BACKGROUND AND PURPOSE Neutrophil overactivation is crucial in the pathogenesis of acute lung injury (ALI). Bletinib (3,3′-dihydroxy-2′,6′-bis(p-hydroxybenzyl)-5-methoxybibenzyl), a natural bibenzyl first extracted from Bletilla striata in 1983, has anti-inflammatory, antibacterial, and antimitotic potential. In this study, we evaluated the therapeutic effects of Bletinib in human neutrophilic inflammation and lipopolysaccharide (LPS)-mediated ALI. EXPERIMENTAL APPROACH We assessed integrin expression, superoxide anion production, degranulation, neutrophil extracellular trap (NET) formation, and adhesion in activated human neutrophils through flow cytometry, spectrophotometry, and immunofluorescence microscopy. Moreover, phosphorylation of Src family kinases (SFKs) and downstream proteins was evaluated through immunoblotting. Finally, a murine LPS-induced ALI model was used to investigate the potential therapeutic effects of Bletinib treatment. KEY RESULTS In activated human neutrophils, Bletinib reduced degranulation, respiratory burst, NET formation, adhesion, migration, and integrin expression; suppressed the enzymatic activity of SFKs, including Src, Lyn, Fgr, and Hck; and inhibited the phosphorylation of SFKs as well as Vav and Bruton’s tyrosine kinase (Btk). In our mice with ALI, the pulmonary sections demonstrated considerable amelioration of prominent inflammatory changes, such as haemorrhage, pulmonary oedema, and neutrophil infiltration, after Bletinib treatment. CONCLUSION AND IMPLICATIONS This is the first study to provide evidence that Bletinib regulates neutrophilic inflammation by inhibiting the SFKs–Btk–Vav pathway and that Bletinib ameliorates LPS-induced ALI in mice. Further biochemical optimisation of Bletinib may be a promising strategy for the development of novel therapeutics for inflammatory diseases.
Background The covid 19 positive patient who is subject to a hyperinflammatory condition associated with lung injury with the development of pneumonia is hospitalized in the intensive care unit. Before resolving and overcoming the “cytokine storm”, with overexpression of pro-inflammatory interleukins (IL-, Il-6), this patient will be intubated for more than 48 hours and therefore needs adequate nutrition. Experimental approach Malnutrition can lead to sarcopenia with a decrease in lean body mass and worsening of the inflammatory state underway. In addition, severe debilitation, if not corrected with adequate nutrition, can greatly lengthen rehabilitation times with prolonged hospitalization, increased costs and reduced turn over already in crisis due to the health emergency caused by coronavirus. Key Results The aim of this study is to focus attention on the nutritional importance that must be provided in case of covid 19 together with pharmacological treatments to lower the number of circulating proinflammatory cytokines. Conclusions Oral, enteral and parenteral nutrition should always be carried out according to the patient’s condition and, in the case of a hyperinflammatory patient, such as the one affected by covid 19, it has been shown that the supplementation of amino acids helps to lower the inflammatory state and promotes normal recovery physiological. Keywords: amino acids, nutrition, covid, glutamine, hyperinflammatory, sarcopenia, cytokine.
