Gliomas are aggressive central nervous system tumours. The emergence and recent widespread adoption of 5-aminolevulinic acid and fluorescence guided surgery have improved the extent of resection, with implications for improved survival and progression-free survival. This review describes the history, rationale and mechanism behind the use of 5-aminolevulinic acid and fluorescence-guided surgery. We also discuss current limitations and future directions for this important adjunct to glioma surgery. This review aims to provide readers with an up-to-date overview and evidence base on this important topic.Gliomas are central nervous systems tumours which are diffusely infiltrative and difficult to treat. The extent of surgical resection is correlated with improved outcomes, including survival and disease-free progression. Cancerous tissue can be directly visualised intra-operatively under fluorescence by administration of 5-aminolevulinic acid to the patient. The adoption of this technique has allowed surgeons worldwide to achieve greater extents of resection, with implications for improved prognosis. However, there are practical limitations to use of 5-aminolevulinic acid. New adjuncts in the field of fluorescence-guided surgery aim to improve recognition of the interface between tumour and brain with the objective of improving resection and patient outcomes. Gliomas are intrinsic tumours of the brain, accounting for the majority of malignant intra-axial central nervous system lesions . The preThe 2016 revision of the WHO 2007 classification of brain tumours  has incoThe inherently infiltrative nature of gliomas through the surrounding brain parenchyma poses a particular challenge for neurosurgeons and neuro-oncologists. Delineation between tumour and normal brain parenchyma is often difficult to achieve both pre-operatively and intra-operatively. Despite modern advances in imaging, it is often difficult to differentiate between peri-lesional oedema and non-enhancing tumour on magnetic resonance imaging (MRI) , as demoGlioma cells invade through the extra-cellular matrix along white matter tracts and perivascular spaces. This invasive growth occurs through a combination of changes induced in its microenvironment including modification of the stromal architecture, active degradation of normal brain matrix and immune modulation to bypass surveillance, detection and destruction . InfiltrThe importance of an adequate resection, removing as much tumour tissue as is safely possible without causing a neurological deficit to the patient, has long been recognised . The thrIncreased resection rates also increase tumour response to chemo-radiotherapy . This grThe total volume of resection to achieve an optimum trade-off between safety and good clinical outcome remains debated . The genIf indeed there is a “surgical minimum” threshold that confers clinical benefit it is likely to differ between tumours with different molecular characteristics, as well as patient factors and oncological treatment. Various molecular markers ,29 demonOver the last two decades, there has been an exploration of intra-operative adjuncts designed to optimise safe glioma resections, led by fluorescence-guided surgery (FGS). Fluorescence refers to the emission of light by a substance which has absorbed electromagnetic radiation. It is commonly seen throughout nature and has a number of industrial uses including the ubiquitous fluorescent light bulbs that grace most hospitals. The first application of fluorescence in surgery dates back to 1948, when fluorescein was used in the localisation of intracranial tumours . In thisMore recently, intra-operative fluorescence guidance using 5-aminolevulinic acid (5-ALA) has become widely trialled and adopted ,33,34. 5The exact mechanism by which 5-ALA specifically targets tumour cells remains unclear. 5-ALA delivery into proliferating cells is facilitated by disturbance of the blood–brain barrier in tumours . Intracr5-ALA is administered orally at a dose of 20 mg/kg, 4–6 h prior to surgery. Tumour tissue can be directly visualised by emitting pink fluorescence under blue light, with 85% sensitivity and 100% specificity reported in the initial piloting study . SimilarThe use of 5-ALA improves the extent of resection, demonstrating that real-time intra-operative visualisation of malignant tissue can lead to wider resection margins that often extend beyond the limit of contrast enhancement on pre-operative imaging ,50. ThisThe now widely accepted notion that gross total resections and radiologically complete resections of enhancing tumour carry the most favourable survival and disease-free progression outcomes has transformed clinical practice in recent years. The increased adoption of routine 5-ALA use worldwide is a testament to this new method of surgical thinking.Reports on how much additional improvement in EOR achieved with 5-ALA vary, though since 2006 there has been an increasing trend of higher gross total resections demonstrating growing familiarity with the technique. Stummer reported complete resection of 65% in patients undergoing 5-ALA aided resection, versus 36% in those operated under standard white light conditions. Figures as high as 93% for GTR > 98% and 100% for GTR > 90% have been reported .The prospective trial data ,34 has lNearly a decade after European approval, 5-ALA use was approved by the FDA as an intraoperative optical imaging agent; the relative delay here was due to the initial interpretation of 5-ALA as a therapeutic rather than adjunctive agent, therefore requiring a novel regulatory process .There are practical limitations of intra-operative 5-ALA use. Normal appearances during 5-ALA-assisted surgery are of a core of highly fluorescent ‘red’ tumour, surrounded by a rim of more faintly fluorescing ‘pink’ tumour which reflects the ‘infiltrating zone’ of a tumour. The main factor limiting achievement of gross total resection is resection of this ill-defined zone. Furthermore, the majority of resected glioblastomas undergo local recurrence at or near to the previous resection cavity ,57. PoteHowever, infiltrating tumour is not always reliably identified by 5-ALA. There are lower levels of PpIX and fluorescence in the infiltrating zones of tumours . Non-fluThe utility of 5ALA as a surgical adjunct is also much more limited in low-grade gliomas (LGGs). With modern treatment regimes, patients with LGGs may survive for many years. If they are exposed to morbidity from surgery, this can lead to long-term disability and reduced quality of life ,64,65; tLimited visible fluorescence in LGG is well recognised , with reFinally, 5-ALA has financial implications which may hinder adoption in some countries. The cost of administering 5-ALA along with the specialist fluorescence intra-operative equipment required has been quoted to approximate £1 million per year for a neurosurgical unit . Cost-efNew adjuncts in the field of fluorescence-guided surgery aim to improve recognition of the interface between tumour and brain with the objective of improving resection, minimising damage to surrounding normal brain, and improving patient outcomes.The practical application of 5-ALA has changed little since its introduction and approval by the EMA in 2007, and the intra-operative evaluation of fluorescence still relies on subjective evaluation with the naked eye. The degree of visualised fluorescence is related to the degree of PpIX accumulation and therefore the density of tumour cells . HoweverThere is scope to refine and enhance the current use case of 5-ALA, either by improving differentiation between areas of minimal fluorescence and normal brain or by enhancing the degree of fluorescence, for example with laser excitation . CurrentNumerous groups ,77,78,79Spectroscopic techniques are providing promising results in terms of improving sensitivity of PpIX fluorescence. However, there are limitations to this approach. There is currently no standardised ‘threshold level’ for what differentiates tumour from normal tissue. While sensitivity for PpIX fluorescence does improve, this has yet to translate into compelling evidence that these techniques improve rates of gross total resection and patient outcomes. There are also operative considerations; the use of handheld probes that only sample a small area of tissue may be disruptive to the surgical workflow, and its applicability to an entire resection cavity is limited. Widefield techniques that can interrogate the entire surgical field are limited in their ability to provide instantaneous quantification that would allow the surgeon to respond in real time .Fluorescence-guided surgery is a rapidly developing field; a recent review identified 39 new agents undergoing 85 clinical trials for all tumours . The ideWhile macroscopically similar, glial tumours and normal brain parenchyma differ significantly in terms of their microscopic structure and behaviour. These differences can be exploited to provide contrast between the tissue types, and this is reflected in the diverse range of techniques and targets being used by novel fluorescent probes. There are a number of novel fluorescent probes undergoing early clinical trials for use in glial tumours see  and a hoEGFR is overexpressed in a majority of primary glioblastomas  but not Alternatively, improved contrast between tumour and normal cells can be achieved via use of an activatable probe. This would eliminate the background signal of normal tissue. 