different mechanisms preserve translation of programmed cell death 8 and JunB in virus-infected endothelial cells.

OBJECTIVE: Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRESs). Whether IRES-mediated translation occurs in stressed primary human endothelial cells (ECs) is unknown. METHODS AND RESULTS: We performed microarray analysis of polyribosomal mRNA from ECs to identify IRES-containing mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks, detection of eukaryotic initiation factor (eIF) 4G cleavage, and decreased protein synthesis. We found that 87.4% of mRNAs were dissociated from polysomes in virus-infected ECs. Twelve percent of mRNAs remained associated with polysomes, and 0.6% were enriched ≥2-fold in polysome fractions from infected ECs. Quantitative reverse transcription-polymerase chain reaction confirmed the microarray findings for 31 selected mRNAs. We found that enriched polysome associations of programmed cell death 8 (PDCD8) and JunB mRNA resulted in increased protein expression in PV-infected ECs. The presence of IRESs in the 5' untranslated region of PDCD8 mRNA, but not of JunB mRNA, was confirmed by dicistronic analysis. CONCLUSIONS: We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected ECs successfully identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.

Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates.

Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs). NETs are lattices of extracellular DNA, chromatin, and antibacterial proteins that mediate extracellular killing of microorganisms and are thought to form via a unique death pathway signaled by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated reactive oxygen species (ROS). We found that neutrophils from term and preterm infants fail to form NETs when activated by inflammatory agonists-in contrast to leukocytes from healthy adults. The deficiency in NET formation is paralleled by a previously unrecognized deficit in extracellular bacterial killing. Generation of ROSs did not complement the defect in NET formation by neonatal neutrophils, as it did in adult cells with inactivated NADPH oxidase, demonstrating that ROSs are necessary but not sufficient signaling intermediaries and identifying a deficiency in linked or downstream pathways in neonatal leukocytes. Impaired NET formation may be a critical facet of a common developmental immunodeficiency that predisposes newborn infants to infection.

Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling.

Tight regulation of COX-2 expression is a key feature controlling eicosanoid production in atherosclerosis and other inflammatory syndromes. Adhesive interactions between platelets and monocytes occur in these conditions and deliver specific signals that trigger inflammatory gene expression. Using a cellular model of monocyte signaling induced by activated human platelets, we identified the central posttranscriptional mechanisms that regulate timing and magnitude of COX-2 expression. Tethering of monocytes to platelets and to purified P-selectin, a key adhesion molecule displayed by activated platelets, induces NF-kappaB activation and COX-2 promoter activity. Nevertheless, COX-2 mRNA is rapidly degraded, leading to aborted protein synthesis. Time-dependent signaling of monocytes induces a second phase of transcript accumulation accompanied by COX-2 enzyme synthesis and eicosanoid production. Here, generation of IL-1beta, a proinflammatory cytokine, promoted stabilization of COX-2 mRNA by silencing of the AU-rich mRNA decay element (ARE) in the 3'-untranslated region (3'UTR) of the mRNA. Consistent with observed mRNA stabilization, activated platelets or IL-1beta treatment induced cytoplasmic accumulation and enhanced ARE binding of the mRNA stability factor HuR in monocytes. These findings demonstrate that activated platelets induce COX-2 synthesis in monocytes by combinatorial signaling to transcriptional and posttranscriptional checkpoints. These checkpoints may be altered in disease and therefore useful as targets for antiinflammatory intervention.

Respiratory immunity is an important component of protection elicited by subunit vaccination against pneumonic plague.

Mice were vaccinated with a recombinant fusion protein, rF1-V, by an intramuscular prime followed by an intranasal boost, to evaluate protection against pneumonic plague. Forty-two days after the intranasal boost, the mice were challenged by aerosol exposure to Yersinia pestis. Survival after exposure depended upon the dose of rF1-V given i.n. with > or = 80% survival in the highest dose groups. Pulmonary and serum antibody titers to V were the best predictors of outcome. For vaccinated mice that succumbed to the infection, death was delayed by 1-2 days compared to sham-inoculated controls. Weight loss early after exposure correlated with outcome. Pathology studies indicated a severe, necrotizing bronchopneumonia in vaccinated mice that succumbed to the infection, compatible with a prolonged disease course, while the lungs of sham-inoculated mice had only mild pneumonia, which is compatible with a more rapid disease course. Immunity in the respiratory tract appears to be critical for protection against primary pneumonia caused by Y. pestis.

Exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox.

