bluuebunny/crossref_metadata_2025 huggingface dataset:
Microsurgical Reconstruction of Foot Defects: A Case Series with Long-Term Follow-Up
Authors: Breidung David , Fikatas Panagiotis , Mandal Patrick , Berns Maresa D. , Barth Andrè A. , Billner Moritz , Megas Ioannis-Fivos , Reichert Bert
Published: April 2022

(1) Background: Microsurgical reconstruction of foot defects with free flaps is rare as it is a challenging task for a surgeon. For extensive defects, advanced surgical procedures, such as free flap transfer with microsurgical anastomosis, may be the last chance to avoid major amputation. The aim of the study was to examine the opportunities and risks posed by free flap reconstruction of foot defects and to illustrate in which situations reconstruction is useful on the basis of case characteristics. (2) Methods: In this study, we retrospectively analyzed data of cases with free flap reconstruction of the foot from 2007 to 2022. Therefore, demographic data, comorbidities, information about the defect situation, data on the operational procedure, and complications were evaluated. (3) Results: A total of 27 cases with free flap coverage of foot defects could be included. In 24 of these cases (89%), defect coverage was successful. In 18 patients, some form of complication occurred in the postoperative stage. The most frequently used flap was the latissimus dorsi flap, with 13 procedures. (4) Conclusions: Foot reconstruction using free flaps is a proven procedure for the treatment of larger foot defects and can offer a predominantly good functional outcome. The lengthy process and possible complications should be thoroughly discussed in advance so as to provide criteria, suitably adjusted to the individual prerequisites of the patients, for deciding whether limb salvage using advanced surgical procedures should be attempted.
Optimization of Callus and Cell Suspension Cultures of Lycium schweinfurthii for Improved Production of Phenolics, Flavonoids, and Antioxidant Activity
Authors: Mamdouh Diaa , Smetanska Iryna
Published: April 2022

Lycium schweinfurthii is a traditional medicinal plant grown in the Mediterranean region. As it is used in folk medicine to treat stomach ulcers, it took more attention as a source of valuable secondary metabolites. The in vitro cultures of L. schweinfurthii could be a great tool to produce secondary metabolites at low costs. The presented study aimed to introduce and optimize a protocol for inducing callus and cell suspension cultures as well as estimating phenolic, flavonoid compounds, and antioxidant activity in the cultures of the studied species. Three plant growth regulators (PGRs) were supplemented to MS medium solely or in combination to induce callus from leaf explants. The combination between 2,4-dichlorophenoxy acetic acid (2,4-D) and 1-naphthyl acetic acid (NAA) induced callus in all explants regardless of the concentration. The highest fresh weight of callus (3.92 g) was obtained on MS medium fortified with 1 mg L−1 of both 2,4-D and NAA (DN1) after 7 weeks of culture. DN1 was the best medium for callus multiplication regarding the increase in fresh weight and size of callus. Otherwise, the highest phenolics, flavonoids, and antioxidant activity against DPPH free radicals were of callus on MS fortified with 2 mg L−1 NAA (N2). The cell suspension cultures were cultivated on a liquid N2 medium with different sucrose concentrations of 5–30 g L−1 to observe the possible effects on cells’ multiplication and secondary metabolite production. The highest fresh and viable biomass of 12.01 g was obtained on N2 containing 30 g L−1 sucrose. On the other hand, the cell cultures on N2 medium of 5 and 30 g L−1 sucrose produced phenolics and flavonoids, and revealed antioxidant activity against DPPH and ABTS+ free radicals more than other sucrose concentrations. The presented protocol should be useful in the large-scale production of phenolic and flavonoid compounds from callus and cell cultures of L. schweinfurthii.
Factors Associated with Colorectal Cancer Screening in Spain: Results of the 2017 National Health Survey
Authors: Nouni-García Rauf , Lara-López Ángela , Carratalá-Munuera Concepción , Gil-Guillén Vicente F. , López-Pineda Adriana , Orozco-Beltrán Domingo , Quesada Jose A.
Published: April 2022

This study aimed to determine the CRC screening coverage of people aged between 50 and 69 years who were living in Spain in 2017 and describe the factors associated with not having had a faecal occult blood test (FOBT). A cross-sectional study was performed using data from the Spanish National Health Survey 2017. We analysed 7568 individuals between the ages of 50 and 69 years. The proportion of respondents between 50 and 69 years old who had had an FOBT was 29.0% (n = 2191). The three autonomous communities with the lowest proportion of respondents who had had an FOBT were Extremadura (8.7%, n = 16), Ceuta–Melilla (10.4%, n = 3), and Andalucia (14.1%, n = 186). The variables associated with not having had an FOBT were being 50–54 years old (PR = 1.09; 95% CI 1.04–1.14), having been born outside of Spain (PR = 1.11; 95% CI 1.06–1.16), not having been vaccinated against the flu (PR = 1.09; 95% CI 1.04–1.15), never having had a colonoscopy (PR = 1.49; 95% CI 1.40–1.59), not having had an ultrasound scan in the last year (PR = 1.09; 95% CI 1.04–1.14), and not having seen a primary care physician in the last month (PR = 1.08; 95% CI 1.04–1.12). The factors associated with not getting an FOBT were young age, having been born outside of Spain, not having been vaccinated against the flu in the last campaign, and not making frequent use of healthcare services.
Nursing Students’ Retention of Knowledge by Basic Knowledge Type: An Exploratory Study
Authors: Kawasaki Hiromi , Yamasaki Satoko , Fukita Susumu , Iwasa Mika , Iki Tomoko
Published: April 2022