5-ALA and PpIX preferentially accumulate in tumour cells; however, the synthesis of PpIX happens in all cell types as it is part of normal biosynthetic pathways. Activatable fluorescent probes remain quenched until they are activated in specific ways within tumour cells. LUM015 is a protease-activated fluorescent probe undergoing investigation in a number of cancers, including glioma. It consists of a fluorescence-quenching molecule and a fluorophore bound by a peptide backbone . This peThese findings also open the door to intriguing applications of fluorescent agents. EGFR-targeting fluorescent probes have demonstrated improved accuracy over 5-ALA in EGFR-positive tumours but not in wildtype tumours . This raAn important consideration with the use of multiple fluorescent agents is one of safety. The previous study did not raise any specific safety concerns with combination therapy; however, liver function tests (which can become deranged after 5-ALA administration) were not routinely monitored, and the power of the study would be insufficient to detect rare adverse events. Further, it is a reasonable assumption that the use of dual fluorescent agents may also increase the rate of false positives in fluorescing tissues, potentially increasing the risk of inadvertent resection of normal brain and its associated complications. Studies designed specifically to assess the safety of combined therapy will therefore be required.www.nice.org.uk/guidance/NG99, accessed on 1 May 2021) supports its use in patients with suspected high-grade gliomas for which complete resection is possible [The evidence base for routine use of 5-ALA in glioma surgery is increasing; however, the quality of clinical data remains low . To datepossible . Similarpossible  license Fluorescence-directed resection comprises part of an armoury of operating tools used in neuro-surgical oncology. It improves the contrast between tumour and normal tissue, thereby improving rates of resection. However, it should not be used in isolation because the overriding surgical objective is to maximise resection whilst minimising the risk of any neurological deficit to the patient. To this end, fluorescence-directed resection must be combined with other intra-operative adjuncts, rigorous pre-operative planning and a detailed understanding of functional neuroanatomy.The addition of 5-ALA and FGS has proved an invaluable tool in helping surgeons worldwide in achieving maximal safe resections for glioma surgery. The ability to directly visualise malignant tumour tissue intra-operatively has strong implications for maximising extent of resection, improving survival and progression-free survival. Despite limitations by the inherent heterogeneity of gliomas and patient populations, the body of literature behind its influential role in glioma surgery is growing. Recent advancements in refining the use of 5-ALA as well as discoveries in novel fluorescence techniques hold future promises for FGS. It is our hope, as well as that of the wider scientific community, that this important field will continue to grow, flourish and benefit patients in generations to come.


Progress in developing fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, is inseparable from the advancement in optical fluorescence microscopy. Super-resolution microscopy, or optical nanoscopy, overcame the far-field optical resolution limit, known as Abbe’s diffraction limit, by taking advantage of the photophysical properties of fluorescent probes. Therefore, fluorescent probes for super-resolution microscopy should meet the new requirements in the probes’ photophysical and photochemical properties. STED optical nanoscopy achieves super-resolution by depleting excited fluorophores at the periphery of an excitation laser beam using a depletion beam with a hollow core. An ideal fluorescent probe for STED nanoscopy must meet specific photophysical and photochemical properties, including high photostability, depletability at the depletion wavelength, low adverse excitability, and biocompatibility. This review introduces the requirements of fluorescent probes for STED nanoscopy and discusses the recent progress in the development of fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, for the STED nanoscopy. The strengths and the limitations of the fluorescent probes are analyzed in detail. An optical fluorescence microscopy is a powerful tool for studying microscopic objects in biological systems due to its ability to observe living cells, reasonable resolution 200–250 nm) for observing many subcellular features, noninvasive image acquisition, an option of multiplexed operation, and most importantly, chemical, and molecular specificity at single-molecule sensitivity. Sensitive and specific fluorescent labeling of biomolecules including proteins, nucleic acids, and lipids is indispensable for the success of modern fluorescence microscopy. Initially, fluorescent staining with molecular specificity was limited to several small molecules with intrinsic affinity to specific biomolecules and cell membrane permeability. A prominent example is the development of the DNA-specific stains DAPI  and Hoec00–250 nmAlthough the resolution in optical microscopy is limited by Abbe’s optical diffraction limit , a serieIn contrast to SMLM -based approaches such as PALM, STORM, or dSTORM, STED optical nanoscopy takes a somewhat orthogonal approach rooted in point scanning confocal microscopy. When a fluorophore gets excited from the ground state to the excited state, it can release the absorbed energy as a fluorescent photon via spontaneous emission. Alternatively, it can release its energy via stimulated emission if there is a light beam carrying the same energy. It is, therefore, possible to force a fluorophore to cycle only in a transition involving stimulated emission but not in a transition involving fluorescence emission; this is called stimulated emission depletion (STED) . In pracansition c 22]. T. TI is h·(2NA)−1 .4 to 105 times stronger than that of the excitation beam. Decreasing the photobleaching in STED nanoscopy has been an important research direction . D. D27]. Dsis cost ,109. CsPm yields . By virtm yields . Pdots cm yields . On the Progress in STED optical nanoscopy was inseparable from the development of fluorescent probes for STED. In some cases, new methodologies for STED nanoscopy were developed to overcome the limitation of the available probes. On the other hand, probes with better photophysical and photochemical characteristics for STED nanoscopy were developed. This review discussed the requirements of probes to be suitable for STED nanoscopy. A proper STED nanoscopy dye must meet four major requirements: high photostability, good depletability, low adverse excitability, and good biocompatibility. These requirements are closely related to the nature of STED nanoscopy and the use of a strong STED beam. The methods that can circumvent the background noise problem arising from low depletability or high adverse excitability have been developed. Although there are technical measures to ease the probe requirements, this review showed that it is essential to have probes with good properties in the first place. Different kinds of probes that have been developed and implemented in STED nanoscopy so far have been identified and discussed. It was noted that fluorescent proteins were developed for STED nanoscopy but were not heavily used due to their relatively low photostability and brightness. Nevertheless, they have been actively developed for RESOLFT nanoscopy, which shares a similar working principle with STED nanoscopy. The review also discussed the development of organic dyes as the most widely used fluorescent probes for STED nanoscopy. Several developments to enhance the brightness, photostability, and biocompatibility of organic dyes have been reported. Adjusting the probe’s excitation and emission spectrum was an important research direction for better multicolor STED imaging. Lastly, different kinds of fluorescent nanoparticles have been introduced. AIE NPs were bright and highly photostable but have compromised biocompatibility. Qdots were bright and photostable but had strong adverse excitation. Methods to overcome the noise originating from Qdots’ adverse excitation were also discussed. Qdots tend to have high cytotoxicity, similar to AIE NP. We introduced recently developed Cdots and Pdots, which are biocompatible, photostable under intense STED laser, and easily depletable, making them potentially suitable STED probes.Interestingly, no single fluorescent probe meets all the requirements simultaneously for the STED nanoscopy. Therefore, there seems to be ample room for further development of FPs, organic dyes, and FNP and the development of STED techniques to compensate for the probes’ weaknesses and achieve high-quality STED imaging.


