Targeting of the Nasal Mucosa by Japanese Encephalitis Virus for Non-Vector-Borne Transmission.

The mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases and cycles between Culex mosquitoes and different vertebrates. For JEV and some other flaviviruses, oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33, and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector-borne direct transmission of Japanese encephalitis virus in pigs.IMPORTANCE JEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving pigs, which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines and therefore infection and replication in macrophages, which is favored by epithelial-cell-derived cytokines. The results are relevant to understand the mechanism of non-vector-borne direct transmission of JEV.

Synthesis and anticancer activity of some 5-fluoro-2'-deoxyuridine phosphoramidates.

Two series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3'-O-(t-butoxycarbonyl)-5-fluoro-2'-deoxyuridine (3'-BOC-FdU) (9a-9j) and 5-fluoro-2'-deoxyuridine (FdU) (10a-10j) were synthesized by means of phosphorylation of 3'-BOC-FdU (4) with 4-chlorophenyl phosphoroditriazolide (7), followed by a reaction with the appropriate amine. Phosphoramidates 9a-9j were converted to the corresponding 10a-10j by removal of the 3'-t-butoxycarbonyl protecting group (BOC) under acidic conditions. The synthesized phosphoramidates 9a-9j and 10a-10j were evaluated for their cytotoxic activity in five human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), liver (HepG2), osteosarcoma (143B) and normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Two phosphoramidates 9b and 9j with the N-ethyl and N-(methoxy-(S)-alaninyl) substituents, respectively, displayed remarkable activity in all the investigated cancer cells, and the activity was considerably higher than that of the parent nucleoside 4 and FdU. Among phosphoramidates 10a-10j compound 10c with the N-(2,2,2-trifluoroethyl) substituent showed the highest activity. Phosphoramidate 10c was more active than the FdU in all the cancer cell lines tested.

[Male contraception - the current state of knowledge]. / Antykoncepcja meska - aktualny stan wiedzy.

Contraception is important from a health, psychological and socioeconomic point of view. Due to the fact that male-based contraceptive methods are mostly represented by condoms and vasectomy, researchers are working on the new solutions, which could let the men be more involved in a conscious family planning. In this review we will present the current state of knowledge on this subject. There is a lot going on in the field of hormonal contraception. Studies including testosterone, progestins, synthetic androgens and other derivatives are on a different stages of clinical trials and mostly demonstrate high efficacy rates. Recent discovers of Izumo and Juno proteins, essential for the fertilization process, give hope for an easily reversible, non-hormonal method. Researchers are also trying to interfere with the process of spermatogenesis using BRDT inhibitor - JQ1, or neutralize the sperm by injecting styrene maleic anhydride (SMA) into the lumen of the vas deferens. The other studies explore processes involved in proper sperm motility. A vaccine which induces an immune response to the reproductive system is also an interesting method. The latest research use ultrasound waves and mechanical device which blocks the patency of vas deferens. The aim of the study current state of knowledge male contraception.

A 1024-Channel CMOS Microelectrode Array With 26,400 Electrodes for Recording and Stimulation of Electrogenic Cells In Vitro.

To advance our understanding of the functioning of neuronal ensembles, systems are needed to enable simultaneous recording from a large number of individual neurons at high spatiotemporal resolution and good signal-to-noise ratio. Moreover, stimulation capability is highly desirable for investigating, for example, plasticity and learning processes. Here, we present a microelectrode array (MEA) system on a single CMOS die for in vitro recording and stimulation. The system incorporates 26,400 platinum electrodes, fabricated by in-house post-processing, over a large sensing area (3.85 × 2.10 mm2) with sub-cellular spatial resolution (pitch of 17.5 µm). Owing to an area and power efficient implementation, we were able to integrate 1024 readout channels on chip to record extracellular signals from a user-specified selection of electrodes. These channels feature noise values of 2.4 µVrms in the action-potential band (300 Hz-10 kHz) and 5.4 µVrms in the local-field-potential band (1 Hz-300 Hz), and provide programmable gain (up to 78 dB) to accommodate various biological preparations. Amplified and filtered signals are digitized by 10 bit parallel single-slope ADCs at 20 kSamples/s. The system also includes 32 stimulation units, which can elicit neural spikes through either current or voltage pulses. The chip consumes only 75 mW in total, which obviates the need of active cooling even for sensitive cell cultures.