Students’ interests help determine their learning effectiveness and knowledge acquisition and retention. It is necessary to confirm whether there is a difference in the way in which content being learned is remembered by the content type. In this study, we examined the characteristics of nursing students’ retention of physiological knowledge and environmental knowledge by utilizing scores obtained in class. The participants comprised 57 nursing students who had taken a class twice—once in their second year and once in their third year. Before and after each class, students completed an 11-question survey with human health and comfort items based on nursing core competencies and Sphere standards. The correct answer rate was calculated using a logistic regression model to account for inter- and intra-individual variations. The estimated correct answer rate per individual showed one of three trends: (1) increasing and decreasing depending on the lesson topic (knowledge type), (2) increasing overall after decreasing, and (3) increasing gradually. Physiological knowledge was retained well, whereas knowledge pertaining to the environment was retained poorly. Even with knowledge of the environment, the knowledge that students apply to their daily lives and social events was maintained.
Antimicrobial Activity of Sunflower (Helianthus annuus) Seed for Household Domestic Water Treatment in Buhera District, Zimbabwe
Authors: Mahamba Caston , Palamuleni Lobina G.
Published: April 2022

Various plants have been used by humans for a very long time, and the uses vary, including food, medicine, toothpaste, dyes, food preservatives, water treatment, and beer brewing, among others. For food preservation and water treatment, the plant must have antimicrobial properties which are biocidal. For this research, extracts were obtained from sunflower (Helianthus annuus) seeds. The extracts were assessed for the presence of antimicrobial properties against three groups of bacteria, including faecal coliforms, total coliforms, and Escherichia coli (E. coli). Dosages of ground sunflower seeds ranging from 0.5 g to 4 g were administered to the three bacterial species and their susceptibilities to the antimicrobial agents were measured and recorded. The results indicate the presence of antimicrobial properties in sunflower. The antimicrobial activities were more effective on E. coli, with an average zone of inhibition of 12 mm with a 3 g dosage of sunflower seed extract. This was followed by total coliforms (11 mm) and lastly faecal coliforms (11 mm). These findings suggested that sunflower seeds proved to be potentially effective in treating water against microbial contaminants.
Young People in the Social World of Physical Activities: Meanings and Barriers
Authors: Koski Pasi , Hirvensalo Mirja , Villberg Jari , Kokko Sami
Published: April 2022

Physical inactivity has become one of the leading risk factors for non-communicable diseases (NCDs) and death worldwide. From the future perspective it is alarming that in the group of young people few meet the recommendations. In this respect, physical activity promotion in general and physical education have challenges and new approaches are needed. In this study, the theoretical framework is based on the physical activity relationship (PAR) approach and the barriers were grouped according to the ecological model. The aim of the study was firstly to present both the meanings and barriers of physical activity in a comprehensive Finnish population of 11–15 year old (n = 2728) and secondly to examine how the number of important meanings and mentioned barriers associate with each other with physical activity levels. Data were collected using a questionnaire. To examine how the meanings and barriers associate with each other and with the PA level, chi-squared test (χ2), Pearson correlation and General linear model (ANCOVA) were used. Logistic regression was applied to estimate effect sizes by odds ratios and 95% confidence intervals. According to the results, the associations between physical activity with the meanings and barriers were reverse and linear. The more important the meanings were found to be, the more likely the study participants were physically active, whereas the more barriers participants reported, the less active they were. The approach which utilizes meanings and barriers has a lot of untapped potential for the promotion of physical activity and physical education. With the right actions, some barriers could be removed or dampened, and by opening up and deepening meanings, PAR could be strengthened.
Surveillance of Indoor Air Concentration of Volatile Organic Compounds in Luxembourgish Households
Authors: Alvarez-Vaca Daniel , Duca Radu Corneliu , Borras-Santos Alicia , Hardy Emilie , Creta Matteo , Eicher Carole , Wurth Laurence , Vergison Anne , Van Nieuwenhuyse An
Published: April 2022

Exposure to air pollution is a well-known health risk. For instance, volatile and very volatile organic compounds (VOCs and VVOCs) are known to cause respiratory, haematologic or immune diseases, and even cancer. Based on the Luxembourgish indoor pollution surveillance program, we performed an exploratory analysis for the period 2014–2019, in order (1) to evaluate the prevalence of VOCs and VVOCs in households, and (2) to estimate the risks of lifelong exposure to selected VOCs on the health of the adult population. The database included 715 indoor air samples from 159 different households. Observed VOC and VVOC levels were similar to those in neighbouring countries. Our health impact assessment identified some health risks associated with the observed concentrations in Luxembourg. Furthermore, this study shows the major public health importance of having a national indoor pollution surveillance system in place. Highlights: (1) This study provides an overview of the domestic indoor pollution in Luxembourg. (2) (V)VOCs levels in Luxembourg were similar to those in neighbouring countries. (3) The results clearly show the importance of having a surveillance system in place.
Alleviation of Tris(2-chloroethyl) Phosphate Toxicity on the Marine Rotifer Brachionus plicatilis by Polystyrene Microplastics: Features and Molecular Evidence
Authors: Ma Wenqian , Sun Zijie , Zhang Xin , Tang Xuexi , Zhang Xinxin
Published: April 2022