Electron tomography (ET) has become an indispensable tool for structural biology studies. In ET, the tilt series alignment and the projection parameter calibration are the key steps toward high-resolution ultrastructure analysis. Usually, fiducial markers are embedded in the sample to aid the alignment. Despite the advances in developing algorithms to find correspondence of fiducial markers from different tilted micrographs, the error rate of the existing methods is still high such that manual correction has to be conducted. In addition, existing algorithms do not work well when the number of fiducial markers is high.In this article, we try to completely solve the fiducial marker correspondence problem. We propose to divide the workflow of fiducial marker correspondence into two stages: (i) initial transformation determination, and (ii) local correspondence refinement. In the first stage, we model the transform estimation as a correspondence pair inquiry and verification problem. The local geometric constraints and invariant features are used to reduce the complexity of the problem. In the second stage, we encode the geometric distribution of the fiducial markers by a weighted Gaussian mixture model and introduce drift parameters to correct the effects of beam-induced motion and sample deformation. Comprehensive experiments on real-world datasets demonstrate the robustness, efficiency and effectiveness of the proposed algorithm. Especially, the proposed two-stage algorithm is able to produce an accurate tracking within an average of https://github.com/icthrm/auto-tilt-pair. Additionally, the detailed original figures demonstrated in the experiments can be accessed at https://rb.gy/6adtk4.The code is available at Bioinformatics online. Before reconstruction, an accurate tilt series alignment is required. Usually, gold beads are used as fiducial markers to assist the alignment . So far,Finding the correspondence of the fiducial markers on different tilted micrographs is the first and most important step in tilt series alignment. After many years of effort, several automated methods have been proposed: RAPTOR  uses theRecently, non-linear alignment and reconstruction have been further proposed in ET . AccordiIn this article, we propose to divide the determination of fiducial marker correspondence into two stages: (i) initial transformation determination and (ii) local correspondence refinement. A novel two-stage algorithm by considering the local geometric constraint is proposed to ensure the efficient fiducial marker tracking while keeping robustness and accuracy. In the first stage, we model the initial transformation estimation as a correspondence pair inquiry and verification problem. The local invariant features are used for the fast comparison of the geometric similarity between different fiducial marker positions on different micrographs. Within each similarity inquiry, we limit the range of local feature extraction and propose a multiple-check technique of the extracted features for fast consistency verification. In the second stage, we encode the geometric distribution of the fiducial markers by a weighted Gaussian mixture model and solve the correspondence of distributions by an expectation-maximization algorithm, where the parameters about non-uniform drift are introduced to correct the potential distortion. The obvious outliers are pre-excluded based on the solved initial transformation.With the two-stage algorithm, our aim is to completely solve the fiducial marker correspondence and tracking problem in ET. The utilization of local geometric constraints further reduces the computational complexity and the introduction of drift parameters ensures the accuracy in fiducial marker correspondence determination. Comprehensive experiments on real-world datasets demonstrate the robustness, efficiency and effectiveness of the proposed algorithm. Especially, the proposed algorithm is able to produce an accurate tracking within an average of First, we would like to redefine the problem under the terminology of point set registration:Denoting the positions of fiducial markers from a micrograph as the fixed ‘scene’ point set A is a 2 × 2 affine matrix and t is a 2 × 1 translation vector. Without considering the sample deformation, an affine transformation is able to describe the correspondence of the fiducial markers extracted from two different tilted micrographs . Various local geometric constraints have been proposed to reduce the complexity, within which the nearest neighbor constraint is the most popular one .minl is related to the numerical stability of a given coordinate system, which depends on the nanoscale of fiducial markers and the pixel size of the micrograph. Here, we call this local geometry constraint the min&max rule for the 4-point invariant feature.We further exclude the 4-point invariant feature with too small diagonal lengths: if the localization error for a fiducial marker is 5 pixels, a 4-point invariant feature with a diagonal length of 20 pixels will suffer from minl = gbl and maxl = hgl for the convenience of demonstration. Here, for the points selected in 2.2.2We could extract a ‘query’ subset from the ‘model’ point set Partial inquiry for a certain feature: For a 4-point invariant feature r1 and r2, we try to check its correspondence in e.r1, and r2  time complexity, building the nearest search tree has O(K) for the number of tries). Therefore, the total complexity of the algorithm is Because point pairs 2.2.3Considering the effect of noise and the missed detection of fiducial markers, multiple trials of congruent subset inquiry is necessary. Here, we adopt a random sample consensus (RANSAC) procedure  to robusBased on the min&max rule, the sets of range limited point pairs Compose a random 4-point set For each congruent subset maxc, save the current value as maxc and update the termination condition I.If the number of congruent points between Repeat Steps 1 ∼4 until termination.β1, β2 as input, the distance threshold d, the area ratio relaxation ξ as parameters, and outputs the estimated transformation d can be set according to the value of fiducial marker diameter and the relaxation ξ can be estimated based on the experiments of mechanical instability. Generally, d is set to D is the fiducial marker diameter and λ is not essential to the system. In the algorithm, we denote the operation to count the corresponding points between d as sp is the required success probability, gp is the percentage of traceable fiducial markers that appear in both k is set to 4 as the inquiry of 4-point invariant subset.By considering the local geometric constraint, the optimized randomized algorithm is able to produce a fast and robust estimation of the initial transformation. However, the distortion caused by sample deformation or beam-induced motion is non-negligible  .