The Impact of Stopping Medications and Introducing a Whole Food Plant-Based Diet on Patients Living with Multiple Sclerosis - A Report of Two Cases.

More than 2 million people live with multiple sclerosis worldwide and the prevalence has been increasing over time. Patients living with multiple sclerosis often explore diet and lifestyle interventions as a means of managing their symptoms and reducing reliance on medication; yet, these approaches are rarely discussed with their physicians. Currently, there is a lack of evidence on when to stop disease-modifying therapies (DMT), and recent research showed no statistically significant difference in the time between relapses when comparing participants who stopped DMT to those who did not, especially over the age of 45. This case report presents 2 patients with multiple sclerosis who made an informed decision to stop their DMT medications and have been managing their condition with a whole food plant-based diet and a healthy lifestyle approach. Over the period of 5 to 6 years since stopping the medications, each patient only had 1 multiple sclerosis flare-up to date. In the report, the focus is on the impact of diet on multiple sclerosis. It adds to currently available literature and encourages further research in the field of managing multiple sclerosis with lifestyle interventions.

Risk Factors and Clinicopathological Features for Developing a Subsequent Primary Cutaneous Squamous and Basal Cell Carcinomas.

BACKGROUND: Patients with diagnosed keratinocyte carcinomas (KCs) have an increased risk of subsequent skin cancers development. Current studies indicate that patients with subsequent tumors should be followed up regularly. However, none of the studies indicate the connection between the specific subtypes and an increased risk for further KCs development. The study assesses the differences in the risk of developing a subsequent skin cancer after a previous diagnosis of KC, especially considering individual types of skin malignances, and identifies potential factors associated with an increased risk of new cutaneous tumor describing non-invasive diagnosis and monitoring. METHODS: Pathology and medical records were examined to identify the characteristics of patients with multiple KCs diagnosed between 1999 and 2019. RESULTS: The study group comprised 13,913 KCs occurring in 10,083 patients. Multiple KCs were observed in 2300 patients (22.8%). The analysis showed aggressive subtypes, multiple tumors, and male sex as significant prognostic factors. CONCLUSIONS: The most crucial risk factors for developing subsequent KC are being of a male gender, an aggressive tumor subtype, and previous history of multiple skin cancers. Basal cell carcinoma subtypes, such as infiltrative basosquamous, with aggressive growth patterns predispose not only to increased risk for the recurrence but are also expected to be at higher risk of subsequent KCs.

The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics.

Planar microelectrode arrays (MEAs) are devices that can be used in biomedical and basic in vitro research to provide extracellular electrophysiological information about biological systems at high spatial and temporal resolution. Complementary metal oxide semiconductor (CMOS) is a technology with which MEAs can be produced on a microscale featuring high spatial resolution and excellent signal-to-noise characteristics. CMOS MEAs are specialized for the analysis of complete electrogenic cellular networks at the cellular or subcellular level in dissociated cultures, organotypic cultures, and acute tissue slices; they can also function as biosensors to detect biochemical events. Models of disease or the response of cellular networks to pharmacological compounds can be studied in vitro, allowing one to investigate pathologies, such as cardiac arrhythmias, memory impairment due to Alzheimer's disease, or vision impairment caused by ganglion cell degeneration in the retina.

Managing Psoriatic Arthritis With a Whole Food Plant-Based Diet: A Case Study.

A 40-year-old female teacher presented to the rheumatology clinic in 2003 with nonspecific back, knees, and right ankle pain. She was subsequently diagnosed with psoriatic arthritis and was taking methotrexate to control her disease. Over the years, her symptoms were mostly under control. However, in 2018, after adopting a whole food plant-based diet free of added salt, oil, and sugar, she was able to stop taking methotrexate. She was discharged from the rheumatology clinic and has remained symptom-free since. The available literature on managing psoriatic arthritis with diet shows that less than 2% of patients with psoriatic arthritis are able to discontinue medication as a result of disease remission. This case report adds to support and encourages further research in the field of managing inflammatory polyarthropathies with diet and lifestyle.

Animal board invited review: Risks of zoonotic disease emergence at the interface of wildlife and livestock systems.