As emerging pollutants, microplastics (MPs) and organophosphorus esters (OPEs) coexist in the aquatic environment, posing a potential threat to organisms. Although toxicological studies have been conducted individually, the effects of combined exposure are unknown since MPs can interact with OPEs acting as carriers. In this study, we assessed the response of marine rotifer, Brachionus plicatilis, to co-exposure to polystyrene MPs and tris(2-chloroethyl) phosphate (TCEP) at different concentrations, including population growth, oxidative status, and transcriptomics. Results indicated that 0.1 μm and 1 μm MPs were accumulated in the digestive system, and, even at up to 2000 μg/L, they did not exert obvious damage to the stomach morphology, survival, and reproduction of B. plicatilis. The presence of 1 μm MPs reversed the low population growth rate and high oxidative stress induced by TCEP to the normal level. Some genes involved in metabolic detoxification and stress response were upregulated, such as ABC and Hsp. Subsequent validation showed that P-glycoprotein efflux ability was activated by combined exposure, indicating its important role in the reversal of population growth inhibition. Such results challenge the common perception that MPs aggravate the toxicity of coexisting pollutants and elucidate the molecular mechanism of the limited toxic effects induced by MPs and TCEP.
Foliar Infiltration of Virus-Derived Small Hairpin RNAs Triggers the RNAi Mechanism against the Cucumber Mosaic Virus
Authors: Villegas-Estrada Bernardo , Sánchez Manuel Alejandro , Valencia-Jiménez Arnubio
Published: April 2022

Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs) and test for their potential to trigger the RNAi mechanism in Nicotiana benthamiana plants against CMV after their foliar infiltration. This approach allowed for the production of significant amounts of shRNAs (60-mers) quickly and easily. The gene silencing was confirmed using polymerase chain reaction (PCR), immunological-based assays, and real-time PCR (qPCR). The highest levels of gene silencing were recorded for mRNAs coding for replication protein (ORF1a), the viral suppressor of RNA silencing (ORF2b), and the capsid protein (ORF3b), with 98, 94, and 70% of total transcript silencing, respectively. This protocol provides an alternative to producing significant shRNAs that can effectively trigger the RNAi mechanism against CMV.
Adiponectin Deficiency Alters Placenta Function but Does Not Affect Fetal Growth in Mice
Authors: Mohan Shrestha Man , Wermelin Sanne , Stener-Victorin Elisabet , Wernstedt Asterholm Ingrid , Benrick Anna
Published: April 2022

Adiponectin administration to pregnant mice decreases nutrient transport and fetal growth. An adiponectin deficiency, on the other hand, as seen in obese women during pregnancy, alters fetal growth; however, the mechanism is unclear. To determine the role of adiponectin on placenta function and fetal growth, we used adiponectin knockout, adiponectin heterozygote that displays reduced adiponectin levels, and wild-type mice on a control diet or high fat/high sucrose (HF/HS) diet. Triglycerides (TGs) in the serum, liver, and placenta were measured using colorimetric assays. Gene expression was measured using quantitative RT-PCR. Adiponectin levels did not affect fetal weight, but it reduced adiponectin levels, increased fetal serum and placenta TG content. Wildtype dams on a HF/HS diet protected the fetuses from fatty acid overload as judged by increased liver TGs in dams and normal serum and liver TG levels in fetuses, while low adiponectin was associated with increased fetal liver TGs. Low maternal adiponectin increased the expression of genes involved in fatty acid transport; Lpl and Cd36 in the placenta. Adiponectin deficiency does not affect fetal growth but induces placental dysfunction and increases fetal TG load, which is enhanced with obesity. This could lead to imprinting effects on the fetus and the development of metabolic dysfunction in the offspring.
Peripheral Serotonin Deficiency Affects Anxiety-like Behavior and the Molecular Response to an Acute Challenge in Rats
Authors: Sbrini Giulia , Hanswijk Sabrina I. , Brivio Paola , Middelman Anthonieke , Bader Michael , Fumagalli Fabio , Alenina Natalia , Homberg Judith R. , Calabrese Francesca
Published: April 2022

Serotonin is synthetized through the action of tryptophan hydroxylase (TPH) enzymes. While the TPH2 isoform is responsible for the production of serotonin in the brain, TPH1 is expressed in peripheral organs. Interestingly, despite its peripheral localization, alterations of the gene coding for TPH1 have been related to stress sensitivity and an increased susceptibility for psychiatric pathologies. On these bases, we took advantage of newly generated TPH1−/− rats, and we evaluated the impact of the lack of peripheral serotonin on the behavior and expression of brain plasticity-related genes under basal conditions and in response to stress. At a behavioral level, TPH1−/− rats displayed reduced anxiety-like behavior. Moreover, we found that neuronal activation, quantified by the expression of Bdnf and the immediate early gene Arc and transcription of glucocorticoid responsive genes after 1 h of acute restraint stress, was blunted in TPH1−/− rats in comparison to TPH1+/+ animals. Overall, we provided evidence for the influence of peripheral serotonin levels in modulating brain functions under basal and dynamic situations.
HPV16 and HPV18 Genome Structure, Expression, and Post-Transcriptional Regulation
Authors: Yu Lulu , Majerciak Vladimir , Zheng Zhi-Ming
Published: April 2022

