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The aim of the study was to perform treatment of juniper wood to obtain wood material with a density and mechanical properties comparable to bone, thus producing a potential material for use in osteosynthesis bone implants. In the first step, partial delignification of wood sample was obtained by Kraft cooking. The second step was extraction with ethanol, ethanol–water mixture, saline, and water to prevent the release of soluble compounds and increase biocompatibility. In the last step, the thermal densification at 100 °C for 24 h was implemented. The results obtained in the dry state are equivalent to the properties of bone. The swelling of chemically pre-treated densified wood was reduced compared to chemically untreated densified wood. Samples showed no cytotoxicity by in vitro cell assays. The results of the study showed that it is possible to obtain noncytotoxic wood samples with mechanical properties equivalent to bones by partial delignification, extraction, and densification. However, further research is needed to ensure the material’s shape stability, water resistance, and reduced swelling.

In the context of growing environmental concerns and the need to promote sustainable living, this study explores the potential of the Lucy Veg App, a digital solution under development, in contributing to the achievement of the Sustainable Development Goals (SDGs) in Valencia. The app is designed to empower the local vegan community by providing resources such as plant-based recipes, eco-friendly product recommendations, and a platform for connecting like-minded individuals. This research utilizes a review of relevant literature and examines the theoretical implications of the app on the achievement of SDGs in Valencia. Methodologically, this study draws on existing research related to digital technologies, sustainable living, and SDGs, focusing on the potential of the Lucy Veg App to promote plant-based diets and sustainable lifestyles. By analyzing the app's features and exploring its potential contributions to multiple SDGs, the research studies the app's alignment with various sustainability dimensions. The findings suggest that the Lucy Veg App has the potential to contribute to several SDGs by fostering community engagement, driving positive behavior change, and addressing key sustainability challenges related to food production, consumption, and their associated environmental and health impacts. The study also identifies promising avenues for future research and practical implementation of the app, including evaluating its effectiveness, expanding its features, analyzing user data, and assessing its broader contribution to SDGs in Valencia. In conclusion, the Lucy Veg App presents a promising opportunity to support Valencia's efforts to achieve the SDGs by promoting plant-based diets, sustainable lifestyles, and leveraging digital technologies. Further research and ongoing evaluation of the app's impact on users' behavior and the achievement of SDGs are necessary to maximize its potential in fostering a more sustainable future.

One of the most important steps in the synthesis of 1,4-dihydropyridine (1,4-DHP) amphiphiles is the bromination of methyl groups in positions 2 and 6 of the entire ring. However, up to now, only N-bromosuccinimide was mainly used for bromination 1,4-DHPs. In this work, the synthesis of bis-1,4-DHP derivatives with ethyl and dodecyl ester groups attached to 1,4-DHP ring at positions 3 and 5 was performed by Hantzsch synthesis. The experimental studies were carried out to find out the best conditions and the agent for the tetra bromination of bis-1,4-DHP methyl groups at positions 2 and 6. Four different brominating agents were screened. The use of pyridinium bromide–perbromide in ethyl acetate was found to be optimal for the bromination of methyl groups. The bromination reaction was followed by the synthesis of cationic pyridine moiety containing amphiphilic bis-1,4-DHP derivatives. By nucleophilic substitution of bromine with various substituted pyridines, 12 new amphiphilic bis-1,4-DHP derivatives were obtained. Evaluation of self-assembling properties of tetracationic bis-1,4-dihydropyridine derivatives by dynamic light scattering (DLS) measurements was also performed.

Enzymatic, de novo XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3′-O-modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties. While the resulting ester-modified nucleotides appear to be poor substrates for polymerases, ether-blocked LNA and DNA nucleotides are readily incorporated into DNA. However, removal of the protecting groups and modest incorporation yields represent obstacles for LNA synthesis via this route. On the other hand, we have also shown that the template-independent RNA polymerase PUP represents a valid alternative to the TdT and we have also explored the possibility of using engineered DNA polymerases to increase substrate tolerance for such heavily modified nucleotide analogs.

A set of styrylpyridinium (SP) compounds was synthesised in order to study their spectroscopic and cell labelling properties. The compounds comprised different electron donating parts (julolidine, p-dimethylaminophenyl, p-methoxyphenyl, 3,4,5-trimethoxyphenyl), conjugated linkers (vinyl, divinyl), and an electron-withdrawing N-alkylpyridinium part. Geminal or bis-compounds incorporating two styrylpyridinium (bis-SP) moieties at the 1,3-trimethylene unit were synthesised. Compounds comprising a divinyl linker and powerful electron-donating julolidine donor parts possessed intensive fluorescence in the near-infrared region (maximum at ~760 nm). The compounds had rather high cytotoxicity towards the cancerous cell lines HT-1080 and MH-22A; at the same time, basal cytotoxicity towards the NIH3T3 fibroblast cell line ranged from toxic to harmful. SP compound 6e had IC50 values of 1.0 ± 0.03 µg/mL to the cell line HT-1080 and 0.4 µg/mL to MH-22A; however, the basal toxicity LD50 was 477 mg/kg (harmful). The compounds showed large Stokes’ shifts, including 195 nm for 6a,b, 240 nm for 6e, and 325 and 352 nm for 6d and 6c, respectively. The highest photoluminescence quantum yield (PLQY) values were observed for 6a,b, which were 15.1 and 12.2%, respectively. The PLQY values for the SP derivatives 6d,e (those with a julolidinyl moiety) were 0.5 and 0.7%, respectively. Cell staining with compound 6e revealed a strong fluorescent signal localised in the cell cytoplasm, whereas the cell nuclei were not stained. SP compound 6e possessed self-assembling properties and formed liposomes with an average diameter of 118 nm. The obtained novel data on near-infrared fluorescent probes could be useful for the development of biocompatible dyes for biomedical applications.

Peptide nucleic acid (PNA) is arguably one of the most successful DNA mimics, despite a most dramatic departure from the native structure of DNA. The present review summarizes 30 years of research on PNA’s chemistry, optimization of structure and function, applications as probes and diagnostics, and attempts to develop new PNA therapeutics. The discussion starts with a brief review of PNA’s binding modes and structural features, followed by the most impactful chemical modifications, PNA enabled assays and diagnostics, and discussion of the current state of development of PNA therapeutics. While many modifications have improved on PNA’s binding affinity and specificity, solubility and other biophysical properties, the original PNA is still most frequently used in diagnostic and other in vitro applications. Development of therapeutics and other in vivo applications of PNA has notably lagged behind and is still limited by insufficient bioavailability and difficulties with tissue specific delivery. Relatively high doses are required to overcome poor cellular uptake and endosomal entrapment, which increases the risk of toxicity. These limitations remain unsolved problems waiting for innovative chemistry and biology to unlock the full potential of PNA in biomedical applications.

