Key Points: • We describe a novel procedure, Endopharyngeal Ultrasound (EPhUS) and EPhUS-guided FNA • EPhUS requires an operator and an assistant, can be performed transnasal or transoral, and utilizes a Endoscopic Ultrasonography Bronchoscope • EPhUS is a safe and effective method for biopsy of deep space neck masses inaccessible to transcutaneous FNA
The ethanol concentration in batch cultivation with the yeast S. cerevisiae was predicted on-line using a gas sensor array. Head space samples were pumped past the gas sensors array every five minutes for 10 seconds and the voltage changes of the sensors were measured. For the calibration procedure no off-line sampling was used. Instead, a theoretical model of the process has been applied to simulate the ethanol production at any given time. However, the kinetic parameters of the simulation model are unknown at the beginning of the calibration. It will be demonstrated that these kinetic parameters of the theoretical process model can be acquired from the response of the gas sensor array alone. The calculated parameters result in a simulation model that is at least as accurate as a model whose parameters are acquired by least squares fitting to off-line measurements. The root mean square error of calibration as well as the percentage error for validation sets was below 0.2 g/L and 7 %, respectively. The obtained results indicate that, the model-based calibrated gas sensor array can be a cheap alternative to other tools that are used for monitoring yeast cultivations such as spectroscopy based methods.
Cel9B, an endocellulase produced by Thermobifida fusca YX, contains a number of structural domains, including carbohydrate binding modules 2 and 4 (CBM2 and CBM4), a fibronectin-like (Fn3) domain, an Eset domain (an Ig-like domain that may play a role in enzyme folding), a catalytic domain, and a fibronectin-like (Fn3) domain. To elucidate the roles of these domains with respect to Cel9B function, a series of truncation mutants were designed and examined for their binding properties and activities on different substrates. Different binding properties of CBM2 and 4 with a variety of substrates distinguish important roles for these domains and provide insight as to how distinct domains interact with each other during substrate degradation. The results of this study implicate the collective roles of the non-catalytic domains with respect to Cel9B function, and in turn, this information can be incorporated into protein engineering strategies for improved biomass conversion.
Terrestrial squamate reptiles from the Galápagos archipelago have limited gene flow among islands, providing an opportunity to test paleogeographic models. Previous work suggests that Pleistocene glaciations had a strong influence on the evolution of Galápagos’ land-locked vertebrates, such as lizards and snakes, by allowing dispersal and contact among populations from different islands or islets through land connections. One prediction of this model is that extant populations, despite being isolated at present, are genetically similar due to recent (Pleistocene) gene flow. Here we test this prediction with a simple comparative phylogeographic analysis of two sympatric lizards from Floreana island and surrounding islets. Based on two mitochondrial genes, we show that Floreana lava lizards (Microlophus grayii) and leaf-toed geckos (Phyllodactylus baurii) from Floreana Island are very similar genetically to conspecifics from Champion, an islet in the Floreana group that was connected to Floreana during Pleistocene glacial maxima. Moreover, they are significantly less similar to conspecifics from Gardner, an islet in the Floreana group that was not in contact with Floreana during Pleistocene glacial maxima. Thus, our results support the idea of Pleistocene glaciation-driven contact among populations from different islands in the Floreana cluster with no identifiable subsequent dispersal. These results also show that Floreana and Champion populations are part of the same evolutionary significant unit for both species, which might be at risk due to an upcoming invasive mammal eradication program in Floreana. Therefore, Champion represents a reasonable source for potential reintroductions of both lava lizards and leaf-toed geckos into Floreana.
SARS-CoV-2 has infected millions of people around the world, with most cases recorded among adults. The cases reported among children have been acknowledged to be minimal in comparison to adults. Nevertheless, COVID-19 has been reported to affect children at all ages, including newborns. The symptoms among children have also been identified to be similar to those observed among adults, although pediatric patients have been noted to display spectrum of clinical features ranging from asymptomatic through mild to moderate symptoms. Despite ample publications on the ongoing pandemic, the literature is only replete with guidelines on treating SARS-CoV-2 infection among older people. In this narrative review, comprehensive updates on the infection in children have been discussed. The latest information on the spread of the disease among children around the world, the clinical features observed among the pediatric population, as well as recommended pharmaceutical treatments of COVID-19 among this special group of patients have been covered. Further, expert consensus statements regarding the management of this highly contagious disease among pregnant women and neonates have been discussed. It is believed that this comprehensive review will provide updated information on the epidemiology and clinical features of the ongoing pandemic among pediatric patients. Additionally, the guidelines for handling SARS-CoV-2 among pregnant women and children, as reviewed in this article, are anticipated to be useful to frontline clinicians battling this fatal disease around the globe.