Human papillomaviruses (HPV) are a group of small non-enveloped DNA viruses whose infection causes benign tumors or cancers. HPV16 and HPV18, the two most common high-risk HPVs, are responsible for ~70% of all HPV-related cervical cancers and head and neck cancers. The expression of the HPV genome is highly dependent on cell differentiation and is strictly regulated at the transcriptional and post-transcriptional levels. Both HPV early and late transcripts differentially expressed in the infected cells are intron-containing bicistronic or polycistronic RNAs bearing more than one open reading frame (ORF), because of usage of alternative viral promoters and two alternative viral RNA polyadenylation signals. Papillomaviruses proficiently engage alternative RNA splicing to express individual ORFs from the bicistronic or polycistronic RNA transcripts. In this review, we discuss the genome structures and the updated transcription maps of HPV16 and HPV18, and the latest research advances in understanding RNA cis-elements, intron branch point sequences, and RNA-binding proteins in the regulation of viral RNA processing. Moreover, we briefly discuss the epigenetic modifications, including DNA methylation and possible APOBEC-mediated genome editing in HPV infections and carcinogenesis.
Microrheological Phenomenon and Mechanical Properties of High-Aspect-Ratio Microgroove Injection Moulding of Kaolin/PP Composites
Authors: Lou Yan , Zhou Xiangwei , Zhang Dongyue , Cheng Fengyu
Published: April 2022

The microrheological phenomenon of kaolin-filled polypropylene (kaolin/PP) composites was investigated for the first time. The microviscosity of kaolin/PP composites was studied by changing the melt temperature and shear rate. Then, injection moulding experiments of rectangular microgrooves with different aspect ratios using kaolin/PP composites and mechanical property tests of the samples were carried out. The results showed that with increasing kaolin content, the microviscosity of the kaolin/PP composites gradually increases. The shear rate had the greatest influence on the microviscosity, and the kaolin content had the least influence. When the aspect ratio of rectangular microgrooves is small, with an increasing kaolin content, the microgroove filling rate increases, and the microstructured sample geometric shape replication effect is good; however, when the aspect ratio reaches 10:1, the microgroove filling rate decreases with an increasing kaolin content. The microstructured sample geometric shape replication effect is also poor, and size effects appear. Different factors control the microrheological morphology of composites with different aspect ratios, including the shear deformation and viscous flow of composites. The increase in kaolin content leads to a decrease in the friction coefficient and an increase in the wear resistance of the composites. We concluded that the best composite formulation for kaolin/PP composites in microinjection is the 7KL/PP composite with 7% kaolin. When the aspect ratio is 5:1, the reproduction of the microstructured sample geometry is the best, and the comprehensive mechanical properties of the sample are the best.
Transduction of Brain Neurons in Juvenile Chum Salmon (Oncorhynchus keta) with Recombinant Adeno-Associated Hippocampal Virus Injected into the Cerebellum during Long-Term Monitoring
Authors: Pushchina Evgeniya V. , Bykova Maria E. , Shamshurina Ekaterina V. , Varaksin Anatoly A.
Published: April 2022

Corpus cerebelli in juvenile chum salmon is a multiprojective region of the brain connected via afferent and efferent projections with the higher regions of the brainstem and synencephalon, as well as with multiprojection regions of the medulla oblongata and spinal cord. During the postembryonic development of the cerebellum in chum salmon, Oncorhynchus keta, the lateral part of the juvenile cerebellum gives rise to the caudomedial part of the definitive cerebellum, which is consistent with the data reported for zebrafish and mouse cerebellum. Thus, the topographic organization of the cerebellum and its efferents are similar between fish (chum salmon and zebrafish) and mammals, including mice and humans. The distributions of recombinant adeno-associated viral vectors (rAAVs) after an injection of the base vector into the cerebellum have shown highly specific patterns of transgene expression in bipolar neurons in the latero-caudal lobe of the juvenile chum tectum opticum. The distribution of rAAVs in the dorsal thalamus, epithalamus, nucleus rotundus, and pretectal complex indicates the targeted distribution of the transgene via the thalamo-cerebellar projections. The detection of GFP expression in the cells of the epiphysis and posterior tubercle of juvenile chum salmon is associated with the transgene’s distribution and with the cerebrospinal fluid flow, the brain ventricles and its outer surface. The direct delivery of the rAAV into the central nervous system by intracerebroventricular administration allows it to spread widely in the brain. Thus, the presence of special projection areas in the juvenile chum salmon cerebellum, as well as outside it, and the identification of the transgene’s expression in them confirm the potential ability of rAAVs to distribute in both intracerebellar and afferent and efferent extracerebellar projections of the cerebellum.
Nanoparticulate Photoluminescent Probes for Bioimaging: Small Molecules and Polymers
Authors: Lee Sanghyuck , Park Chul Soon , Yoon Hyeonseok
Published: April 2022

Recent interest in research on photoluminescent molecules due to their unique properties has played an important role in advancing the bioimaging field. In particular, small molecules and organic dots as probes have great potential for the achievement of bioimaging because of their desirable properties. In this review, we provide an introduction of probes consisting of fluorescent small molecules and polymers that emit light across the ultraviolet and near-infrared wavelength ranges, along with a brief summary of the most recent techniques for bioimaging. Since photoluminescence probes emitting light in different ranges have different goals and targets, their respective strategies also differ. Diverse and novel strategies using photoluminescence probes against targets have gradually been introduced in the related literature. Among recent papers (published within the last 5 years) on the topic, we here concentrate on the photophysical properties and strategies for the design of molecular probes, with key examples of in vivo photoluminescence research for practical applications. More in-depth studies on these probes will provide key insights into how to control the molecular structure and size/shape of organic probes for expanded bioimaging research and applications.
Effects of Pituitary Adenylate Cyclase Activating Polypeptide on Cell Death
Authors: Horvath Gabriella , Reglodi Dora , Fabian Eszter , Opper Balazs
Published: April 2022