Synthesis of a double-charged cationic amphiphilic 1,4-dihydropyridine derivative with dodecyl ester groups at positions 3 and 5 of the 1,4-DHP ring was performed starting from Hantzsch type cyclization of dodecyl acetoacetate, 2-naphthaldehyde and ammonium acetate. Bromination of this compound followed by nucleophilic substitution of bromine with (E)-4-(2-(naphthalen-2-yl)vinyl)pyridine gave the desired cationic amphiphilic 1,1′-{[3,5-bis(dodecyloxycarbonyl)-4-(naphthalen-2-yl)-1,4-dihydropyridine-2,6-diyl]bis(methylene)}bis{4-[(E)-2-(naphthalen-2-yl)vinyl]pyridin-1-ium}dibromide. The obtained target compound was fully characterized by IR, UV, 1H-NMR, 13C-NMR, HRMS and microanalysis. The characterization of the cationic 1,4-DHP nanoparticles in an aqueous solution was performed by DLS measurements. The obtained results showed that the compound formed nanoparticles with an average diameter of around 300 nm, a PDI value of around 490 and a zeta-potential of around 20 mV for freshly prepared samples. However, after one week of storage at room temperature, an aggregation of nanoparticles was detected.

Abstract Background Up to 30% or even more of all infective endocarditis (IE) cases are recognized as blood culture negative, meaning that the causative agent is left unidentified. The prompt diagnosis together with the identification of causative microorganism and targeted antibiotic treatment can significantly impact the prognosis of the disease and further patient’s health status. In some studies, blood culture negative endocarditis has been shown to be associated with delayed diagnosis, worse outcome and course of the disease, and a greater number of intra and postoperative complications. Methods We retrospectively analysed the medical records of all patients who underwent cardiac surgery for endocarditis between years 2016 and 2019. The aim of this study was to analyse short and long-term mortality and differences of laboratory, clinical and echocardiography parameters in patients with blood culture positive endocarditis (BCPE) and blood culture negative endocarditis (BCNE) and its possible impact on the clinical outcome. Results In our study population were 114 (55.1%) blood culture positive and 93 (44.9%) blood culture negative cases of infectious endocarditis. The most common pathogens in the blood culture positive IE group were S.aureus in 36 cases (31.6%), Streptococcus spp. in 27 (23.7%), E.faecalis in 24 (21.1%), and other microorganisms in 27 (23.7%). Embolic events were seen in 60 patients (28.9%). In univariate analyses, detection of microorganism, elevated levels of procalcitonin were found to be significantly associated with intrahospital death, however it did not reach statistical significance in multivariate analyses. Among microorganisms, S.aureus was significantly associated with intrahospital death in both univariate and multivariate analyses. Conclusions There are no statistically significant differences between groups of BCPE and BCNE in terms of intrahospital mortality, hospital and ICU stay or 3-year mortality. There were higher levels of procalcitonin in BCPE group, however procalcitonin failed to show independent association with mortality in multivariate analysis. The most common microorganism in the BCPE group was S.aureus. It was associated with independently higher intrahospital mortality when compared to other causative microorganisms.

Bone fractures and bone defects affect millions of people every year. Metal implants for bone fracture fixation and autologous bone for defect reconstruction are used extensively in treatment of these pathologies. Simultaneously, alternative, sustainable, and biocompatible materials are being researched to improve existing practice. Wood as a biomaterial for bone repair has not been considered until the last 50 years. Even nowadays there is not much research on solid wood as a biomaterial in bone implants. A few species of wood have been investigated. Different techniques of wood preparation have been proposed. Simple pre-treatments such as boiling in water or preheating of ash, birch and juniper woods have been used initially. Later researchers have tried using carbonized wood and wood derived cellulose scaffold. Manufacturing implants from carbonized wood and cellulose requires more extensive wood processing—heat above 800 °C and chemicals to extract cellulose. Carbonized wood and cellulose scaffolds can be combined with other materials, such as silicon carbide, hydroxyapatite, and bioactive glass to improve biocompatibility and mechanical durability. Throughout the publications wood implants have provided good biocompatibility and osteoconductivity thanks to wood’s porous structure.

Removal of nitrogen from wastewaters (WW) represents a global problem. The low nitrification rate during WW treatment is often caused by ecotoxicity. This problem is attributed mostly to the industrial WW. Our study was focused on the testing of industrial WW and activated sludge (AS) with the aim to reveal the abundance of nitrifiers and increase their biomass, thus, providing the additional step, i.e., bioaugmentation, within the technological process of WW treatment. Plating of AS on the selective solidified media designated for the 1st and 2nd nitrification stages, resulted in the shift in bacterial community structure with dominated Alcaligenaceae and Alcanivorax for the 1st stage, and Alcanivorax-for the 2nd stage of nitrification, respectively. Incubation of AS in the presence of real WW and selective nitrification broth resulted in a considerable increase (one or two magnitudes in the presence of the 1st and 2nd stage nitrification broth, respectively) of culturable nitrifiers after 5 days incubation under aerated conditions. The obtained data provide with evidence about a possibility to strengthen the role of heterotrophic nitrifiers in the treatment of industrial WW, where toxicity obstacles inhibited nitrification under conventional conditions.

Objectives. The study aimed to investigate the long-term outcomes of a double stent scaffold strategy in patients with left main (LM) bifurcation lesions involving the ostium of the left circumflex artery (LCX), utilizing a drug-eluting stent (DES) in the LM extending into the left anterior descending artery (LAD) and a bioresorbable vascular scaffold (BVS) in the LCX ostium. Background. The high occurrence of in-stent restenosis of the LCX ostium is the major limitation of percutaneous coronary intervention (PCI) for LM lesions with a two-stent strategy. Methods. This was a single-center, prospective, single-arm study of 46 consecutively enrolled patients with a stable coronary artery disease and significant unprotected LM distal bifurcation disease. Patients underwent imaging-guided PCI using DES in the LM-LAD and BVS in the LCX using a T-stent or mini-crush technique. The primary outcome at four years was the composite of death, myocardial infarction, stroke, and target lesion revascularization (TLR). Results. At four years, the primary outcome was identified in 9 patients (19.6%). All events were TLRs except one myocardial infarction due to BVS thrombosis. Seven of the eight TLRs were a result of side branch BVS restenosis. Univariate predictors of the 4-year outcome were higher LDL cholesterol and BVS size ≤2.5 mm. On multivariate analysis, LCX lesion preparation with a cutting balloon and post-procedure use of intravascular ultrasound for optimization were found to be independent protective factors of MACE. Conclusions. In selected patients with LM distal bifurcation disease, an imaging-guided double stent scaffold strategy with DES in the LM and BVS in the LCX ostium was technically successful in all patients and was reasonably safe and effective for four years.