σ is a parameter to describe the instability of the system. Similarly, the probability that the point x belongs to the point set P(m) is the prior of x under the condition of the mth point Given the fixed ‘scene’ point set x either belongs to outliers with w probability or sampled from the point set mth point σ applied to all the points P(m) is the reweighted i.d.d prior and Considering a drift transform 2.3.2A negative log-likelihood objective function could be effectively solved by the expectation-maximization (E-M) optimization, where the algorithm iterates between the E-step and the M-step until convergence. With Jensen’s inequality , and M-step: Here we model the transform 1—the column vector of all ones;a;4. P—the matrix that is composed by Q: λ is a trade-off parameter.Based on the Tikhonov regularization framework , a non-rIf we model the regularization function e (RKHS) , the negY under the Euler-Lagrange differential equation: L. By rewriting the equation and using an integral of a Green’s function G is an M × M kernel matrix with elements To minimize T back into Q and solving the partial derivation, Consequently, the transform d. With the initial transformation inherited from the first stage, Algorithm 3 is able to quickly converge. At the same time, because the initial transformation is very close to the global optimum, Algorithm 3 will converge to the global optimum in a high probability.The overall iterative optimization for the GMM-based drift correction is summarized in Six real-world datasets are used to evaluate the proposed method. The first dataset is a tilt series that has been used in the previous studies, provided by the Institute of Biophysics, Chinese Academy of Sciences .2. There are 95 images with the tilt ranging from The first dataset, Hemocyanin, is a tilt series of vitrified keyhole limpet hemocyanin solution . It is ahttps://bit.ly/35HJoWS; Vibrio2 is downloaded from https://bit.ly/3kGvl8l.), are two cryo-ET datasets of isolated Vibrio cholerae cells , are three cryo-ET datasets of isolated Nitrosopumilus maritimus cells  in the space, they may result in a very different drift magnitude, even though both markers appear next to each other in the projection image, which could be observed in nth and nth and In practice, for the tilt series alignment, we may track several neighbors of the micrographs and then compose the fiducial marker correspondences into the fiducial marker tracks. Here, for all the datasets, we matched the fiducial marker positions within 2-neighbors micrographs, i.e. the Considering that the deformation usually happens in high tilt angles, we calculated the average detection ratio of micrograph pairs with tilt angle The efficiency is also a critical factor when a new algorithm is applied to the real-world datasets. For each matching operation, the runtime of different methods against the number of fiducial markers is recorded. As shown in 3.2.3Finally, we integrated the two-stage algorithm into the fully automatic alignment scheme  Office of Sponsored Research (OSR) under Awards No. FCC/1/1976-17, FCC/1/1976-23, FCC/1/1976-26, URF/1/4098-01-01, URF/1/4352-01-01, URF/1/4379-01-01, REI/1/0018-01-01 and REI/1/4473-01-01.Conflict of Interest: none declared.btaa1098_Supplementary_DataClick here for additional data file.


Enterobacterales isolates. The use of these agents led to an increase of carbapenem resistance among Enterobacterales. Monitoring isolates that carry β-lactamases is important to understand their prevalence and susceptibility to clinically available antimicrobial agents. We evaluated the prevalence of β-lactamases and the activity of antimicrobial agents against 1,209 isolates collected in 69 US hospitals.Carbapenems are broadly used for the treatment of ESBL-producing Enterobacterales isolates collected during 2019 were susceptibility (S) tested by reference broth microdilution methods. Isolates submitted to whole genome sequencing were: (1) Escherichia coli (EC) and Klebsiella pneumoniae  displaying MIC values ≥2 mg/L for at least 2 of the following β-lactams: ceftazidime, ceftriaxone, aztreonam, or cefepime; (2) Enterobacter cloacae (ECL) and Citrobacter spp.  displaying MIC values ≥16 mg/L for ceftazidime and/or ≥2 mg/L for cefepime; and (3) Enterobacterales (n=118) displaying elevated carbapenem (meropenem and/or imipenem) MIC results at >1 mg/L.A total of 9,686 blaCTX-M-15 (n=516), followed by blaCTX-M-14 (n=153). Most of these isolates were EC (278/147 for blaCTX-M-15/blaCTX-M-14), but 220 KPN harbored blaCTX-M-15. A total of 302 EC and KPN isolates carried blaOXA-1. Among ECL and CIT, blaCTX-M-15 and SHV genes encoding ESBLs were noted among 18 and 18 isolates. Carbapenemase genes were noted among 77 isolates, including 65 blaKPC, 3 blaSME, 6 blaOXA-48-like, and 3 blaNDM. Ceftazidime-avibactam (CAZ-AVI) was the only agent active against all ESBL-producers that did not carry carbapenemases (Table). CAZ-AVI was active against 90.9% of the isolates producing carbapenemases. Isolates resistant to this combination included 3 NDM-producers and 1 isolate harboring blaKPC-31.A total of 723 isolates harbored ESBL genes but did not carry carbapenemases. The most common ESBL gene was Enterobacterales isolates carrying ESBLs, mainly blaCTX-M-15, were very prevalent in this collection of US isolates. CAZ-AVI was very active against isolates tested, including isolates producing carbapenemases that displayed resistance to many comparator agents.Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support Lalitagauri M. Deshpande, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Pfizer, Inc. (Research Grant or Support) Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Helio S. Sader, MD, PhD, FIDSA, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)


The present article deals with a continuum mechanics-based method to model an electro-magneto-rheological (EMR) fluid deformation subjected to an electromagnetic field. The proposed method follows the fundamental laws of physics, including the principles of thermodynamics. We start with the general balance laws for mass, linear momentum, angular momentum, energy, and the second law of thermodynamics in the form of Clausius–Duhem inequality with Maxwell’s equations. Then, we formulated a generalized constitutive model for EMR fluids following the representation theorem. Later, we validate the model with the results of an EMR rheometer and ER fluid valve system-based configurations. At last, the possible simulation-based velocity profiles are also discussed for parallel plate configuration. As a result, we succeed in providing more physics-based analytical findings than the existing studies in the literature. At the same time, there is a specific need to understand the deformation of such smart fluids due to their usage finding in various applications like clutches and brakes in cars, vibration-dampers, and absorbers, lubricating fluids in bearings, smart transducers for medical purposes, etc. In engineering applications, EMR fluids are generally used in hydraulic systems, which are inherently nonlinear. Therefore, their accurate model prediction became so essential that it will directly affect the system performance. The current work is motivated by our previous works on electro-magneto-active (EMA) solids7. In brief, EMR fluids are colloidal suspensions of polar particles. Similarly, the particles are added as impurities in EMA solids. In EMR fluid, the particles undergo Brownian motion without external fields. While in EMA solids, the filler particles are randomly oriented in the absence of external fields. On the application of external field, the EMR fluid behaves as near-solid but not like a solid. This exceptional nature demarcates the EMR fluid from EMA solid. Thus, thermodynamic pressure is inevitable in the case of EMR fluid, irrespective of whether an external field is applied or not. One may readily appreciate that the constitutive relation of EMR fluid will be governed by the stretch rate, which is time-dependent. Conversely, the EMA solids are considered incompressible hyperelastic, which essentially exhibits the time-independent nature and constitutive relation associated with a Lagrange multiplier to address the incompressibility9. In the current work, we focus on the constitutive modeling of EMR fluids specifically used in smart hydraulic systems. In general, EMR fluids are characterized by their ability to vary their mechanical properties with an electromagnetic field significantly. EMR fluids are smart, synthetic fluids that change their viscosity from liquid to semi-solid state within milliseconds if a sufficiently strong electric or magnetic field is applied11. These fluids usually consist of micron-sized electro-magneto-active filler particles dissolved in a non-conducting liquid like mineral or silicone oil. EMR fluids also offer an innovative potential for quick and adaptively controllable electro-magneto-mechanical