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated as a hypothalamic peptide based on its efficacy to increase adenylate cyclase (AC) activity. It has a widespread distribution throughout the body including the nervous system and peripheral organs, where PACAP exerts protective effects both in vivo and in vitro through its anti-apoptotic, anti-inflammatory, and antioxidant functions. The aim of the present paper was to review the currently available literature regarding the effects of PACAP on cell death in vitro in neural and non-neural cells. Among others, its effect on apoptosis can be detected in cerebellar granule cells against different toxic stimuli. Different neural cell types from the cerebral cortex are also prevented from cell death. PACAP also shows effects on cell death in cells belonging to the peripheral nervous system and protects both neural and non-neural cells of sensory organs. In addition, cell survival-promoting effect can be observed in different peripheral organ systems including cardiovascular, immune, respiratory, gastrointestinal, urinary, and reproductive systems. The studies summarized here indicate its noteworthy effect on cell death in different in vitro models, suggesting PACAP’s potential therapeutic usage in several pathological conditions.
Roles of Abscisic Acid and Gibberellins in Stem/Root Tuber Development
Authors: Chen Peilei , Yang Ruixue , Bartels Dorothea , Dong Tianyu , Duan Hongying
Published: April 2022

Root and tuber crops are of great importance. They not only contribute to feeding the population but also provide raw material for medicine and small-scale industries. The yield of the root and tuber crops is subject to the development of stem/root tubers, which involves the initiation, expansion, and maturation of storage organs. The formation of the storage organ is a highly intricate process, regulated by multiple phytohormones. Gibberellins (GAs) and abscisic acid (ABA), as antagonists, are essential regulators during stem/root tuber development. This review summarizes the current knowledge of the roles of GA and ABA during stem/root tuber development in various tuber crops.
Molecular Dynamics Studies on the Structural Stability Prediction of SARS-CoV-2 Variants Including Multiple Mutants
Authors: Choi Kwang-Eun , Kim Jeong-Min , Rhee Jee Eun , Park Ae Kyung , Kim Eun-Jin , Yoo Cheon Kwon , Kang Nam Sook
Published: April 2022

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused the Coronavirus Disease (COVID-19) pandemic worldwide. The spike protein in SARS-CoV-2 fuses with and invades cells in the host respiratory system by binding to angiotensin-converting enzyme 2 (ACE2). The spike protein, however, undergoes continuous mutation from a D614G single mutant to an omicron variant, including multiple mutants. In this study, variants, including multiple mutants (double, triple mutants, B.1.620, delta, alpha, delta_E484Q, mu, and omicron) were investigated in patients. The 3D structure of the full-length spike protein was used in conformational analysis depending on the SARS-CoV-2 variants. The structural stability of the variant types was analyzed based on the distance between the receptor-binding domain (RBD) of each chain in the spike protein and the binding free energy between the spike protein and bound ACE2 in the one-, two-, and three-open-complex forms using molecular dynamics (MD) simulation. Omicron variants, the most prevalent in the recent history of the global pandemic, which consist of 32 mutations, showed higher stability in all open-complex forms compared with that of the wild type and other variants. We suggest that the conformational stability of the spike protein is the one of the important determinants for the differences in viral infectivity among variants, including multiple mutants.
New Insights into and Emerging Roles of Animal Models for Neurological Disorders
Authors: Moon Changjong
Published: April 2022

Many researchers rely on animal studies to elucidate the mechanisms underlying diverse disease processes and to test the safety of emerging medical interventions [...]
An In Silico Methodology That Facilitates Decision Making in the Engineering of Nanoscale Protein Materials
Authors: Parladé Eloi , Voltà-Durán Eric , Cano-Garrido Olivia , Sánchez Julieta M. , Unzueta Ugutz , López-Laguna Hèctor , Serna Naroa , Cano Montserrat , Rodríguez-Mariscal Manuel , Vazquez Esther , Villaverde Antonio
Published: April 2022

Under the need for new functional and biocompatible materials for biomedical applications, protein engineering allows the design of assemblable polypeptides, which, as convenient building blocks of supramolecular complexes, can be produced in recombinant cells by simple and scalable methodologies. However, the stability of such materials is often overlooked or disregarded, becoming a potential bottleneck in the development and viability of novel products. In this context, we propose a design strategy based on in silico tools to detect instability areas in protein materials and to facilitate the decision making in the rational mutagenesis aimed to increase their stability and solubility. As a case study, we demonstrate the potential of this methodology to improve the stability of a humanized scaffold protein (a domain of the human nidogen), with the ability to oligomerize into regular nanoparticles usable to deliver payload drugs to tumor cells. Several nidogen mutants suggested by the method showed important and measurable improvements in their structural stability while retaining the functionalities and production yields of the original protein. Then, we propose the procedure developed here as a cost-effective routine tool in the design and optimization of multimeric protein materials prior to any experimental testing.
Mono-Sized Anion-Exchange Magnetic Microspheres for Protein Adsorption
Authors: Wang Zhe , Wang Wei , Meng Zihui , Xue Min
Published: April 2022