3D printing can be of great use, particularly the production of personal medical products and devices such as scaffolds. In this study, the main aim is to develop propolis (Ps) containing wound dressings by making use of 3D printing technology. Different combinations and structures of propolis (Ps) incorporated sodium alginate (SA) scaffolds are developed. The morphological studies show that the porosity of developed scaffolds was optimized when 20% (v/v) of Ps was added in the solution. The pore sizes decreased by increasing Ps concentration up to a certain level due to its adhesive properties. The mechanical, swelling-degradation (weight loss) behaviors and Ps release kinetics were highlighted for the scaffold stability. The antimicrobial assay was employed to test and screen antimicrobial behaviour of Ps against Escherichia coli and Staphylococcus aureus strains. The results show that the Ps added scaffolds have an excellent antibacterial activity because of Ps’s compounds. The in-vitro cytotoxicity test was also applied on the scaffold by using the extract method on the human dermal fibroblasts (HFFF2) cell line. It is clearly found that the control SA and Ps added SA are non-toxic. The 3D printed SA-Ps scaffolds are very effective structures for wound dressing applications with unique properties.
A recent commentary published in BJCP used lopinavir/ritonavir as an example to highlight the importance of the clinical pharmacology principles in the repurposing of old drugs for therapeutic use against Coronavirus disease 19 (COVID-19).1 Here, we provide another example to support this point.A recent study found that ivermectin, an FDA-approved anti-parasitic drug, has inhibitory effects on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).2 Ivermectin has broad anti-viral activity through inhibition of viral proteins including importin α/β1 heterodimer and integrase protein.3 In the in vitro study reported by Caly and colleagues, the addition of ivermectin at a concentration of 5 micromolar (μM) (twice the reported IC50) to Vero-hSLAM cells 2 hours post infection of with SARS-CoV-2 resulted in a reduction in the viral RNA load by 99.98% at 48 hours.2Large trials of mass drug administration of ivermectin in adults and children have shown that ivermectin is well tolerated.4 Even at doses that are 10 times greater than the highest FDA-approved dose of 200 μg/kg, central nervous system toxicity has not been reported.5 However, following the oral administration of supra-therapeutic doses of ivermectin (i.e. 120 mg) the maximum plasma concentration achieved was 0.28 ± 0.18 (standard deviation) μM, a value 18 times lower than the reported 5 μM ivermectin concentration used by Caly et al in their SARS-CoV-2 experiment.5 To date, the clinical effects of ivermectin at a concentration of 5 μM range are unknown, but likely to be toxic. Furthermore, ivermectin is only commercially available as a 3 mg oral tablet.6 These factors hinder our ability to immediately repurpose ivermectin in its current form for the treatment of COVID-19.While the findings by Caly and colleagues provide some promise, viral suppression was not seen at concentrations observed with standard doses in humans. Further preclinical in vivo studies should evaluate the pharmacokinetics and pharmacodynamics to determine the kill pattern of ivermectin. A potential alternate solution may be to develop an inhaled formulation of ivermectin to efficiently deliver a high local concentration in the lung, whilst minimising systemic toxicity. As therapeutic agents to tackle the COVID-19 pandemic are urgently sought, careful consideration of the pharmacokinetics of these drugs should be considered to guide in vitro testing.