Em Angola, mais concretamente na província do Namibe, existem várias fontes termais que podem ser utilizadas para diversos fins, mas não são realizadas acções de preservação e beneficiação da sua exploração; nestas condições, no concelho da Bibala, existe uma nascente termal denominada Montipa. O presente trabalho visa avaliar a qualidade físico-química, microbiológica e radiológica das águas termais de Montipa, município da Bibala-Namibe, aspectos socio ambientais da área em estudo; Para o qual foi desenvolvido um estudo analítico não experimental baseado em métodos e técnicas de análises físico-químicas, microbiológicas e radiológicas de águas. Conclui-se que, de um modo geral, as fontes termais de Montipa apresentam boas características físico-químicas, microbiológicas e radiológicas de acordo com as normas internacionais e nacionais. É possível aproveitar melhor esse recurso para consumo humano e terapêutico, cochos para gado, irrigação agrícola e ecoturismo.

Current research is devoted to the investigation of spelt husk (SH) and nanoclay-modified compatibilised polypropylene (PP) binary and ternary composites for injection-moulding applications. PP composites were obtained using twin-screw extrusion. The content of mechanically milled SH microfiller with aspect ratio within 2 and 6 was fixed at 40 wt.%, whereas the amount of nanoclay functional filler in the polypropylene matrix was changed in the range from 0.5 to 5 wt.%. Nanoclay filler was introduced in the polypropylene matrix either in the form of nanoclay powder (C) or as a masterbatch (M). Regular distribution of the clay nanofiller within the polymer matrix has been observed, disregarding its form and concentration. The effects of the individual or combined addition of SH microreinforcement and nanoclay fillers on the rheological, mechanical, calorimetric, and thermal properties of the developed PP composites were investigated. It is revealed that the addition of the nanoclay fillers insignificantly influences the viscosity of both PP nanocomposites and hybrid composites with SH. Additionally, for PP nanocomposites, remarkable increases in tensile and flexural modules and strength are observed by maintaining considerable ultimate deformations, mainly in the case of M-containing systems. Concomitantly, because of the addition of the nanoclay filler, the improvement in thermal stability of PP nanocomposites and PP hybrid composites with SH is observed. As a result of SH addition, considerable increases in tensile and flexural modules are also observed. Results of the research demonstrate the potential of the use of natural materials (agricultural residues and clay minerals) for the development of PP composites with increased stiffness and thermal properties.

A água é bem indispensável para todos os seres vivos, serve de suporte às atividades económicas, à preservação do meio ambiente e é factor de equilíbrio dos ecossistemas. Nos últimos anos, está a acontecer uma grande pressão sobre este recurso, com impactos na sua escassez e na contaminação química, biológica, etc. Em Angola, concretamente na província do Namibe, existem várias fontes termais que podem ser aproveitadas para diversos fins, mas não existem atitudes na vertente preservação e valorização. Nestas condições, no municipio da Bibala, existe uma fonte termal chamada de Montipa. Neste sentido, o presente trabalho tem como objectivo analisar de que forma a educação ambiental pode influenciar no sentido da protecção e conservação das águas termais da Montipa, no municipio da Bibala, província do Namibe-Angola. Para se atingir este objectivo, realizou-se uma revisão bibliográfica sobre a temática educação ambiental e a caracterização socioambiental da área objecto de investigação. Os trabalhos de campo permitiram a caracterização físico e química da água termal da Montipa, a observação do local, a georreferenciação, a recolha de amostras de água e o registo fotográfico, assim como se aplicou questionários à algumas entidades locais estatais e a estudantes de duas escolas do segundo ciclo do ensino secundário, nomeadamente Liceu e Magistério Primário, nos princípios de Março de 2019. Os resultados propiciaram a sugestão de acções de educação ambiental no sentido da preservação, protecção, conservação e valorização da área das Termas da Montipa aos estudantes e a população da comunidade.

The accumulation of lipid intermediates may interfere with energy metabolism pathways and regulate cellular energy supplies. As increased levels of long-chain acylcarnitines have been linked to insulin resistance, we investigated the effects of long-chain acylcarnitines on key components of the insulin signalling pathway. We discovered that palmitoylcarnitine induces dephosphorylation of the insulin receptor (InsR) through increased activity of protein tyrosine phosphatase 1B (PTP1B). Palmitoylcarnitine suppresses protein kinase B (Akt) phosphorylation at Ser473, and this effect is not alleviated by the inhibition of PTP1B by the insulin sensitizer bis-(maltolato)-oxovanadium (IV). This result indicates that palmitoylcarnitine affects Akt activity independently of the InsR phosphorylation level. Inhibition of protein kinase C and protein phosphatase 2A does not affect the palmitoylcarnitine-mediated inhibition of Akt Ser473 phosphorylation. Additionally, palmitoylcarnitine markedly stimulates insulin release by suppressing Akt Ser473 phosphorylation in insulin-secreting RIN5F cells. In conclusion, long-chain acylcarnitines activate PTP1B and decrease InsR Tyr1151 phosphorylation and Akt Ser473 phosphorylation, thus limiting the cellular response to insulin stimulation.

An increased demand for new and improved coating systems, for environmental & health & safety and performance reasons, have appeared during the recent decades. Currently, there is new interest in preparation of thin UV curable urethane acrylate (UA) composite coatings with short-term properties. Cellulose based additives: nitrocellulose, cellulose acetate butyrate, sucrose benzoate and silica were evaluated to determine their influence on unreacted composite characteristics (viscosity, pigment suspension stability) and characteristics of cured film (ultimate tensile strength, elongation at break, surface gloss, surface scratch resistance and film adhesion loss time). The most suitable additive content was found to provide required viscosity. All additives increase surface scratch resistance, but cellulose based additives increase surface gloss values and decrease the time of adhesion loss. Silica has great effect on the interaction between linear and hyperbranched urethane acrylates, which has crucial influence on the stability of uncured pigmented mixture samples.