In this study, mono-sized anion-exchange microspheres with polyglycidylmethacrylate were engineered and processed to introduce magnetic granules by penetration–deposition approaches. The obtained magnetic microspheres showed a uniform particle diameter of 1.235 μm in average and a good spherical shape with a saturation magnetic intensity of 12.48 emu/g by VSM and 12% magnetite content by TGA. The magnetic microspheres showed no cytotoxicity when the concentration was below 10 μg/mg. The magnetic microspheres possess respective adsorption capacity for three proteins including Bovine albumin, Hemoglobin from bovine blood, and Cytochrome C. These magnetic microspheres are also potential biomaterials as targeting medicine carriers or protein separation carriers at low concentration.
The Role of ATF3 in Neuronal Differentiation and Development of Neuronal Networks in Opossum Postnatal Cortical Cultures
Authors: Petrović Antonela , Ban Jelena , Ivaničić Matea , Tomljanović Ivana , Mladinic Miranda
Published: April 2022

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding (CREB) family, is upregulated by various intracellular and extracellular signals such as injury and signals related to cell proliferation. ATF3 also belongs to the regeneration-associated genes (RAG) group of transcription factors. RAG and ATF/CREB transcription factors that play an important role in embryonic neuronal development and PNS regeneration may also be involved in postnatal neuronal differentiation and development, as well as in the regeneration of the injured CNS. Here we investigated the effect of ATF3 in differentiation, neural outgrowth, network formation, and regeneration after injury using postnatal dissociated cortical neurons derived from neonatal opossums (Monodelphis domestica). Our results show that RAG and ATF genes are differentially expressed in early differentiated neurons versus undifferentiated neurospheres and that many members of those families, ATF3 in particular, are upregulated in cortical cultures obtained from younger animals that have the ability to fully functionally regenerate spinal cord after injury. In addition, we observed different intracellular localization of ATF3 that shifts from nuclear (in neuronal progenitors) to cytoplasmic (in more mature neurons) during neuronal differentiation. The ATF3 inhibition, pharmacological or by specific antibody, reduced the neurite outgrowth and differentiation and caused increased cell death in early differentiating cortical neuronal cultures, suggesting the importance of ATF3 in the CNS development of neonatal opossums. Finally, we investigated the regeneration capacity of primary cortical cultures after mechanical injury using the scratch assay. Remarkably, neonatal opossum-derived cultures retain their capacity to regenerate for up to 1 month in vitro. Inhibition of ATF3 correlates with reduced neurite outgrowth and regeneration after injury. These results indicate that ATF3, and possibly other members of RAG and ATF/CREB family of transcription factors, have an important role both during cortical postnatal development and in response after injury.
Genome-Wide Characterization of High-Affinity Nitrate Transporter 2 (NRT2) Gene Family in Brassica napus
Authors: Du Run-Jie , Wu Ze-Xuan , Yu Zhao-Xi , Li Peng-Feng , Mu Jian-Yu , Zhou Jie , Li Jia-Na , Du Hai
Published: April 2022

Nitrate transporter 2 (NRT2) plays an essential role in Nitrogen (N) uptake, transport, utilization, and stress resistance. In this study, the NRT2 gene family in two sequenced Brassica napus ecotypes were identified, including 31 genes in ‘Zhongshuang11’ (BnaZSNRT2s) and 19 in ‘Darmor-bzh’ (BnaDarNRT2s). The candidate genes were divided into three groups (Group I−III) based on phylogenetic analyses, supported by a conserved intron-exon structure in each group. Collinearity analysis revealed that the large expansion of BnaZSNRT2s attributed to allopolyploidization of ancestors Brassica rapa and Brassica oleracea, and small-scale duplication events in B. napus. Transcription factor (TF) binding site prediction, cis-element analysis, and microRNA prediction suggested that the expressions of BnaZSNRT2s are regulated by multiple factors, and the regulatory pattern is relatively conserved in each group and is tightly connected between groups. Expression assay showed the diverse and differentiated spatial-temporal expression profiles of BnaZSNRT2s in Group I, but conserved patterns were observed in Group II/III; and the low nitrogen (LN) stress up-regulated expression profiles were presented in Group I−III, based on RNA-seq data. RT-qPCR analyses confirmed that BnaZSNRT2.5A-1 and BnaZSNRT2.5C-1 in Group II were highly up-regulated under LN stress in B. napus roots. Our results offer valid information and candidates for further functional BnaZSNRT2s studies.
Characteristics of microRNAs and Target Genes in Maize Root under Drought Stress
Authors: Tang Qi , Lv Haozhe , Li Qimeng , Zhang Xiaoyue , Li Le , Xu Jie , Wu Fengkai , Wang Qingjun , Feng Xuanjun , Lu Yanli
Published: April 2022