Paraganglioma is a benign neuro-endocrine neoplasm rarely localized on the lumbar spine. A 50-year-old male who presented for paraparesis and urinary leakage. Lumbar spine MRI showed a lesion compressing the dural sac from L1 to L5. The patient was operated, and the pathologic examination concluded to a cauda equine paraganglioma
Analysis of biodiversity in natural environments based on environmental DNA (eDNA) has been applied to a wide range of ecosystems and species. The combination of high-throughput sequencing technologies and eDNA analysis is a powerful tool that enables comprehensive non-invasive monitoring of species present in the environment. Quantitative data of the eDNA from each species is essential for understanding species abundance but until recently required individual assays targeting each species. Recently developed quantitative sequencing (qSeq) allows simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. The eDNA extracted from aquaria with five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus) across 4 days was quantified by microfluidic digital PCR using a TaqMan probe and qSeq. The eDNA abundance quantified by qSeq was consistent with dPCR for each fish species at each sampling time. However, the relative abundances of sequences obtained from high throughput sequencing did not follow the same trend as the quantitative analyses, probably due to different PCR amplification efficiencies for each species. The correlation coefficients between qSeq and dPCR were 1.052, 1.074, and 1.114 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA. The application of qSeq to eDNA of other species will provide comprehensive quantitative data that could deepen our knowledge of natural ecosystems.
N6-methylated adenosine (m6A) and N1-methylated adenosine (m1A) are two epi-transcriptomic modifications on eukaryotic mRNA which have recently been rediscovered and are generating considerable interest. M6A methylation impacts on all aspects of cellular RNA metabolism and numerous physiological processes. Although less abundant than the m6A epitranscriptomic mark, m1A methylation has recently also attracted interest due to its dynamic nature in response to physiological changes. We investigated the role of the m6A and m1A methylation regulators on the expression of a transgene in Chinese Hamster Ovary (CHO) cells - the host cell of choice in producing biopharmaceutical proteins commercially. Using siRNA-mediated gene depletion and methylation-specific RNA immunoprecipitation with anti-m6A or m1A-antibodies, we show that (i) knock-down of the m6A ‘reader’ YTHDF2 or the m1A ‘eraser’ ALKBH3 dramatically impacts transgene expression; (ii) the effects of YTHDF2 and ALKBH3 depletion on transgene expression are m6A- and m1A-mediated. We conclude that the expression of transgenes in CHO cells can be subjected to regulation by both m6A and m1A regulators. These findings open up the prospect of previously unexplored epi-transcriptomic-based approaches to CHO cell line engineering for improved recombinant protein production.
The cleavage of heparin by heparin lyases showed great potential as a cost-effective and innoxious method for producing heparin with low molecular weight (LMWH). One of the most studied and sought heparin lyase is heparinase I (HepI). However, the industrial use of HepI was largely hampered by its low specific activity and thermal stability. In this article we describe increasing in specific heparinase I activity by stepwise site-directed mutagenesis. Thus after two cycles of mutagenesis, we obtained mutant heparinase I Flavobacterium heparinum with significantly increased specific activity (25%).
Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established either by antibiotic and/or metabolic selection. Here we report a novel technology, PTSelect™ that utilizes an siRNA cloned upstream of the gene of interest (GOI) that is processed to produce functional PTSelect™-siRNAs, which enable cell selection. Cells with stably integrated GOI are selected and separated from cells without GOI by transfecting CD4/siRNA mRNA regulated by PTSelect™-siRNAs and exploiting the variable expression of CD4 on the cell surface. This study describes the PTSelect™ principle and compares the productivity, doubling time and stability of clones developed by PTSelect™ with conventionally developed clones. PTSelect™ rapidly established a pool population with comparable stability and productivity to pools generated by traditional methods and can further be used to easily monitor productivity changes due to clonal drift, identifying individual cells with reduced productivity.
Objectives: To investigate how surgeons interpret the ATA 2015 and BTA 2014 guidelines for low risk well differentiated thyroid cancers (LRDTCs) and how they impact patient experiences across the UK. Design: Three nationally disseminated anonymised questionnaires. Setting: A nationwide snapshot of LRDTC management. Participants: Thyroid surgeons and their respective thyroid cancer multidisciplinary teams (MDTs) and thyroid cancer patients. Main outcome measures: The outcomes of interest were how surgeons/MDTs are managing LRDTCs and patient perspectives on ‘shared-decision-making’ and their ideal surgical management for LRDTCs. Results: 74 surgeons responded. 88% utilised BTA guidelines to assess recurrence risk. Tumour size, histology, stage T3b and central nodal involvement were important for >85%, but age (>45 years) only for 50%. In T1 (2cm), Thy5 solitary nodule, 58% supported hemi-thyroidectomy (HT), with 33% for total thyroidectomy (TT). In T2 (3cm) PTC, 54% opted for TT, with 24% favouring HT. Over 90% recommended TT for any incidentally excised microscopically positive lymph nodes. In T1a(m) multifocal micro-PTC, 63% suggested HT, but with contralateral benign nodules, 66% supported TT. 40% of patients felt ‘pros and cons’ of different managements were not fully explained. 47% felt they didn’t have significant input in their management, with 53% feeling final management was clinician’s choice. 60% preferred TT, with 80% wanting to ensure there was no cancer left and avoid recurrence. 20% preferred HT, with 46% wishing to avoid lifelong thyroxine. Conclusions: There is variation in risk assessment and management of LRDTCs nationally, with contrasting views of optimum treatment between patients and clinicians. These variations in practice are affecting patient experiences nationally.