The ongoing coronavirus disease 19s pandemic has yet again demonstrated the importance of the human-animal interface in the emergence of zoonotic diseases, and in particular the role of wildlife and livestock species as potential hosts and virus reservoirs. As most diseases emerge out of the human-animal interface, a better understanding of the specific drivers and mechanisms involved is crucial to prepare for future disease outbreaks. Interactions between wildlife and livestock systems contribute to the emergence of zoonotic diseases, especially in the face of globalization, habitat fragmentation and destruction and climate change. As several groups of viruses and bacteria are more likely to emerge, we focus on pathogenic viruses of the Bunyavirales, Coronaviridae, Flaviviridae, Orthomyxoviridae, and Paramyxoviridae, as well as bacterial species including Mycobacterium sp., Brucella sp., Bacillus anthracis and Coxiella burnetii. Noteworthy, it was difficult to predict the drivers of disease emergence in the past, even for well-known pathogens. Thus, an improved surveillance in hotspot areas and the availability of fast, effective, and adaptable control measures would definitely contribute to preparedness. We here propose strategies to mitigate the risk of emergence and/or re-emergence of prioritized pathogens to prevent future epidemics.

A randomised controlled trial evaluating the effectiveness of Facebook compared to leaflets in raising awareness of melanoma and harmful sun-related behaviour among young adults.

This study describes a randomised control trial investigating whether printed leaflets or social media are more effective in increasing knowledge of the risks of sun exposure and melanoma in people aged 18-29. The study participants were 18-29-year-old university students or graduates, recruited in London. A baseline level of knowledge was measured using the Skin Cancer and Sun Knowledge questionnaire. Study participants were then randomised into either a leaflet arm or Facebook arm. Identical information was delivered through a SunSafe campaign via either posted leaflets or Facebook during a 10-day exposure window. Following this, participants repeated the Skin Cancer and Sun Knowledge questionnaire. Following the SunSafe intervention, the mean knowledge score improved in both groups to a statistically significant degree (Facebook = 1.82, leaflets = 3.04, P < 0.001). Moreover, the improvement in knowledge score of the leaflet arm was statistically significantly greater than in the Facebook arm (95% confidence interval: 0.35-2.09, P = 0.0059). Participants of lighter skin colour demonstrated greater levels of knowledge about skin cancer and sun exposure at baseline (P = 0.005; P < 0.05). There was no correlation between sex and baseline knowledge (P = 0.7725). There was no significant effect of skin tone or sex on the knowledge change (P = 0.139 and 0.643). The findings suggest that printed information in the form of leaflets is more impactful in increasing knowledge than online platforms such as Facebook among a young adult demographic in the UK. These findings should be considered when designing public health campaigns, acting as a reminder to not neglect traditional media in health promotion.

Living with cancer and multimorbidity: the role of primary care.

PURPOSE OF REVIEW: As more people live longer with cancer, the number of patients with cancer and multiple other chronic conditions (multimorbidity) has increased. The presence of multimorbidity impacts on all stages of cancer care, from prevention and early detection through to end of life care, but research into cancer and multimorbidity is in its infancy. This review explores the impact of multimorbidity on adults living with (and beyond) cancer, with particular attention paid to the role of primary care in supporting patients in this situation. RECENT FINDINGS: Patterns of multimorbidity vary depending on cancer type and stage, as well as population characteristics and available data (e.g. number of conditions assessed). Cancer survivors are at increased risk of developing other chronic conditions, due to a combination of shared risk factors (e.g. smoking and obesity), effects of cancer treatments and psychosocial effects. SUMMARY: Primary care has a central role to play in supporting multimorbid adults living with cancer, providing holistic care of physical and mental well being, while taking treatment burden and social circumstances into account. New models of person-centred and personalized cancer care include holistic needs assessments, care planning, treatment summaries and cancer care reviews, and depend on improved communication between oncologists and primary care colleagues.

Monocyte-Derived Dendritic Cells as Model to Evaluate Species Tropism of Mosquito-Borne Flaviviruses.