Maize (Zea mays) is an important multi-functional crop. The growth and yield of maize are severely affected by drought stress. Previous studies have shown that microRNAs (miRNAs) in maize play important roles in response to abiotic stress; however, their roles in response to drought stress in maize roots is unclear. In our study, we found 375 miRNAs in the roots of 16 inbred lines. Of the 16 lines, zma-MIR168, zma-MIR156, and zma-MIR166 were highly expressed, whereas zma-MIR399, zma-MIR2218, and zma-MIR2275 exhibited low expression levels. The expression patterns of miRNA in parental lines and their derived RILs are different. Over 50% of miRNAs exhibited a lower expression in recombinant inbred lines than in parents. The expression of 50 miRNAs was significantly altered under water stress (WS) in at least three inbred lines, and the expression of miRNAs in drought-tolerant lines changed markedly. To better understand the reasons for miRNA response to drought, the degree of histone modifications for miRNA genes was estimated. The methylation level of H3K4 and H3K9 in miRNA precursor regions changed more noticeably after WS, but no such phenomenon was seen for DNA methylation and m6A modification. After the prediction of miRNA targets using psRNATarget and psRobot, we used correlation analysis and qRT-PCR to further investigate the relationship between miRNAs and target genes. We found that 87 miRNA–target pairs were significantly negatively correlated. In addition, a weighted gene co-expression network analysis using miRNAs, as well as their predicted targets, was conducted to reveal that miR159, miR394, and miR319 may be related to maize root growth. The results demonstrated that miRNAs might play essential roles in the response to drought stress.
Elevated Expression of the Immune Checkpoint Ligand CD276 (B7-H3) in Urothelial Carcinoma Cell Lines Correlates Negatively with the Cell Proliferation
Authors: Harland Niklas , Maurer Florian B. , Abruzzese Tanja , Bock Cornelia , Montes-Mojarro Ivonne A. , Fend Falko , Aicher Wilhelm K. , Stenzl Arnulf , Amend Bastian
Published: April 2022

The cell surface molecule CD276 (B7-H3) is an immune checkpoint antigen. The elevated expression of CD276 on tumors contributes to the suppression of anti-tumor T-cell responses and correlates with poor prognosis. Methods: The expression of CD276 was explored in vitro on eight urothelial carcinoma cell lines (UM-UC) in comparison to eight normal urothelial cells (NUCs) by RT-qPCR, Western blotting, and flow cytometry. Cell proliferation was enumerated over consecutive passages. The expression of cancer stem cell markers CD24 and CD44, cytokeratins, and vimentin was investigated by immunofluorescence. The expression of CD276 in bladder tumor samples and metastases was explored by immunohistochemistry. Results: Expression of CD276 on cell surfaces was elevated on UM-UCs when compared to NUCs. In UM-UCs, CD276 transcripts correlated moderately positive with CD276 protein expression (ρ = 0.660) and strongly positive with CD276 surface-expression (ρ = 0.810). CD276 mRNA expression (ρ = −0.475) and CD276 protein expression (ρ = −0.417) had a significant negative correlation with proliferation, while a significant correlation between proliferation and cell surface expression was not observed in UM-UCs. Conclusion: The expression of CD276 on UM-UC bladder tumor cell surfaces is elevated. Slow proliferating UM-UC cells express more CD276 mRNA and protein than fast proliferating cells. In patients, slow proliferating CD276high tumor (stem) cells may evade immune surveillance. However, cancer therapy targeting CD276 may be effective in the treatment of slow proliferating tumor cells.
Capsule-Targeting Depolymerases Derived from Acinetobacter baumannii Prophage Regions
Authors: Drobiazko Alena Y. , Kasimova Anastasia A. , Evseev Peter V. , Shneider Mikhail M. , Klimuk Evgeniy I. , Shashkov Alexander S. , Dmitrenok Andrei S. , Chizhov Alexander O. , Slukin Pavel V. , Skryabin Yuriy P. , Volozhantsev Nikolay V. , Miroshnikov Konstantin A. , Knirel Yuriy A. , Popova Anastasia V.
Published: April 2022

In this study, several different depolymerases encoded in the prophage regions of Acinetobacter baumannii genomes have been bioinformatically predicted and recombinantly produced. The identified depolymerases possessed multi-domain structures and were identical or closely homologous to various proteins encoded in other A. baumannii genomes. This means that prophage-derived depolymerases are widespread, and different bacterial genomes can be the source of proteins with polysaccharide-degrading activities. For two depolymerases, the specificity to capsular polysaccharides (CPSs) of A. baumannii belonging to K1 and K92 capsular types (K types) was determined. The data obtained showed that the prophage-derived depolymerases were glycosidases that cleaved the A. baumannii CPSs by the hydrolytic mechanism to yield monomers and oligomers of the K units. The recombinant proteins with established enzymatic activity significantly reduced the mortality of Galleria mellonella larvae infected with A. baumannii of K1 and K92 capsular types. Therefore, these enzymes can be considered as suitable candidates for the development of new antibacterials against corresponding A. baumannii K types.
Non-Traditional Pathways for Platelet Pathophysiology in Diabetes: Implications for Future Therapeutic Targets
Authors: Sagar Rebecca C. , Ajjan Ramzi A. , Naseem Khalid M.
Published: April 2022

Cardiovascular complications remain the leading cause of morbidity and mortality in individuals with diabetes, driven by interlinked metabolic, inflammatory, and thrombotic changes. Hyperglycaemia, insulin resistance/deficiency, dyslipidaemia, and associated oxidative stress have been linked to abnormal platelet function leading to hyperactivity, and thus increasing vascular thrombotic risk. However, emerging evidence suggests platelets also contribute to low-grade inflammation and additionally possess the ability to interact with circulating immune cells, further driving vascular thrombo-inflammatory pathways. This narrative review highlights the role of platelets in inflammatory and immune processes beyond typical thrombotic effects and the impact these mechanisms have on cardiovascular disease in diabetes. We discuss pathways for platelet-induced inflammation and how platelet reprogramming in diabetes contributes to the high cardiovascular risk that characterises this population. Fully understanding the mechanistic pathways for platelet-induced vascular pathology will allow for the development of more effective management strategies that deal with the causes rather than the consequences of platelet function abnormalities in diabetes.
Intra-Laboratory Evaluation of DNA Extraction Methods and Assessment of a Droplet Digital PCR for the Detection of Xanthomonas citri pv. citri on Different Citrus Species
Authors: Pucci Nicoletta , Scala Valeria , Tatulli Giuseppe , L’Aurora Alessia , Lucchesi Simone , Salustri Manuel , Loreti Stefania
Published: April 2022