Background and Objectives: Over the last five decades cardiac implantable electronic devices (CIED) have become established as the mainstay for the treatment of permanent bradycardias, chronic heart failure and dangerous heart rhythm disturbances. These devices improve survival and quality of life in many patients. However, infections associated with CIED implantation, particularly lead-related infective endocarditis (LRIE), can offset all benefits and make more harm than good for the patient. To date, there are no other studies in Latvia, addressing patients with lead-related infective endocarditis. The objective of this study was to identify the most common pathogens associated with LRIE and their antimicrobial resistance and to identify possible risk factors of patients who present with LRIE. Materials and Methods: The study was performed retrospectively at Pauls Stradins Clinical University Hospital (PSCUH). The study included patients who were referred to PSCUH due to LRIE for lead extraction. Patients were identified from procedural journals. Information about isolated microorganisms, patient comorbidities and visual diagnostics data was taken from patient records. Results: Forty-nine patients with CIED related infective endocarditis were included in the study, 34 (69.4%) were male, median age of all patients was 65.0 (50.5−73.0) years, median hospital stay was 15.5 (22.0−30.5) days. Successful and complete lead extraction was achieved in all patients. Thirty-two (65.3%) had received antibiotics prior to blood sample. Only in 31 (63.3%) positive culture results were seen. The most common isolated pathogens were Staphylococcus aureus (23.5%) and coagulase negative staphylococci (23.5%). Other bacteria were isolated considerably less often. The atrial lead was most common location for lead vegetations, seen in 50.0% of cases. Five (10.2%) patients have died due to the disease. Conclusions: Lead-related infective endocarditis is a major complication of cardiac implantable electronic devices with considerable morbidity and mortality, which in our study was as high as 10.2%.

Modification of properties of conventional thermoplastics with thermotropic liquid crystal polymers, from one hand, allows decrease their viscosities, substantially facilitating processing conditions, and, from another hand, allows increase their exploitation properties. Orientation of the labile structure of liquid crystal polymer in extrusion or injection moulding causes specific reinforcement (so-called self-reinforcement) to occur in the blends containing liquid crystal polymer. Up to now the effect of self-reinforcement is mostly investigated in the blends, containing considerable amount of liquid crystal polymer. In this research the effect of minor amounts of liquid crystalline co-polyester modifier on the properties of polyethylene is investigated. Various compositions of laboratory synthesized hydroxybenzoic acid /polyethylene terephtalate copolymer containing polyethylene composites have been manufactured by thermoplastic blending. It has been observed that 1) modulus of elasticity, yield strength and ultimate strength increase with raising the content of liquid crystalline modifier; 2) void content in the investigated polyethylene/liquid crystal copolymer composites is not greater that 1 %; 3) addition of liquid crystalline co-polyester modifier improves arrangement of PE crystalline phase.http://dx.doi.org/10.5755/j01.ms.17.2.483

Effective cell invasion into thick electrospun biomimetic scaffolds is an unsolved problem. One possible strategy to biofabricate tissue constructs of desirable thickness and material properties without the need for cell invasion is to use thin (

Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.

Mature microRNAs (miRNAs), derived through cleavage of pre-miRNAs by the Dicer1 enzyme, regulate protein expression in many cell-types including cells in the pancreatic islets of Langerhans. To investigate the importance of miRNAs in mouse insulin secreting β-cells, we have generated mice with a β-cells specific disruption of the Dicer1 gene using the Cre-lox system controlled by the rat insulin promoter (RIP). In contrast to their normoglycaemic control littermates (RIP-Cre(+/-) Dicer1(Δ/wt)), RIP-Cre(+/-)Dicer1(flox/flox) mice (RIP-Cre Dicer1(Δ/Δ)) developed progressive hyperglycaemia and full-blown diabetes mellitus in adulthood that recapitulated the natural history of the spontaneous disease in mice. Reduced insulin gene expression and concomitant reduced insulin secretion preceded the hyperglycaemic state and diabetes development. Immunohistochemical, flow cytometric and ultrastructural analyses revealed altered islet morphology, marked decreased β-cell mass, reduced numbers of granules within the β-cells and reduced granule docking in adult RIP-Cre Dicer1(Δ/Δ) mice. β-cell specific Dicer1 deletion did not appear to disrupt fetal and neonatal β-cell development as 2-week old RIP-Cre Dicer1(Δ/Δ) mice showed ultrastructurally normal β-cells and intact insulin secretion. In conclusion, we have demonstrated that a β-cell specific disruption of the miRNAs network, although allowing for apparently normal β-cell development, leads to progressive impairment of insulin secretion, glucose homeostasis and diabetes development.

BackgroundThe FFAR1 receptor is expressed mainly in pancreatic beta cells and is activated by medium to long chain free fatty acids (FFAs), as well as by thiazolidinediones, resulting in elevated Ca(2+) concentrations and promotion of insulin secretion. These properties suggest that FFAR1 could be a mediator of lipotoxicity and a potential candidate gene for Type 2 diabetes (T2D). We therefore investigated whether variations at the FFAR1 locus are associated with T2D and beta cell function.Methodology/principal findingsWe re-sequenced the FFAR1 region in 96 subjects (48 healthy and 48 T2D individuals) and found 13 single nucleotide polymorphisms (SNPs) 8 of which were not previously described. Two SNPs located in the upstream region of the FFAR1 gene (rs1978013 and rs1978014) were chosen and genotyped in 1929 patients with T2D and 1405 healthy control subjects. We observed an association of rs1978013 and rs1978014 with insulinogenic index in males (p = 0.024) and females (p = 0.032), respectively. After Bonferroni corrections, no association with T2D was found in the case-control material, however a haplotype consisting of the T-G alleles conferred protection against T2D (p = 0.0010).Conclusions/significanceVariation in the FFAR1 gene may contribute to impaired beta cell function in T2D.