interfaces in control units when used in suitable devices13. A coupled electro-magneto-rheological theory is needed to model such EMR fluids within the framework of the continuum mechanics-based approach. Also, the coupled theory must describe the electromagnetic field interaction with the moving deformable media16 such as EMR fluids.In the current scenario, electro-magneto-rheology established a new research direction, which potentially provides fruitful ideas for modern engineering and medical field applications. In line with that, new hybrid electro-magneto-rheological (EMR) fluid systems that couple electro-magneto-mechanical properties of smart fluids17 made an initial attempt to understand the behavior of ER fluids under external gradient. He17 investigated an electrically induced fibration of small particles in fluid liquid suspension. Nevertheless, their applicability in fluid system modeling has not been fully explored for many years. Later, the first standardized approach to model such rheological behaviours under external gradients were presented by various researchers, namely, Atkin and Bullogh18, Abu-Jdayil and Brunn21, Rajagopal10 along-with Yalamanchili22 and Wineman11. Herein, they22 adopted the classical continuum mechanics-based approach to model the rheological behaviours of ER fluids under external gradient. Further, some researchers, namely, Conard et al.23, and Jordan et al.24 also proposed different one-dimensional models for ER fluids. Furthermore, Rajagopal and Wineman10 developed three-dimensional models satisfying the appropriate invariance requirements. However, in these models10, the electric field was treated as a constant field. In addition to the deformation modeling of ER fluid under external gradient, similar studies on the deformation modeling of MR fluid were also focused by the researchers26 followed by similar approaches. To mention some earlier works on EMR fluids, the theoretical foundations for the analysis of electro-magnetomechanical interactions in EMR fluids were developed in part during the 1990s and 2020s, and a detailed summary may be found, for example, in Hutter et al.27 and references therein. In addition, Fujita et al.1 investigated a combined electromagnetic field control effect on the rheological properties of a fluid. However, they1 have not fully utilized the magnetic field for fine control of the rheological properties. Next, Minagawal et al.2 developed a new technique to measure the rheological properties of fluids under electric and magnetic fields. Further, Koyama et al.3 compared an EMR effect observed in fluids with the parallel and cross-field systems. Moreover, they3 suggested that the parallel-field effect was more significant than the crossed one. At last, we observe that many works on the modeling of ER and MR fluids have been devoted. However, the deformation mechanics dedicated to an EMR fluid continua under an electromagnetic field is still not been reported to the best of our knowledge.To mention some earlier works on electro-rheological (ER) and magneto-rheological (MR) fluids, Winslow28 is adopted. Additionally, the developed model is also experimentally validated in contrast to the existing works22 aimed at the pure theoretical models for smart fluids.The present article aims to generalize the deformation concept of fluid continua to electro-magneto-rheology in contrast to the existing studies on ER and MR fluids. A constitutive model is developed here for an incompressible isotropic non-Newtonian EMR fluid subjected to an electromagnetic field. To develop the same, a thermodynamically consistent classical continuum mechanics-based approachThe further part of the paper is organized as follows. In “In this section, a brief review on electro-magneto-rheology related to the deformation of EMR fluid continua is presented, followed by the fundamental laws of physics. These fundamental laws provide initial steps to model the exact electro-magneto-rheological behavior of EMR fluids under an electromagnetic field application.X with respect to an origin. With an application of electromagnetic field, the material deforms and a point X in the material occupies a new position v and acceleration a of that material point in three-dimensional Euclidean space, at the instant of time t are defined asd and anti-symmetric part W asConsider an EMR fluid occupying the material space E, the electric displacement vector D and the polarization density P as the electric field variables. Similarly, the magnetic field vector H, the magnetic displacement vector B and the magnetization density vector M are considered as the magnetic field variables in the current configuration 29 under our constant electric field as well as constant magnetic field application assumption asE and D, respectively.At reference configuration The mechanical balance laws for fluids are expressed in the local form asS and 2932 and references therein.U is the internal energy (per unit mass) and 29rom Eqs. , the aboIn this section, the concepts of an electro-magneto-rheological deformation of fluid continua presented in previous “27k defined asS as an isotropic in nature. In this regard, we may define a thermodynamic pressure acting on a homogeneous EMR fluid with an application of electromagnetic field as S is an isotropic function of its arguments and has various mini-subspaces. This mini-subspaces span can be utilized for the span required to map other tensors that functionally depend on the tensor S. Now, from the representation theorem33, we consider that the tensors S. We may here now link our previous physical assumption (isotropic Cauchy stress) that is verified from the relation , Case-II) and Case-III), which are discussed in Table Case-I, the applied electric and magnetic fields are mutually parallel and normal to the EMR fluid flow direction. Next, in Case-II, the applied electric field is mutually perpendicular to the magnetic field and parallel to the EMR fluid flow direction. At last, in Case-III, the applied electric and magnetic fields are mutually perpendicular and normal to the EMR fluid flow direction. In addition, we herein assume that the fluid velocity varies only in one direction significantly, and the other directional variations are negligible.In line with the shear flow applications, we consider three different standard cases as shown in Figs. 34 is modeled through the developed constitutive relation  channel of height h formed by two electrodes as shown in Fig. L and width B. The ER fluid in the channel is assumed to be isotropic, incompressible, and isothermal. Herein, the volume flow was driven by the pressure difference between inlet and outlet pressures. With an application of electrical voltage U at a single electrode and by earthening the other electrode, an electric field was generated that changes the rheological properties of the ER fluid.To check the validity of constitutive relation  derived 34 as shown in Fig. 34 had developed an analytical model similar to our 34. In ad3 They3 carried out rheological measurements of EMR fluid by the use of a parallel-plate rheometer equipped with electrodes and magnetic coils as shown in Fig. To further access the validity of constitutive relation , we compThe electric field was applied to the system by the electrodes attached to the surfaces of the plates. At the same time, the magnetic field was generated through the motion of the coils toward the plates. The rheological measurements were taken as the stress increase induced by electric and magnetic fields.3 as shown in Fig. Firstly, we model the above EMR fluid-based rheometer configuration used by Koyama et al.nt}α5 in  based ons tensor  through bove Eq.  for an E3 in parallel-field condition, we plot the same on Fig. H at different applied electric field intensities. Herein, an increase in the slope of the lines under higher electric fields indicates the ‘synergistic