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.
Dear Editor,Since December 2019, coronavirus 2019 (COVID-19) has spread worldwide.1 Some data have suggested that the prevalence and mortality of COVID-19 are different among races. 2However, this analysis did not account for potential confounding factors.Since chronic kidney disease (CKD) is common, the number of COVID-19 patients with CKD will increase. However, there are scarce data about outcomes in CKD patients. We herein investigated the outcomes from COVID-19 in AAs compared to those in whites.We analyzed Mount Sinai Health System (MSHS) medical records up to April 5, 2020, using Epic SlicerDicer software. We extracted data from patients who had positive for the COVID-19 reverse-transcription polymerase chain reaction (RT-PCR) test. Sex, age, race, and comorbidities (hypertension, diabetes mellitus, ischemic heart disease, heart failure, and atrial fibrillation) were extracted using the 10th revision of the International Statistical Classification of Diseases code. Mortality and intensive care unit (ICU) admission were tracked through April 12, 2020. Relative risks (RR) and 95 % confidence interval (CI) in each race stratified by age groups and comorbidities were calculated using a Fisher’s exact test. MSHS waived Institutional Review Board approval since this research used only deidentified, aggregate-level data.During the study period, 1,269 AAs COVID-19 patients with 105 CKD patients and 1,450 whites COVID-19 patients with 80 CKD patients were detected. AAs were younger (median 66, IQR 55-76) than whites (median 75, IQR 65-83) (p< 0.001). There was no significant difference in mortality between AAs and whites (0.65 [0.36-1.15]). This tendency was observed after stratification by age and medical conditions. Similarly, AAs did not have an increased risk of ICU admission (0.84 [0.6-1.18])) even after stratification by age and comorbidities (Table).To the best of our knowledge, this is the first study that compared the risk of severe outcomes among races in CKD patients. Although it has been suggested that there might be racial disparity in COVID-19, our study did not show any significant differences in outcomes, even after stratifying patients by age and comorbidities. Our data suggested that we do not need to stratify these patients by race.The racial and ethnic diversity in NYC enabled us to investigate differences in outcomes among races in the same cohort. However, our study has several limitations. First, the number of patients was relatively small. Second, we did not access individual data, which prevented us from performing multivariate analyses. The fact that AAs were younger might mask differences among races.In conclusion, AAs with CKD did not have a higher risk of mortality or ICU admission than whites with CKD. This trend was consistent after stratification by age, sex, or comorbidities.Acknowledgements: noneConflict of Interest Disclosures: TY reports no conflict of interest. TM reports no conflict of interest. NC reports no conflict of interest. HM reports no conflict of interest. SC repots no conflict of interest. SM reports no conflict of interest.Reference1. Team CC-R. Preliminary Estimates of the Prevalence of Selected Underlying Health Conditions Among Patients with Coronavirus Disease 2019 - United States, February 12-March 28, 2020. MMWR Morb Mortal Wkly Rep. 2020;69(13):382-386.2. Health N. Age adjusted rate of fatal lab confirmed COVID-19 cases per 100,000 by race/ethnicity group as of April 6, 2020 (Accessed Aprio 12, 2020). 2020.
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.