Smallpox virus (variola) poses a significant threat as an agent of bioterrorism. To mitigate this risk, antiviral drugs and an improved vaccine are urgently needed. Satisfactory demonstration of protective efficacy against authentic variola will require development of an animal model in which variola produces a disease course with features consistent with human smallpox. Toward this end, cynomolgus macaques were exposed to several variola strains through aerosol and/or i.v. routes. Two strains, Harper and India 7124, produced uniform acute lethality when inoculated i.v. in high doses (10(9) plaque-forming units). Lower doses resulted in less fulminant, systemic disease and lower mortality. Animals that died had profound leukocytosis, thrombocytopenia, and elevated serum creatinine levels. After inoculation, variola was disseminated by means of a monocytic cell-associated viremia. Distribution of viral antigens by immunohistochemistry correlated with the presence of replicating viral particles demonstrated by electron microscopy and pathology in the lymphoid tissues, skin, oral mucosa, gastrointestinal tract, reproductive system, and liver. These particles resembled those seen in human smallpox. High viral burdens in target tissues were associated with organ dysfunction and multisystem failure. Evidence of coagulation cascade activation (D dimers) corroborated histologic evidence of hemorrhagic diathesis. Depletion of T cell-dependent areas of lymphoid tissues occurred, probably as a consequence of bystander apoptotic mechanisms initiated by infected macrophages. Elaboration of cytokines, including IL-6 and IFN-gamma, contribute to a cytokine storm formerly known as "toxemia." A more precise understanding of disease pathogenesis should provide targets for therapeutic intervention, to be used alone or in combination with inhibitors of variola virus replication.

Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler.

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.

Modeling of occupant-generated CO2 dynamics in naturally ventilated classrooms.

A numerical method is presented to estimate the concentration of occupant-generated CO(2) for the (time-varying) occupancy typically found in nonforced ventilated elementary school classrooms. Here, the governing mass balance was solved numerically and compared to experimental measurements in order to estimate the respiration and (time-varying) infiltration rates. For the cases studied, we estimate an average CO(2) generation rate per child as 404 mg/min(-1). This is similar to estimates found in the literature for the age and activity level of elementary students, the classroom occupants. The average estimated infiltration rates were found to be larger than those measured from the decay of the tracer gas SF(6) under closed-door static conditions. The in-use infiltration rates were increased by additional air exchange due to people entering and leaving the room. In addition, we show that the difference (or error) between the instantaneous concentration of CO(2) and the time-averaged value recorded by a data-logging CO(2) monitor varies depending on the infiltration rate and sampling time. Therefore, the time interval selected for averaging may increase the overall error of the apparent CO(2) concentration. We conclude that the methods used to measure air exchange rates in naturally ventilated rooms underestimate the actual ventilation rate of a room under "in-use" conditions. However, even with the addition of uncontrolled outdoor air, the concentration of CO(2) in the classrooms studied was higher than recommended to meet air quality objectives.

Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox.

The potential use of smallpox as a biological weapon has led to the production and stockpiling of smallpox vaccine and the immunization of some healthcare workers. Another public health goal is the licensing of a safer vaccine that could benefit the millions of people advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates. Here we compare the highly attenuated modified vaccinia virus Ankara (MVA) with the licensed Dryvax vaccine in a monkey model. After two doses of MVA or one dose of MVA followed by Dryvax, antibody binding and neutralizing titres and T-cell responses were equivalent or higher than those induced by Dryvax alone. After challenge with monkeypox virus, unimmunized animals developed more than 500 pustular skin lesions and became gravely ill or died, whereas vaccinated animals were healthy and asymptomatic, except for a small number of transient skin lesions in animals immunized only with MVA.

Detection and identification of Variola virus in fixed human tissue after prolonged archival storage.

Smallpox disease has been eradicated from the human population since 1979, but is again a concern because of its potential use as an agent of bioterrorism or biowarfare. World Health Organization-sanctioned repositories of infectious Variola virus are known to occur in both Russia and the United States, but many believe other undeclared and unregulated sources of the virus could exist. Thus, validation of improved methods for definitive identification of smallpox virus in diagnostic specimens is urgently needed. In this paper, we describe the discovery of suspected Variola infected human tissue, fixed and preserved for decades in largely unknown solutions, and the use of routine histology, electron microscopy, and ultimately DNA extraction and fluorogenic 5' nuclease (TaqMan) assays for its identification and confirmation.

Pulmonary gene expression profiling of inhaled ricin.

Aerosol exposure to ricin causes irreversible pathological changes of the respiratory tract resulting in epithelial necrosis, pulmonary edema and ultimately death. The pulmonary genomic profile of BALB/c mice inhalationally exposed to a lethal dose of ricin was examined using cDNA arrays. The expression profile of 1178 mRNA species was determined for ricin-exposed lung tissue, in which 34 genes had statistically significant changes in gene expression. Transcripts identified by the assay included those that facilitate tissue healing (early growth response gene (egr)-1), regulate inflammation (interleukin (IL)-6, tristetraproline (ttp)), cell growth (c-myc, cytokine-inducible SH2-containing protein (cish)- 3), apoptosis (T-cell death associated protein (tdag)51, pim-1) and DNA repair (ephrin type A receptor 2 (ephA2)). Manipulation of these gene products may provide a means of limiting the severe lung damage occurring at the cellular level. Transcriptional activation of egr-1, cish-3, c-myc and thrombospondin (tsp)-1 was already apparent when pathological and physiological changes were observed in the lungs at 12 h postexposure. These genes may well serve as markers for ricin-induced pulmonary toxicity. Ongoing studies are evaluating this aspect of the array data and the potential of several genes for clinical intervention.