Importance: Some tick species are competent to transmit more than one pathogen while other species are, until now, known to be competent to transmit only one single or any pathogen. Such a difference in vector competence for one or more pathogens might be related to the microbiome, and understanding what differentiates these two groups of ticks could help us control several diseases aiming at the bacteria groups that contribute to such a broad vector competence. Using 16S rRNA from tick species that could be classified into these groups, genera such as Rickettsia and Staphylococcus seemed to be associated with such a broad vector competence. Our results highlight differences in tick species when they are divided based on the number of pathogens they are competent to transmit. These findings are the first step into understanding the relationship between one single tick species and the pathogens it transmits., Competing Interests: The authors declare no conflict of interest.

Although there are now approved treatments and vaccines for Ebola virus disease, the case fatality rate remains unacceptably high even when patients are treated with the newly approved therapeutics. Furthermore, these countermeasures are not expected to be effective against disease caused by other filoviruses. A meeting of subject-matter experts was held during the 10th International Filovirus Symposium to discuss strategies to address these gaps. Several investigational therapeutics, vaccine candidates, and combination strategies were presented. The greatest challenge was identified to be the implementation of well-designed clinical trials of safety and efficacy during filovirus disease outbreaks. Preparing for this will require agreed-upon common protocols for trials intended to bridge multiple outbreaks across all at-risk countries. A multinational research consortium including at-risk countries would be an ideal mechanism to negotiate agreement on protocol design and coordinate preparation. Discussion participants recommended a follow-up meeting be held in Africa to establish such a consortium., Competing Interests: Potential conflicts of interest. T. C. is a full-time employee of Gilead Sciences and holds stock or stock options in the company. T. N. holds stock or stock options in Mapp Biopharmaceutical. H. F. and T. W. G. claim intellectual property for VSV-based viral hemorrhagic fever vaccines. G. K. has received grants or contracts from the Defense Advanced Research Projects Agency for a phase 1 clinical trial of an Ebola vaccine boost (ended in March 2023). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2023.)

Lutzomyia longipalpis is known as one of the primary insect vectors of visceral leishmaniasis. For such ectothermic organisms, the ambient temperature is a critical life factor. However, the impact of temperature has been ignored in many induced-stress situations of the vector life. Therefore, this study explored the interaction of Lu. longipalpis with temperature by evaluating its behaviour across a thermal gradient, thermographic recordings during blood-feeding on mice, and the gene expression of heat shock proteins (HSP) when insects were exposed to extreme temperature or infected. The results showed that 72 h after blood ingestion, Lu. longipalpis became less active and preferred relatively low temperatures. However, at later stages of blood digestion, females increased their activity and remained at higher temperatures. Real-time imaging showed that the body temperature of females can adjust rapidly to the host and remain constant until the end of blood-feeding. Insects also increased the expression of HSP90(83) during blood-feeding. Our findings suggest that Lu. longipalpis interacts with temperature by using its behaviour to avoid temperature-induced physiological damage during the gonotrophic cycle. However, the expression of certain HSP might be triggered to mitigate thermal stress in situations where a behavioural response is not the best option., (© 2022 Royal Entomological Society.)

Objective: To investigate if the diet quality and its components are associated with breast cancer risk., Methods: A case-control study was conducted with 332 women, 114 who were diagnosed with breast cancer, and 218 control individuals. Groups were matched for age, body mass index, and menopausal status. The quality of diet was assessed using Brazilian Healthy Eating Index Revised (BHEI-R) and its components. Food consumption was measured through three 24-h dietary recalls and assessed using the NDS-R software. For statistical analyses, it was performed an adjusted logistic regression, estimation of the Odds Ratio (OR), and 95% confidence interval (95%CI), with a p-value <0.05., Results: The BHEI-R score, classified into quartiles, did not differ between groups in the lowest quartile of diet quality (p=0.853). The components total cereals (p=0.038), saturated fat (p=0.039) and Gord&#95;AA (fat, alcohol, and added sugar) (p=0.023) had higher scores among the case group. The scores for total fruits (p=0.010) and milk and dairy products (p=0.039) were higher among the control group. The BHEI-R components and the quality of diet were not associated with the outcome., Conclusion: Diet quality, assessed by the BHEI-R and its components, was not associated with breast cancer.

The Ebola virus disease outbreak that occurred in Western Africa from 2013-2016, and subsequent smaller but increasingly frequent outbreaks of Ebola virus disease in recent years, spurred an unprecedented effort to develop and deploy effective vaccines, therapeutics, and diagnostics. This effort led to the U.S. regulatory approval of a diagnostic test, two vaccines, and two therapeutics for Ebola virus disease indications. Moreover, the establishment of fieldable diagnostic tests improved the speed with which patients can be diagnosed and public health resources mobilized. The United States government has played and continues to play a key role in funding and coordinating these medical countermeasure efforts. Here, we describe the coordinated U.S. government response to develop medical countermeasures for Ebola virus disease and we identify lessons learned that may improve future efforts to develop and deploy effective countermeasures against other filoviruses, such as Sudan virus and Marburg virus.

Background: Ebola virus disease (EVD) supportive care strategies are largely guided by retrospective observational research. This study investigated the effect of EVD supportive care algorithms on duration of survival in a controlled nonhuman primate (NHP) model., Methods: Fourteen rhesus macaques were challenged intramuscularly with a target dose of Ebola virus (1000 plaque-forming units; Kikwit). NHPs were allocated to intensive care unit (ICU)-like algorithms (n = 7), intravenous fluids plus levofloxacin (n = 2), or a control group (n = 5). The primary outcome measure was duration of survival, and secondary outcomes included changes in clinical laboratory values., Results: Duration of survival was not significantly different between the pooled ICU-like algorithm and control groups (8.2 vs 6.9 days of survival; hazard ratio; 0.50; P = .25). Norepinephrine was effective in transiently maintaining baseline blood pressure. NHPs treated with ICU-like algorithms had delayed onset of liver and kidney injury., Conclusions: While an obvious survival difference was not observed with ICU-like care, clinical observations from this model may aid in EVD supportive care NHP model refinement., (Published by Oxford University Press for the Infectious Diseases Society of America 2020.)