Several mosquito-borne Flaviviruses such as Japanese encephalitis virus (JEV), West Nile virus (WNV), Dengue Virus (DENV), and Zika virus (ZIKV) can cause severe clinical disease. Being zoonotic, Flaviviruses infect a wide variety of terrestrial vertebrates, which dependent of the virus-host interactions, can enhance ongoing epidemics and maintain the virus in the environment for prolonged periods. Targeted species can vary from amphibians, birds to various mammals, dependent on the virus. For many mosquito-borne flaviviruses the spectrum of targeted species is incompletely understood, in particular with respect to their contribution to the maintenance of virus in certain geographical regions. Furthermore, little is known about virus and host factors contributing to species tropism. The present study utilized human and porcine monocyte-derived dendritic cells (MoDC) as a cell culture model to better understand Flavivirus species tropism and innate immune responses. MoDC were selected based on their presence in the skin and their role as an early target cell for several Flaviviruses and their role as immune sentinels. While differences in viral infectivity and replication were minor when comparing porcine with human MoDC for some of the tested Flaviviruses, a particularly strong replication in human MoDC was found with USUV, while JEV appeared to have a stronger tropism for porcine MoDC. With respect to innate immune responses we found high induction of TNF and IFN-ß in both human and porcine MoDC after infection with JEV, WNV, and USUV, but not with DENV, ZIKV, and Wesselsbron virus. Spondweni virus induced these cytokine responses only in porcine MoDC. Overall, innate immune responses correlated with early infectivity and cytokine production. In conclusion, we demonstrate Flavivirus-dependent differences in the interaction with MoDC. These may play a role in pathogenesis but appear to only partially reflect the expected species tropism.

Corrigendum: Monocyte-Derived Dendritic Cells as Model to Evaluate Species Tropism of Mosquito-Borne Flaviviruses.

[This corrects the article DOI: 10.3389/fcimb.2019.00005.].

Long-Term High-Density Extracellular Recordings Enable Studies of Muscle Cell Physiology.

Skeletal (voluntary) muscle is the most abundant tissue in the body, thus making it an important biomedical research subject. Studies of neuromuscular transmission, including disorders of ion channels or receptors in autoimmune or genetic neuromuscular disorders, require high-spatial-resolution measurement techniques and an ability to acquire repeated recordings over time in order to track pharmacological interventions. Preclinical techniques for studying diseases of neuromuscular transmission can be enhanced by physiologic ex vivo models of tissue-tissue and cell-cell interactions. Here, we present a method, which allows tracking the development of primary skeletal muscle cells from myoblasts into mature contracting myotubes over more than 2 months. In contrast to most previous studies, the myotubes did not detach from the surface but instead formed functional networks between the myotubes, whose electrical signals were observed over the entire culturing period. Primary cultures of mouse myoblasts differentiated into contracting myotubes on a chip that contained an array of 26,400 platinum electrodes at a density of 3,265 electrodes per mm2. Our ability to track extracellular action potentials at subcellular resolution enabled study of skeletal muscle development and kinetics, modes of spiking and spatio-temporal relationships between muscles. The developed system in turn enables creation of a novel electrophysiological platform for establishing ex vivo disease models.

New directions in hepatitis B therapy research.

Chronic hepatitis B treatment is available for a long period, allowing disease control and infection suppression, but it is rarely responsible for HBsAg clearance. None of the drugs available aim at cccDNA, the obstacle in HBV infection eradication. Complications related to CHB, such as liver insufficiency, cirrhosis, and hepatocellular carcinoma are reduced in conditions of good viremia suppression, but still exist even after HBsAg seroclearance, what makes a need for urgent forthcoming of new therapeutics. Recent years brought promising and interesting results of experimental approaches, which are directed against different phases of HBV life cycle, target ccc DNA, or boost, and restore host immune response. Unfortunately, encouraging results in vitro and on animal models are not always reflected in human. Nevertheless, the multiplicity of novel antivirals allows to expect that at least some of them will enter clinical practice and relieve patients from chronic hepatitis B, fatal and devastating disease.

Cortical Axons, Isolated in Channels, Display Activity-Dependent Signal Modulation as a Result of Targeted Stimulation.

Mammalian cortical axons are extremely thin processes that are difficult to study as a result of their small diameter: they are too narrow to patch while intact, and super-resolution microscopy is needed to resolve single axons. We present a method for studying axonal physiology by pairing a high-density microelectrode array with a microfluidic axonal isolation device, and use it to study activity-dependent modulation of axonal signal propagation evoked by stimulation near the soma. Up to three axonal branches from a single neuron, isolated in different channels, were recorded from simultaneously using 10-20 electrodes per channel. The axonal channels amplified spikes such that propagations of individual signals along tens of electrodes could easily be discerned with high signal to noise. Stimulation from 10 up to 160 Hz demonstrated similar qualitative results from all of the cells studied: extracellular action potential characteristics changed drastically in response to stimulation. Spike height decreased, spike width increased, and latency increased, as a result of reduced propagation velocity, as the number of stimulations and the stimulation frequencies increased. Quantitatively, the strength of these changes manifested itself differently in cells at different frequencies of stimulation. Some cells' signal fidelity fell to 80% already at 10 Hz, while others maintained 80% signal fidelity at 80 Hz. Differences in modulation by axonal branches of the same cell were also seen for different stimulation frequencies, starting at 10 Hz. Potassium ion concentration changes altered the behavior of the cells causing propagation failures at lower concentrations and improving signal fidelity at higher concentrations.