Manual assessment of flower abundance of different flowering plant species in grasslands is a time consuming process. We present an automated approach to determine the flower abundance in grasslands from drone images using a deep learning (Faster R-CNN) object detection approach, which is trained and evaluated on data of five flights and two sites. Our deep learning network is able to identify and classify individual flowers. The novel method allows generating spatially explicit maps of flower abundance that meets or exceeds the accuracy of the manually counted extrapolation method and is less labor intensive. The results are very good for some types of flowers with precision and recall being close to or higher than $90\ \%$. Other flowers are detected poorly due to reasons such as lack of enough training data, appearance changes due to phenology or flowers being too small to be reliably distinguishable on the aerial images. The method is able to give precise estimates of the abundance of many flowering plant species. The collection of more training data will allow better predictions in the future for the flowers that are not well predicted yet. The developed pipeline can be applied to any sort of aerial object detection problems.
Methods: We systemically and comprehensively retrieved the eligible study evaluating clinical difference between severe versus non-severe Covid-19. Main effect sizes were demography characteristics, comorbidities, signs and symptoms, laboratory findings as well as radiological features in chest CT. Results: 14 studies with a total of 2,566 individuals (771 in Severe group and 1,795 in Non-severe group) were eligible for this meta-analysis. It was demonstrated that older and male person was more susceptible to severe Covid-19. Patients with underlying comorbidity, such as hypertension, diabetes, heart disease and COPD were associated with significantly increased susceptibility of severe Covid-19. Patients with dyspnea were more likely to be severe illness. Depressed total lymphocytes were observed in this article. Meanwhile, although reticulation (30.8%), intrathoracic lymph node enlargement (56.4%) and pleural effusions (30.8%) were relatively rarely seen, meta-analysis revealed that patients with these presentations in chest CT were associated with increased risks of severe Covid-19. Conclusions: There are significant differences in clinical characteristic between the severe and non-severe Covid-19 patients. Many factors are related to the severity of the disease, which can help clinicians to differentiate severe patient from non-severe patient.
The high genetic diversity of rear-edge refugia has been predicted to result from repeated retreats of species’ ranges to low latitudes during glacial periods in the course of Quaternary climate change. However, a few recent empirical studies of cold tolerant plants revealed an opposite pattern. We investigate whether current populations of the cold-adapted and range-restricted Bupleurum euphorbioides in the Baekdudaegan, South Korea and North Korea, could be interglacial refugia, and document how their rear-edge populations differ genetically from those of typical temperate species. Phylogeographic analysis and ecological niche modeling (ENM) were used. Genetic structure was analyzed using microsatellite markers and chloroplast DNA sequences. The congener B. longiradiatum was included as a typical temperate plant species. Despite having almost identical life history traits, these congeneric species exhibited contrasting patterns of genetic diversity. ENM revealed a wide range expansion along the Korean mountains to northern Far East Russia (Primorsky) during the Last Glacial Maximum, but not during the last interglacial. Thus, we hypothesize that B. euphorbioides retreated to refugia following maximum range contraction during interglacial periods. Unlike populations in the central region, the rear-edge populations were genetically impoverished and uniform, both within populations and in pooled regional populations. The rear-edge has endured at least one past interglacial, contributing to the species’ genetic diversity. We believe that such genetic variation in the cold-adapted B. euphorbioides gives it the necessary adaptations that will enable it to survive an upcoming favorable environment (the next glacial), unless there is artificial environmental change.