effect’ of the electric and magnetic fields on the EMR effect. This effect is taken care through our field dependent material constant 3. The model  induced by the cross-field condition in an EMR fluid system significantly affect the resistance against shear deformation compared to the parallel-field condition. In addition, the analytical predictions of the model  with  that \doCase-I in “11 at zero magnetic field condition.In the given configuration, a constant electric field In summary, we may control the EMR fluid flow situations and play with the corresponding theoretical parameters for the given situation through a specific choice of the relationships between Case-III is independent of an additional magnetic field application. However, for the remaining Case-I and Case-II, this additional magnetic field application significantly affects the EMR fluid deformation. Based on this observation, we may comment that an electric or magnetic field normal to fluid flow direction with different plane does not affect the shear flow deformation in EMR fluids.From (26), (29) and (32) for each of the considered case, we note that the shear response in S with the stretch rate tensor d, electric field vector E and magnetic field vector H. Next, the relation (In the present paper, we develop a thermodynamically consistent generalized constitutive relation  for an irelation  is appliThe major contribution of the present study is an attempt to generalize the deformation concept of fluid continua to electro-magneto-rheology in contrast to the existing works on ER and MR fluids. Moreover, the previous works are also obtainable from the same as a special case. In addition, an analytical model  proposed


Botulinum toxin-A (BoNT-A) blocks acetylcholine release at the neuromuscular junction (NMJ) and is widely used for neuromuscular disorders . However, its therapeutic effects are usually measured by clinical scales of questionable validity. Single-fiber electromyography (SFEMG) is a sensitive, validated diagnostic technique for NMJ impairment such as myasthenia. The jitter parameter (µs) represents the variability of interpotential intervals of two muscle fibers from the same motor unit. This narrative review reports SFEMG use in BoNT-A treatment. Twenty-four articles were selected from 175 eligible articles searched in Medline/Pubmed and Cochrane Library from their creation until May 2020. The results showed that jitter is sensitive to early NMJ modifications following BoNT-A injection, with an increase in the early days’ post-injection and a peak between Day 15 and 30, when symptoms diminish or disappear. The reappearance of symptoms accompanies a tendency for a decrease in jitter, but always precedes its normalization, either delayed or nonexistent. Increased jitter is observed in distant muscles from the injection site. No dose effect relationship was demonstrated. SFEMG could help physicians in their therapeutic evaluation according to the pathology considered. More data are needed to consider jitter as a predictor of BoNT-A clinical efficacy. Botulinum toxin type A (BoNT-A) has been approved by the U.S. Food and Drug Administration and the European regulatory agencies as an effective, validated and safe treatment for neuromuscular disorders such as involuntary spasms including blepharospasm (BSP) , hemifacFor spasticity, clinical scales such as the Ashworth scale ,14, the ® technology. Others report mechanical properties via mechanography and electrophysiological properties via electromyography (EMG) such as the M response and the H and F waves [Several instrumental ,30,31,32 F waves ,48,49,50One solution could be the single-fiber EMG (SFEMG) which was first described in 1963 by Stålberg and Ekstedt, and is considered to be the most sensitive test for diagnosing NMJ disorders ,52,53 suJitter variations are largely influenced by the release of acetylcholine (ACh) within the motor end plate . Thus, aThe purpose of this work was to present a review of the literature of the value of jitter to characterize electrophysiological changes induced by BoNT-A, related to local and remote effects. We also aimed to confirm whether there was any association between jitter and clinical signs, and the treatment procedure (dose and timing). Finally, we wished to make suggestions for future research in this area.Among the 62 articles selected as being potentially eligible, 38 were excluded after analysis of the full text: six articles were withdrawn because the article was written in a language other than French or English: Japanese ,85, ChinOriginally, only studies with high quality data  and prospective studies with robust methodology) were planned to be retained, but due to the sparsity of relevant studies, the filter was voluntarily broadened to include all clinical trials of inferior methodological quality, as well as those concerning fields of application for BoNT-A other than spasticity . From the review of Leonardi et al. , we idenOnly six studies explored the results of SFEMG in the injected muscle with clinical evolution; in orbicularis oculi (OO) ,101,115,Increased jitter is found both in the muscles in the immediate vicinity and more remote from the point of injection, raising the possibility of propagation, diffusion and migration of BoNT-A. When cervical muscles ,97,98,99p < 0.05), contrary to the BoNT-A group whose pre-treatment normal jitter lengthened and decreased respectively at 2 and 12 weeks post-injection with full recovery  and at distance (EDC and BB muscles) . In addip = 0.05) [When adverse effects were observed after BoNT-A treatment for spasticity , SFEMG showed an increased jitter in distant non-injected muscles (EDC and/or OO) ,106 at a = 0.05) .p < 0.02) improvement of BSP symptoms was seen between the first and the second weeks after BoNT-A injection, with jitter significantly higher than the normal pre-treatment value (p < 0.0005), estimated at three times higher than the upper limit of normal value , with aSo far, no other literature review has included SFEMG as a tool for evaluating outcomes following injection of BoNT-A. However, it is essential to characterize the effects of this treatment as it provokes a transitory interruption in neuromuscular transmission and a precise evaluation of its therapeutic efficacy is necessary to guarantee optimal management. Although SFEMG seems to be a pertinent tool for spasticity evaluation, its application for involuntary spasms follow-up is questionable, as clinical evaluation seems to be usual ,3. DevelA narrative review was performed since investigators had no clear question for a systematic review and considering that the included studies had very moderate statistical power due to the low number of subjects and the limited application of statistical methods. The narrow field under study forced us to include a fairly small number of studies, and most often with observational descriptive design and rarely interventional, randomized or controlled, with a high diversity of protocols, neurological disorders ,111,115,Considering the repetitive nature of BoNT-A treatment in the fields of neuromuscular disorders (spasticity and dystonic disorders) and its residual effect with partly reversible modifications in neuromuscular transmission, it is imperative to develop reliable tools to provide objective follow-up. SFEMG appears to fulfil this demand at least partially. Focusing on spasticity treatment, in the majority of cases BoNT-A is renewed every three months, although complete restauration of the NMJ is uncertain ,152 as rSFEMG is an electromyographic technique, which has revealed the mechanisms of NMJ damage. It appears to be a sensitive tool for analyzing the action of BoNT-A toxin by early increase of jitter in the muscle itself and/or at a distance. This narrative review highlights the potential of this tool for clinicians in pharmacological management with BoNT-A. Future