Disease specific enrichment of circulating let-7 family microRNA in MuSK+ myasthenia gravis.

Myasthenia gravis (MG) patients with antibodies against the muscle specific tyrosine kinase (MuSK+) have predominantly involvement of cranio-bulbar muscles and do not display thymus pathology, as do acetylcholine receptor antibody seropositive (AChR+) MG patients. In search of novel biomarkers for MuSK+ MG, we evaluated circulating serum microRNAs. Four analyzed microRNAs were specifically elevated in MuSK+ MG patient serum samples: let-7a-5p, let-7f-5p, miR-151a-3p and miR-423-5p. The circulating microRNA profile in MuSK+ MG differs from the profile previously observed in the serum of AChR+ MG, thus indicating the etiological difference between these two entities. We propose that the identified microRNAs could serve as potential serum biomarkers for MuSK+ MG.

Immobilization of Zidovudine Derivatives on the SBA-15 Mesoporous Silica and Evaluation of Their Cytotoxic Activity.

Novel zidovudine derivatives, able to be covalently conjugated to silica surface, have been obtained and grafted to SBA-15 mesoporous silica. Cytotoxic activity of the hybrid organic-inorganic (zidovudine derivatives-silica) systems against HeLa and KB cell lines has been analyzed. Addition of folic acid had a positive influence on the cytotoxicity. Up to 69% of HeLa and 65% of KB tumor cells growth inhibition has been achieved at low silica concentration used (10 µg/mL).

Recording large extracellular spikes in microchannels along many axonal sites from individual neurons.

The numerous connections between neuronal cell bodies, made by their dendrites and axons, are vital for information processing in the brain. While dendrites and synapses have been extensively studied, axons have remained elusive to a large extent. We present a novel platform to study axonal physiology and information processing based on combining an 11,011-electrode high-density complementary metal-oxide semiconductor microelectrode array with a poly(dimethylsiloxane) channel device, which isolates axons from somas and, importantly, significantly amplifies recorded axonal signals. The combination of the microelectrode array with recording and stimulation capability with the microfluidic isolation channels permitted us to study axonal signal behavior at great detail. The device, featuring two culture chambers with over 30 channels spanning in between, enabled long-term recording of single spikes from isolated axons with signal amplitudes of 100 µV up to 2 mV. Propagating signals along axons could be recorded with 10 to 50 electrodes per channel. We (i) describe the performance and capabilities of our device for axonal electrophysiology, and (ii) present novel data on axonal signals facilitated by the device. Spontaneous action potentials with characteristic shapes propagated from somas along axons between the two compartments, and these unique shapes could be used to identify individual axons within channels that contained many axonal branches. Stimulation through the electrode array facilitated the identification of somas and their respective axons, enabling interfacing with different compartments of a single cell. Complex spike shapes observed in channels were traced back to single cells, and we show that more complicated spike shapes originate from a linear superposition of multiple axonal signals rather than signal distortion by the channels.

Synthesis and biological activity of salinomycin conjugates with floxuridine.

As part of our program to develop anticancer agents, we have synthesized new compounds, which are conjugates between well-known anticancer drug, floxuridine and salinomycin which is able to selectivity kill cancer stem cells. The conjugates were obtained in two ways i.e. by copper(I) catalysed click Huisgen cycloaddition reaction performed between 3'-azido-2',3'-dideoxy-5-fluorouridine and salinomycin propargyl amide, and by the ester synthesis starting from salinomycin and floxuridine under mild condition. The compounds obtained were characterized by spectroscopic methods and evaluated for their in vitro cytotoxicity against seven human cancer cell lines as well as antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE). The conjugate obtained by esterification reaction showed a significantly higher antiproliferative activity against the drug-resistant cancer cells and lower toxicity than those of salinomycin and floxuridine towards normal cells, as well as standard anticancer drugs, such as cisplatin and doxorubicin. The conjugate compound revealed also moderate activity against MRSA and MRSE bacterial strains. Very high activity of floxuridine and 5-fluorouracil against MRSA and MRSE has been also observed.