Since coronavirus disease 2019 (COVID-19) was reported globally pandemic, dentistry is known as one of the most critical disciplines. Teledentistry as a sub branch of telemedicine which can be a useful modality is defined by using any telecommunication technology facility, including digital photo and video to deliver oral care to isolated patients in distant places by low cost(1). Although teledentistry has been introduced as a valid tool to prevent unnecessary referrals, policy makers have not shown much interest on it yet (2,3).Diagnosis, treatment, and monitoring could be performed, meanwhile research and continuing education or public education could be done via teledentistry. In COVID-19 pandemic, teledentistry can prevent human and non-human resources burden. Oral chronic diseases like vesiculobullous and painful conditions are so dreadful and exhausting needing more care. Pediatric patients and those who are suffering from cancer are no more exceptions to this point. Tablets, smart phones, and all types of electronic gadgets can facilitate virtual communication which is helpful in telehealth.Traditional visual examination is a gold standard, in order to reach a better diagnosis and treatment plan. In teledentistry, intra oral or other high quality cameras can provide essential documents. There are so many investigations to compare visual and virtual examination, which showed only some problems in diagnosing interproximal carious lesions which is solved by digital radiography (3).Teledentistry examinations are valid, feasible, and comparable to visual examination for oral screening. Data storage is another crucial part in teledentistry due to privacy policies (2,3).Pediatric dentistry, oral medicine, orthodontics, and periodontics are the most common disciplines that have studied the efficacy of teledentistry rather than effectiveness. In developing countries, where emergency care is more important than preventive care, conservatism of decision makers, lack of resources and ICT infrastructure are the most common problems in providing teledentistry (4).Meanwhile there is a hidden advantage for teledentistry and telemedicine that it can avoid feelings of isolation and stress for patients; so, it can be more useful during COVID-19 quarantine (5).Teledentistry performs in two ways; asynchronous by transmission intraoral or extraoral images that are not used in real time; also, known as the store and forward method, synchronous by use of real-time interactive technologies, such as video or photoconferencing, and mobile health care services via smartphone apps and text messages.All the process should be recorded in patients file for further information. Accurate triage is another prominent advantage of teledentistry, which is so important during COVID-19.For people in need of care like geriatrics or patients with special needs telemedicine can eliminate some barriers (6).Besides to teledentirstry’s advantages, especially in a situation like a pandemic infection even with very primary facilities teledentistry can be performed and be useful for triage. By introducing dentistry as a very dangerous discipline, teledentistry can play a very specific role to solve the majority of patients’ problems as a helpful facility in this crisis and may be it can also persuade policy makers to perform teledentistry in a vast program.
Despite improvement in short-term outcomes, long-term results for kidney transplant recipients remain suboptimal. Immunological rejection is a leading cause of graft failure and recent research points to undetected “silent” subclinical acute rejection as a key component of this problem. While biopsies remain the gold-standard method for detecting silent rejection, non-invasive methods offer significant advantages especially in terms of patient safety and for serial monitoring of stable patients. This manuscript details the real-life challenges involved in the ultimately successful development and commercialization of TruGraf, a clinically validated, blood-based gene expression assay that offers the potential to reduce the use of surveillance (protocol) biopsies in renal transplant recipients with stable renal function.
Phasins are proteins found on the surface of natural polyhydroxyalkanoate (PHA) granules. Due to their high affinity for PHA, they can potentially be used as a fusion partner to immobilize other proteins. In this study, we investigated the immobilization of a lipase onto electrospun polyhydroxybutyrate nanofibers. Due to a superior surface area-to-volume ratio, PHB nanofibers retained much larger amounts of enzyme than conventional immobilization supports. More importantly, when used in combination with a phasin tag, the enzyme immobilized on PHB nanofibers exhibited markedly higher activity and reusability. Our approach combines the advantageous features of nanofibrous materials and the regio-specificity of biomolecular interactions for the efficient use of enzymes.
One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell cultures, is the deregulated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (being both the main nutrients used as a carbon, nitrogen and energy sources) yields to the production and accumulation of large amounts of lactate and ammonia in the culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients are the major causes that triggers the apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line producing an IgG3 (used in the ABO blood testing system) was engineered with BHRF1 protein (KB26.5-BHRF1), an Epstein–Barr virus-encoded early protein homologous to the anti-apoptotic protein Bcl-2, with the aim of protecting the cell line from apoptosis. Surprisingly, besides achieving an effective protection from apoptosis, the expression of BHRF1 modified significantly the metabolism of the hybridoma cell line. The comparison of cell physiology and metabolism analysis of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease on lactate secretion in KB26.5-BHRF1 cells. The flux balance analysis allowed quantifying intracellular fluxes of both cell lines. The main metabolic differences were identified in the glucose consumption and, consequently, the lactate generation. The lactate production flux was reduced by 60% since the need for NADH regeneration in the cytoplasm decreased due to the glucose uptake reduction by more than 50%. In general terms, BHRF1 engineered cell line showed a more efficient metabolism yielding to an increase of the biomass volumetric productivity under identical culture conditions.
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.