studies will be required to certify the reliability of this tool, to develop a predictive model and establish recommendations for the treatment of each muscle with BoNT-A.A narrative review of studies reporting the use of SFEMG during BoNT-A treatment was based on the Preferred Reporting Item for Systematic reviews and Meta-Analysis (PRISMA) statement .Articles were searched for in Medline/Pubmed and Cochrane Library from the date of database creation until May 2020. The research criteria for single keys were used as follows: “single-fiber” OR “SFEMG” OR “single fiber needle electrode” OR “JITTER” OR “electrophysiology” AND “Botulinum”.First, two reviewers , independently screened the titles and abstracts to identify the most relevant studies that satisfied the inclusion and exclusion criteria (see below). When it was not possible to decide based on the title and abstract, the full text was analyzed. Discrepancies between the reviewers were resolved initially via discussion, and persistent disagreement was resolved by a third reviewer (AD) who made the final decision.Articles were retained if BoNT-A treatment was investigated by SFEMG examination . In a first step, only striated skeletal muscle of the limbs, paretic spastic or healthy (to differentiate changes related to spastic paresis) in humans were targeted. In order to interpret the results, all the jitter values found in the various articles were analyzed according to the references calculated by a collaborative committee by the Ad Hoc Committee of the AAEM Singe Fiber Special Interest Group, with a determination of an upper 95% normal limit for jitter measurements (standard electrode) and mean MCD . As CNE


Severe coronavirus disease 2019 (COVID-19) often results from the immune-mediated cytokine storm, triggered by granulocyte macrophage-colony stimulating factor (GM-CSF), potentially leading to respiratory failure and death. Lenzilumab, a novel anti-human GM-CSF monoclonal antibody, neutralizes GM-CSF and demonstrated potential to improve clinical outcomes in a matched case-cohort study of patients with severe COVID-19 pneumonia. This Phase 3 randomized, double-blind, placebo-controlled trial investigated the efficacy and safety of lenzilumab to improve the likelihood of survival without invasive mechanical ventilation (SWOV), beyond available treatments. Hypoxic patients, hospitalized with COVID-19 (n=520), requiring supplemental oxygen, but not invasive mechanical ventilation, were randomized on Day 0 to receive lenzilumab  or placebo (n=259), and available treatments, including remdesivir and/or corticosteroids; and were followed through Day 28. Baseline demographics were comparable between groups: male, 64.7%; mean age, 60.5 years; median CRP, 79.0 mg/L. Patients across both groups received steroids (93.7%), remdesivir (72.4%), or both (69.1%). Lenzilumab improved the primary endpoint, likelihood of SWOV in the mITT population, by 1.54-fold . Lenzilumab improved SWOV by 1.91-fold  and 1.92-fold  in patients receiving remdesivir or remdesivir and corticosteroids, respectively. A key secondary endpoint of incidence of IMV, ECMO or death was also improved in patients receiving remdesivir (p=0.020) or remdesivir and corticosteroids (p=0.0180). Treatment-emergent serious adverse events were similar across both groups.Lenzilumab significantly improved SWOV in hypoxic COVID-19 patients upon hospitalization, with the greatest benefit observed in patients receiving treatment with remdesivir and corticosteroids. NCT04351152Zelalem Temesgem, MD, Humanigen, Inc (Grant/Research Support) Jason Baker, MD, Humanigen, Inc (Grant/Research Support) Christopher Polk, MD, Atea (Research Grant or Support)Gilead Humanigen (Research Grant or Support)Regeneron (Research Grant or Support) Claudia R. Libertin, MD, Gilead (Grant/Research Support) Colleen F. Kelley, MD, MPH, Gilead Sciences  Involved: Self): Grant/Research Support; Moderna  Involved: Self): Grant/Research Support; Novavax  Involved: Self): Grant/Research Support; Viiv  Involved: Self): Grant/Research Support Vincent Marconi, MD, Bayer Eli Lilly Gilead Sciences ViiV  Victoria Catterson, PhD, Humanigen, Inc (Consultant) William Aronstein, MD, PhD, Humanigen, Inc (Consultant) Cameron Durrant, MD, Humanigen, Inc (Employee) Dale Chappell, MD, Humanigen, Inc (Employee) Omar Ahmed, PharmD, Humanigen, Inc (Employee) Gabrielle Chappell, MSc, Humanigen, Inc (Consultant) Andrew Badley, M.D., AbbVie (Consultant) for the LIVE-AIR Study Group, n/a, Humanigen, Inc (Grant/Research Support)


There was no change in patient weight after supplementation (p = 0.63). There was no difference in the median loop diuretic dose before and after supplementation, with the diuretic dose in furosemide equivalents of 3.2 mL/kg/day (2.3–4.4) vs. 3.2 mL/kg/day (2.2–4.7) (p = 0.50). There was no difference in the proportion of patients receiving thiazide diuretics after supplementation (56% before vs. 50% after (p = 0.10)). NaCl supplementation in children receiving loop diuretics increased calculated fluid balance, but weight was unchanged, and this was not associated with an increase in diuretic needs, suggesting clinicians did not consider the increase in fluid balance to be clinically significant.The use of sodium chloride (NaCl) supplementation in children being prescribed diuretics is controversial due to concerns that supplementation could lead to fluid retention. This is a single-center retrospective study in which fluid balance and diuretic dosing was examined in children prescribed enteral NaCl supplements for hyponatremia while receiving loop diuretics. The aim of this study was to determine whether significant fluid retention occurred with the addition of NaCl. Fifty-five patients with 68 events were studied. The median age was 5.2 months, and 82% were hospitalized for cardiac disease. Daily fluid balance the seven days prior to NaCl supplementation was lower than the seven days after, with measurement of: median 17 mL/kg/day (7–26) vs. 22 mL/kg/day (13–35) ( Fluid overload is common in hospitalized children, occurring in over 30% of patients admitted to the pediatric intensive care unit (PICU) ,2. This Loop diuretics are typically the first line of therapy for fluid overload and work by blocking sodium resorption in the thick ascending limb of the loop of Henle, causing sodium loss in the urine, which is followed by water . ThiazidKnowing how to approach diuretic-induced hyponatremia can be difficult. Sodium chloride (NaCl) supplementation is commonly ordered for children on diuretics  but is cThis study was approved by the Institutional Review Board of the University of Nebraska Medical Center (0216-18-EP). We performed a retrospective chart review of children less than 18 years of age admitted to the Children’s Hospital & Medical Center PICU or med/surg floors from 1 January 2015 to 31 December 2017. Children with all diagnoses were eligible for inclusion if they received scheduled or a continuous infusion of loop diuretics for at least 7 days without NaCl supplementation, then received scheduled enteral NaCl for a minimum of 7 days while inpatient in order to compare fluid outcomes before and after supplementation. Children were excluded if they were receiving renal replacement therapy or were discharged less than 7 days after starting NaCl supplementation. In cases where a child had NaCl supplementation discontinued for at least 14 days then reordered, the second instance was analyzed as a separate supplementation event.Presence of acute kidney injury was determined using the Kidney Disease Improving Global Outcomes criteria . ChildreDaily fluid balance was calculated the 7 days before and the 7 days after NaCl supplementation was started. The day NaCl was first ordered was not included in fluid calculations. Daily fluid balance was calculated from the difference between the total fluid intake and the total fluid output. Change