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca), causal agents of citrus bacterial canker, are both regulated by the European Union to prevent their introduction. Xcc is responsible for severe outbreaks of citrus production worldwide, therefore, a prompt and reliable detection is advisable for the early detection of this bacterium either in symptomatic or asymptomatic plant material. The current EPPO (European and Mediterranean Plant Protection Organization) diagnostic protocol, PM 7/44(1), includes several diagnostic tests even if new assays have been developed in the latter years for which validation data are needed. Recently, a test performance study was organized within the Valitest EU Project to validate Xcc diagnostic methods and provide evidence on the most reliable assays; however, the influence of DNA extraction methods (DEM) on the reliability of the detection has never been assessed. In this study we evaluate four different DEM, by following two different approaches: (i) a comparison by real-time PCR standard curves of bacterial DNA versus bacterial DNA added to plant DNA (lemon, leaves and fruit; orange fruit); and (ii) the evaluation of performance criteria of spiked samples (plant extract added with ten-fold diluted bacterial suspensions at known concentrations). Droplet digital PCR is developed and compared with real-time PCR, as the detection method.
Highly Cytotoxic Osmium(II) Compounds and Their Ruthenium(II) Analogues Targeting Ovarian Carcinoma Cell Lines and Evading Cisplatin Resistance Mechanisms
Authors: Hildebrandt Jana , Häfner Norman , Kritsch Daniel , Görls Helmar , Dürst Matthias , Runnebaum Ingo B. , Weigand Wolfgang
Published: April 2022

(1) Background: Ruthenium and osmium complexes attract increasing interest as next generation anticancer drugs. Focusing on structure-activity-relationships of this class of compounds, we report on 17 different ruthenium(II) complexes and four promising osmium(II) analogues with cinnamic acid derivatives as O,S bidentate ligands. The aim of this study was to determine the anticancer activity and the ability to evade platin resistance mechanisms for these compounds. (2) Methods: Structural characterizations and stability determinations have been carried out with standard techniques, including NMR spectroscopy and X-ray crystallography. All complexes and single ligands have been tested for cytotoxic activity on two ovarian cancer cell lines (A2780, SKOV3) and their cisplatin-resistant isogenic cell cultures, a lung carcinoma cell line (A549) as well as selected compounds on three non-cancerous cell cultures in vitro. FACS analyses and histone γH2AX staining were carried out for cell cycle distribution and cell death or DNA damage analyses, respectively. (3) Results: IC50 values show promising results, specifically a high cancer selective cytotoxicity and evasion of resistance mechanisms for Ru(II) and Os(II) compounds. Histone γH2AX foci and FACS experiments validated the high cytotoxicity but revealed diminished DNA damage-inducing activity and an absence of cell cycle disturbance thus pointing to another mode of action. (4) Conclusion: Ru(II) and Os(II) compounds with O,S-bidentate ligands show high cytotoxicity without strong effects on DNA damage and cell cycle, and this seems to be the basis to circumvent resistance mechanisms and for the high cancer cell specificity.
Basophil Activation Test Utility as a Diagnostic Tool in LTP Allergy
Authors: Cañas José A. , Pérez-Sánchez Natalia , Lopera-Doblas Leticia , Palomares Francisca , Molina Ana , Bartra Joan , Torres María J. , Gómez Francisca , Mayorga Cristobalina
Published: April 2022

Plant-food allergy is an increasing problem, with nonspecific lipid transfer proteins (nsLTPs) triggering mild/severe reactions. Pru p 3 is the major sensitizer in LTP food allergy (FA). However, in vivo and in vitro diagnosis is hampered by the need for differentiating between asymptomatic sensitization and allergy with clinical relevance. The basophil activation test (BAT) is an ex vivo method able to identify specific IgE related to the allergic response. Thus, we aimed to establish the value of BAT in a precise diagnosis of LTP-allergic patients. Ninety-two individuals with peach allergy sensitized to LTP, Pru p 3, were finally included, and 40.2% of them had symptoms to peanut (n = 37). In addition, 16 healthy subjects were recruited. BAT was performed with Pru p 3 and Ara h 9 (peanut LTP) at seven ten-fold concentrations, and was evaluated by flow cytometry, measuring the percentage of CD63 (%CD63+) and CD203c (%CD203chigh) cells, basophil allergen threshold sensitivity (CD-Sens), and area under the dose–response curve (AUC). Significant changes in BAT parameters (%CD63+ and %CD203chigh) were found between the controls and patients. However, comparisons for %CD63+, %CD203chigh, AUC, and CD-Sens showed similar levels among patients with different symptoms. An optimal cut-off was established from ROC curves, showing a significant positive percentage of BAT in patients compared to controls and great values of sensitivity (>87.5%) and specificity (>85%). In addition, BAT showed differences in LTP-allergic patients tolerant to peanut using its corresponding LTP, Ara h 9. BAT can be used as a potential diagnostic tool for identifying LTP allergy and for differentiating peanut tolerance, although neither reactivity nor sensitivity can distinguish the severity of the clinical symptoms.