Background: Evidence suggests that chemotherapy (CT) leads to unfavorable outcomes on nutritional and metabolic profile; however, this is poorly understood., Objective: To evaluate the impact of CT on body composition, bone mineral density (BMD), insulin resistance, lipid markers related to atherosclerotic cardiovascular diseases in women recently diagnosed with breast cancer according to menopausal status., Methods: This is a prospective study that enrolled women newly diagnosed with stage II-III breast cancer (2014-18). Body composition were measured by dual-energy x-ray absorptiometry. Blood samples were collected to assess lipid profile, insulin resistance and sensitivity, visceral adiposity index and lipid accumulation product were calculated. Dietary intake, physical activity and function were also evaluated at the time of breast cancer diagnosis and after CT completion., Results: Ninety-nine women (40.4% in the premenopausal stage) aged 51 ± 1 years took part in this study. CT duration was 197 ± 27 days and main regimen was anthracyclines with taxanes (88.9%). CT was associated with an increase in total and central adiposity, insulin resistance, and all lipid-related markers, and a decrease in appendicular lean mass index, BMD and HDL-c concentration. Premenopausal women experienced greater unfavorable outcomes on adiposity markers and BMD compared to postmenopausal women (p < 0.01). No changes were observed in dietary intake and physical activity after CT., Conclusions: Breast cancer CT negatively impacted body composition and metabolic profile. Premenopausal women experienced greater unfavorable impact on adiposity markers and BMD., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The establishment of correlates of protection is particularly relevant in the context of rare, highly lethal pathogens such as filoviruses. We previously demonstrated that an Ebola glycoprotein virus-like particle (VLP) vaccine, when given as two intramuscular doses, conferred protection from challenge in a murine challenge model. In this study, we compared the ability of Advax inulin-based adjuvant formulations (Advax1-4) to enhance Ebola VLP vaccine protection in mice. After two immunizations, Advax-adjuvants that included a TLR9 agonist component induced high IgG responses, with complete protection against Ebola virus challenge. Although anti-Ebola IgG levels waned over time, protection was durable and was still evident 150 days post-immunization. Mice were protected after just a single VLP immunization with Advax-2 or -4 adjuvants. Advax-adjuvanted VLPs induced a stronger IFN-γ, TNF and IL-12 signature and serum transferred from Advax-adjuvanted vaccinees was able to transfer protection to naïve animals, showing that Ebola protection can be achieved by antibodies in the absence of cellular immunity. By contrast, serum from vaccinees incorporating a pICLC adjuvant did not transfer protection despite high IgG levels on ELISA. These data highlight the importance of adjuvant selection for development of a successful Ebola VLP vaccine., Competing Interests: Declaration of Competing Interest NP is affiliated with Vaxine Pty Ltd, a company having a financial interest in Advax adjuvants. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The West Africa Ebola virus disease outbreak of 2014-2016 demonstrated that responses to viral hemorrhagic fever epidemics must go beyond emergency stopgap measures and should incorporate high-quality medical care and clinical research. Optimal patient management is essential to improving outcomes, and it must be implemented regardless of geographical location or patient socioeconomic status. Coupling clinical research with improved care has a significant added benefit: Improved data quality and management can guide the development of more effective supportive care algorithms and can support regulatory approvals of investigational medical countermeasures (MCMs), which can alter the cycle of emergency response to reemerging pathogens. However, executing clinical research during outbreaks of high-consequence pathogens is complicated and comes with ethical and research regulatory challenges. Aggressive care and excellent quality control must be balanced by the requirements of an appropriate infection prevention and control posture for healthcare workers and by overcoming the resource limitations inherent in many outbreak settings. The Joint Mobile Emerging Disease Intervention Clinical Capability was established in 2015 to develop a high-quality clinical trial capability in Uganda to support rigorous evaluation of MCMs targeting high-consequence pathogens like Ebola virus. This capability assembles clinicians, laboratorians, clinical researchers, logisticians, and regulatory professionals trained in infection prevention and control and in good clinical and good clinical laboratory practices. The resulting team is prepared to provide high-quality medical care and clinical research during high-consequence outbreaks.

Following the 2013-2016 Ebola virus outbreak in West Africa, numerous groups advocated for the importance of executing clinical trials in outbreak settings. The difficulties associated with obtaining reliable data to support regulatory approval of investigational vaccines and therapeutics during that outbreak were a disappointment on a research and product development level, as well as on a humanitarian level. In response to lessons learned from the outbreak, the United States Department of Defense established a multi-institute project called the Joint Mobile Emerging Disease Intervention Clinical Capability (JMEDICC). JMEDICC's primary objective is to establish the technical capability in western Uganda to execute clinical trials during outbreaks of high-consequence pathogens such as the Ebola virus. A critical component of clinical trial execution is the establishment of laboratory operations. Technical, logistical, and political challenges complicate laboratory operations, and these challenges have been mitigated by JMEDICC to enable readiness for laboratory outbreak response operations., Competing Interests: The authors have declared that no competing interests exist.

Identification of modifiable risk factors for breast cancer is critical for primary prevention of the disease. The aim of this study was to evaluate how certain lifestyle variables modify the chances of developing breast cancer based on menopausal status. A case-control study was performed in a group of 542 women, 197 who were diagnosed with breast cancer and 344 control individuals. The groups were matched by age, body mass index, and menopausal status. Participants were evaluated for level of physical activity, alcohol consumption, smoking habit, weight, height, and waist circumference (WC). A multivariate logistic regression model was used to estimate odds ratios and 95% confidence intervals (95% CI). Regular consumption of alcoholic beverages (2.91, 95% CI 1.58-5.38 and 1.86, 95% CI 1.15-3.03) and sedentary behavior (2.08; 95% CI 1.12-3.85 and 1.81; 95% CI 1.12-2.94) were associated with breast cancer risk in pre- and postmenopausal women, respectively. High WC (3.31, 95% CI 1.45-7.55) was associated with an increased risk of developing breast cancer in premenopausal women. While in postmenopausal women, current smoking (2.43, 95% CI 1.01-5.83) or previous history of smoking (1.90; 95% CI 1.14-3.14) increased the chances of developing breast cancer. Sedentary behavior and current consumption of alcoholic beverages were more likely to increase the risk of developing breast cancer regardless of menopausal status.

Most alphaviruses are mosquito-borne and can cause severe disease in domesticated animals and humans. The most notable recent outbreak in the Americas was the 2014 chikungunya virus (CHIKV) outbreak affecting millions and producing disease highlighted by rash and arthralgia. Chikungunya virus is a member of the Semliki Forest (SF) serocomplex, and before its arrival in the Americas, two other member of the SF complex, Una (UNAV) and Mayaro (MAYV) viruses, were circulating in Central and South America. This study examined whether antibodies from convalescent CHIKV patients could cross-neutralize UNAV and MAYV. Considerable cross-neutralization of both viruses was observed, suggesting that exposure to CHIKV can produce antibodies that may mitigate infection with UNAV or MAYV. Understanding the impact of CHIKV exposure on population susceptibility to other emerging viruses may help predict outbreaks; moreover, identification of cross-reactive immune responses among alphaviruses may lead to the development of vaccines targeting multiple viruses.

Objective: The objective of this study was to verify possible associations between bone mineral density (BMD) and breast cancer in recently diagnosed women in the Brazilian Mid-west region, considering the menopausal status of patients., Methods: A case-control study was conducted with 142 cases of breast cancer and 234 controls matched by for age, body mass index (BMI), and menopausal status (pre- and postmenopause), performed in a university hospital in the Brazilian Mid-west. Lumbar spine (L1-L4), femoral neck, and total femur BMD were measured by the dual-energy X-ray absorptiometry (DXA) method. For association, a logistic regression analysis was used., Results: Women in the highest lumbar spine BMD quartile presented had a higher chance of developing breast cancer (OR = 2.31; 1.02-5.25; p = 0.045), after adjusting for the confounding variables. Nonetheless, there were no statistically significant differences in the association between pre- and postmenopause in that quartile and breast cancer., Conclusions: High lumbar spine BMD was positively associated with breast cancer in the total sample. In evaluating the BMD of the femoral neck and total femur, such an association was not observed.

Objective:  The aim of the present study was to assess the anthropometric measures, food intake and food cravings during the menstrual cycle of undergraduate students of the faculty of nutrition., Methods:  A cross-sectional study was performed with 27 students from a public university in the state of Mato Grosso do Sul, Brazil, who had their food intake evaluated through a 24-hour food recall, their nutritional status evaluated based on anthropometric measures, and food cravings evaluated using the Food Desire Questionnaire. Data were collected during an evaluation in the follicular phase (between the 5 th and the 9 th day of the menstrual cycle) and another in the luteal phase (LP) (between the 20 th and the 25 th day of the menstrual cycle). For food intake variables, the analysis of variance (ANOVA) test was used, followed by the Tukey test. The Mann-Whitney test was used for the analysis of food cravings, considering a significance level of 5% ( p  < 0.05)., Results:  The desire for foods rich in sugar, salt, and fat, such as chocolate, pastries, snacks and desserts were higher ( p  < 0.05) during the premenstrual period, although it did not reflect neither a higher energy intake nor an alteration in the distribution of macronutrients. A higher intake of carbohydrates, proteins, fibers, and calcium was observed during the LP; however, without statistical difference between the groups. There were no differences either in the intake of any food group or in the anthropometric measurements ( p  > 0.05)., Conclusion:  Food cravings of nutrition students differed between the phases of the menstrual cycle; however, with no difference in food intake and in anthropometric measures., Competing Interests: The authors have stated explicitly that there are no conflicts of interest in connection with the present article., (Thieme Revinter Publicações Ltda Rio de Janeiro, Brazil.)

Objective: To examine whether breast cancer is associated with body composition and level of physical activity, considering the menstrual status., Methods: This was a case-control study with 116 women recently diagnosed with breast cancer and 226 controls. Body composition was assessed by dual-energy X-ray absorptiometry, and cardiometabolic risk was assessed by conicity index and waist-to-height ratio. The short version of the International Physical Activity Questionnaire was used to estimate the level of physical activity. All analyses were adjusted for age and BMI., Results: The total body fat percentage, android body fat, android-gynoid ratio, and waist circumference were positively associated (p < 0.05), whereas the percentage of lean body mass (p <0.05) and the level of physical activity (p < 0.01) were inversely associated with breast cancer in premenopausal women. Among postmenopausal women, physical activity decreased the chance of developing breast cancer by 49% (95% CI = 0.29 to 0.92, p = 0.02)., Conclusion: A low percentage of lean body mass and high abdominal adiposity in the premenopausal period increase the chances of developing breast cancer. Regular physical activity is inversely associated with breast cancer in pre- and postmenopausal women.

Differentiating between illness caused by community-acquired respiratory pathogens versus infection by biothreat agents is a challenge. This review highlights respiratory and clinical features of category A and B potential biothreat agents that have respiratory features as their primary presenting signs and symptoms. Recent world events make such a reminder that the possibility of rare diseases and unlikely events can occur timely for clinicians, policymakers, and public health authorities. Despite some distinguishing features, nothing can replace good clinical acumen and a strong index of suspicion in the diagnosis of uncommon infectious diseases.

Introduction: in recent decades, lifestyle changes in women involving physical inactivity, insulin resistance and body fat distribution have been associated with an increase in breast cancer., Objective: to assess whether insulin resistance, lipid profile, and visceral adiposity are associated with increased risk of breast cancer., Methods: a hospital-based case control study was conducted with 116 women newly diagnosed with breast cancer and 226 controls. Body mass index, waist circumference, total cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL), triglycerides, glycated hemoglobin, HOMA-IR, HOMA-β, lipid accumulation product (LAP), and visceral adiposity index (VAI) were assessed. Logistic regression was adjusted for body mass index and age to quantify the association between breast cancer risk and insulinresistance, dyslipidemias, and visceral adiposity., Results: the case group had higher insulin resistance (p < 0.001), LAP (p = 0.012), and VAI (p = 0.004), and lower concentrations of HDL (p = 0.024) and HOMA-β(p = 0.010) compared to the control. Insulin resistance (OR = 3.00, 95% CI: 1.75-5.17, p < 0.001) and higher VAI (OR = 1.91, 95% CI: 1.17-3.13, p = 0.01) were associated with breast cancer, whereas the highest concentration of HDL reduces the chances of cancer by 53% (95% CI: 0.32-0.86, p = 0.026). In the multivariate analysis, only LAP and VAI were associated to breast cancer., Conclusions: visceral fat accumulation increases the risk of breast cancer.

Humoral responses are essential for the protective efficacy of most Ebola virus (EBOV) candidate vaccines; however, the in vivo development of protective anti-EBOV B-cell responses is poorly defined. Here, by using the virus-like particle (VLP) as a model antigen, we demonstrate that humoral responses are generated through follicular B-cell and T-cell-dependent mechanisms in a mouse model of EBOV infection. In addition, we show that the inclusion of the clinical-grade dsRNA adjuvant known as poly-ICLC in VLP vaccinations both augments and sustains germinal center B-cell reactions, antigen-specific B-cell frequencies and anti-EBOV serum titers. Finally, we used mice that were deficient in either B-cells or T-cell-dependent antibody production to distinguish the contributing roles of EBOV humoral responses. We demonstrate that while anti-EBOV antibody responses promote protection, VLP-vaccinated mice can survive EBOV infection in the absence of detectable anti-EBOV antibodies. Moreover, we found that adjuvant signaling could circumvent the complete requirement for B-cell immunity in protection against EBOV. Collectively, these studies may prove valuable for the characterization and future development of additional EBOV vaccine candidates.

Despite the unprecedented Ebola virus outbreak response in West Africa, no Ebola medical countermeasures have been approved by the US Food and Drug Administration. However, multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk-pathogen setting. Numerous therapeutics were explored or developed during the outbreak, including repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir, favipiravir, and GS-5734), nucleic acid-based drugs (TKM-Ebola and AVI-7537), and immunotherapeutics (convalescent plasma and ZMapp). We review Ebola therapeutics progress in the aftermath of the West Africa Ebola virus outbreak and attempt to offer a glimpse of a path forward.

Background: Ebola virus like particles (EBOV VLPs, eVLPs), are produced by expressing the viral transmembrane glycoprotein (GP) and structural matrix protein VP40 in mammalian cells. When expressed, these proteins self-assemble and bud from 'host' cells displaying morphology similar to infectious virions. Several studies have shown that rodents and non-human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin-like domain of envelope glycoprotein GP1 serves as the major target for a productive humoral immune response. Therefore GP1 concentration is a critical quality attribute of EBOV vaccines and accurate measurement of the amount of GP1 present in eVLP lots is crucial to understanding variability in vaccine efficacy., Methods: After production, eVLPs are characterized by determining total protein concentration and by western blotting, which only provides semi-quantitative information for GP1. Therefore, a liquid chromatography high resolution mass spectrometry (LC-HRMS) approach for accurately measuring GP1 concentration in eVLPs was developed. The method employs an isotope dilution strategy using four target peptides from two regions of the GP1 protein. Purified recombinant GP1 was generated to serve as an assay standard. GP1 quantitation in 5 eVLP lots was performed on an LTQ-Orbitrap Elite and the final quantitation was derived by comparing the relative response of 200 fmol AQUA peptide standards to the analyte response at 4 ppm., Results: Conditions were optimized to ensure complete tryptic digestion of eVLP, however, persistent missed cleavages were observed in target peptides. Additionally, N-terminal truncated forms of the GP1 protein were observed in all eVLP lots, making peptide selection crucial. The LC-HRMS strategy resulted in quantitation of GP1 with a lower limit of quantitation of 1 fmol and an average percent coefficient of variation (CV) of 7.6 %. Unlike western blot values, the LC-HRMS quantitation of GP1 in 5 eVLP vaccine lots exhibited a strong linear relationship (positive correlation) with survival (after EBOV challenge) in mice., Conclusions: This method provides a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP1. By monitoring variability in GP1 content, the eVLP production process can be optimized, and the total amount of GP1 needed to confer protection accurately determined.

Filoviruses are the etiological agents of two human illnesses: Ebola virus disease and Marburg virus disease. Until 2013, medical countermeasure development against these afflictions was limited to only a few research institutes worldwide as both infections were considered exotic due to very low case numbers. Together with the high case-fatality rate of both diseases, evaluation of any candidate countermeasure in properly controlled clinical trials seemed impossible. However, in 2013, Ebola virus was identified as the etiological agent of a large disease outbreak in Western Africa including almost 30,000 infections and more than 11,000 deaths, including case exportations to Europe and North America. These large case numbers resulted in medical countermeasure development against Ebola virus disease becoming a global public-health priority. This review summarizes the status quo of candidate vaccines against Ebola virus disease, with a focus on those that are currently under evaluation in clinical trials., Competing Interests: Declaration of Interests The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Background: Filovirus virus-like particles (VLP) are strong immunogens with the potential for development into a safe, non-infectious vaccine. However, the large size and filamentous structure of this virus has heretofore made production of such a vaccine difficult. Herein, we present new assays and a purification procedure to yield a better characterized and more stable product., Methods: Sonication of VLP was used to produce smaller "nano-VLP", which were purified by membrane chromatography. The sizes and lengths of VLP particles were analyzed using electron microscopy and an assay based on transient occlusion of a nanopore. Using conformationally-sensitive antibodies, we developed an in vitro assay for measuring GP conformational integrity in the context of VLP, and used it to profile thermal stability., Results: We developed a new procedure for rapid isolation of Ebola VLP using membrane chromatography that yields a filterable and immunogenic product. Disruption of VLP filaments by sonication followed by filtration produced smaller particles of more uniform size, having a mean diameter close to 230 nm. These reduced-size VLP retained GP conformation and were protective against mouse-adapted Ebola challenge in mice. The "nano-VLP" consists of GP-coated particles in a mixture of morphologies including circular, branched, "6"-shaped, and filamentous ones up to ~1,500 nm in length. Lyophilization conferred a high level of thermostability on the nano-VLP. Unlike Ebola VLP in solution, which underwent denaturation of GP upon moderate heating, the lyophilized nano-VLP can withstand at least 1 h at 75°C, while retaining conformational integrity of GP and the ability to confer protective immunity in a mouse model., Conclusions: We showed that Ebola virus-like particles can be reduced in size to a more amenable range for manipulation, and that these smaller particles retained their temperature stability, the structure of the GP antigen, and the ability to stimulate a protective immune response in mice. We developed a new purification scheme for "nano-VLP" that is more easily scaled up and filterable. The product could also be made thermostable by lyophilization, which is highly significant for vaccines used in tropical countries without a reliable "cold-chain" of refrigeration.

Pathogen-associated molecular patterns (PAMPs) are stand-alone immunomodulators or 'danger signals,' that are increasingly recognized as critical components of many modern vaccines. Polyinosinic-polycytidylic acid (poly-IC) is a synthetic dsRNA that can activate multiple elements of the host defense in a pattern that parallels that of a viral infection. When properly combined with an antigen, it can be utilized as a PAMP-adjuvant, resulting in modulation and optimization of the antigen-specific immune response. We briefly review the preclinical and clinical uses of poly-IC and two poly-IC derivatives, poly-IC12U (Ampligen) and poly-ICLC (Hiltonol), as vaccine adjuvants.