in weight was also recorded the 7 days before and after the start of NaCl supplementation as another measure of fluid balance . Total lFluid intake and output, urine output, and weight were compared for the seven days before and the seven days after NaCl supplementation. Data were collected in a REDCap database . CategorFifty-six patients were included for a total of 68 supplementation events. The median age was 5.2 months (2.2–10.0) and median weight was 5.6 kg (4.4–8.0). Cardiac disease was the primary diagnosis in 82% of patients . Three (p < 0.0001. Sodium level increased after seven days of NaCl supplementation to 135 ± 4 mmol/L (range 123–148), p < 0.0001. The mean chloride level on the day NaCl was ordered was 92 ± 5 mmol/L (range 78–104) and increased after supplementation to 98 ± 6 mmol/L (range 77–116), p < 0.0001. There was no difference in plasma sodium levels on the day NaCl was ordered between patients that received chlorothiazide or hydrochlorothiazide compared to those that did not ,and no difference between those that received metolazone and those that did not  prior to supplementation. The median starting NaCl dose was 2.5 mEq/kg/day (IQR 1.8–3.0). The mean plasma sodium level at hospital admission was 137 ± 4 mmol/L (range 129–151). The mean plasma sodium level when NaCl was ordered was 130 ± 3 mmol/L (range 122–140), which was significantly different than sodium level at admission, The median daily fluid balance and diuretic dose the seven days before and the seven days after starting NaCl are shown in p = 0.10); metolazone use was 37% before and 25% after (p = 0.10). Use of mineralocorticoid-receptor antagonists was similar before and after supplementation .There was no change in loop diuretic dosing after supplementation . Chlorotp = 0.32) and loop diuretic dosing before supplementation . The sodium <130 group had an increase in fluid balance, with no changes in urine output, weight, or loop diuretic dose  or level at the start of NaCl supplementation .There were no adverse neurologic events reported secondary to hyponatremia. The average length of hospital stay was 83 days (IQR 42–139), and there was no correlation between the admission sodium level [r(61) = 0.03] or sodium level at the start of supplementation [r(61) = −0.14] with length of stay. Survival to hospital discharge was 84%. There was no difference between survivors and non-survivors in the admission sodium level  activation, improving diuretic efficacy and improving infant growth. There may also be a benefit to normalizing chloride levels.While we cannot determine the exact etiology of hyponatremia in individual patients, the cause is likely multifactorial and may not simply be a result of diuretic-induced sodium wasting. The majority of children in this study were hospitalized with cardiac disease (82%). The RAAS is activated both after cardiac surgery and with heart failure ,16,17,18Increasing serum sodium levels has the potential to decrease RAAS activation ,23. SodiSufficient sodium is necessary for infant growth, and milk alone may not provide enough in infants who are wasting sodium due to diuretic use. Sodium supplementation has been found to improve weight gain in premature infants without an increase in edema ,29,30. OAnother likely cause for hyponatremia in this cohort was the increased delivery of free water. The median age in this study was 5 months, indicating that a majority of children were given breast milk or formula for enteral nutrition. Over the fifteen-day study period there was a transition from intravenous fluids (IVF) and total parenteral nutrition (TPN) to enteral hydration and nutrition. By the end of the study period all children were receiving most of their fluids from enteral nutrition. For a child receiving the median post-supplementation feeds (106 mL/kg/day), they would obtain approximately 0.7 mEq/kg/day of sodium from their feeds ,32. ThisNaCl supplements do not just provide sodium. Chloride is the second most common electrolyte in the plasma and plays an important role in acid–base balance and sodium homeostasis. It is wasted along with sodium in diuretic use but has been studied far less. A majority of subjects were also hypochloremic. Hypochloremia has been linked to increased cardiovascular mortality in adults ,34 and mThis study was limited by its retrospective and single-center design and the lack of a control group. There is no standard practice in this institution for the treatment of mild to moderate hyponatremia, so there was considerable variation in the timing of supplementation and the dose ordered. Given local practice, we were unable to build a historic control group that had hyponatremia but were not started on NaCl supplementation. Clinicians may also choose to reduce diuretic use in the face of hyponatremia, even with a positive fluid balance. With the limitations in the electronic medical record, it is not always possible to determine whether diuretics are being decreased due to patient improvement or medication side effects. A prospective randomized study of NaCl supplementation to treat hyponatremia associated with diuretic use would be ideal, but given the potential consequences of hyponatremia, equipoise may be lacking.Total sodium provided by IVF/TPN was not collected due to the high number of infusions many of these patients were on during the study period, so the assumption that the sodium intake decreased as feeds were increased could be incorrect. However, given the low sodium content of infant feeds and the move away from hypotonic fluids in pediatrics , it is hNaCl supplementation increased measured fluid balance but did not increase weight in children receiving diuretics. Diuretic dose did not change in response to the increased fluid balance. Further investigations with a control group would aid in determining whether NaCl supplementation alters outcomes.


Edema in the limbs can arise from pathologies such as elevated capillary pressures due to failure of venous valves, elevated capillary permeability from local inflammation, and insufficient fluid clearance by the lymphatic system. The most common treatments include elevation of the limb, compression wraps and manual lymphatic drainage therapy. To better understand these clinical situations, we have developed a comprehensive model of the solid and fluid mechanics of a lower limb that includes the effects of gravity. The local fluid balance in the interstitial space includes a source from the capillaries, a sink due to lymphatic clearance, and movement through the interstitial space due to both gravity and gradients in interstitial fluid pressure (IFP). From dimensional analysis and numerical solutions of the governing equations we have identified several parameter groups that determine the essential length and time scales involved. We find that gravity can have dramatic effects on the fluid balance in the limb with the possibility that a positive feedback loop can develop that facilitates chronic edema. This process involves localized tissue swelling which increases the hydraulic conductivity, thus allowing the movement of interstitial fluid vertically throughout the limb due to gravity and causing further swelling. The presence of a compression wrap can interrupt this feedback loop. We find that only by modeling the complex interplay between the solid and fluid mechanics can we adequately investigate edema development and treatment in a gravity dependent limb. Unfortunately, there are several pathologies that can disrupt the normal fluid balance leading to fluid accumulation—edema—which if left unchecked can lead to recurrent skin and soft tissue infections, chronic wounds and loss of the limb function. Chronic edema or lymphedema is reported to impact about 2 to 4% of the population4 and 24 to 49% of radical mastectomy patients5. Lymphedema accounted for an estimated 165,055 hospital admissions in the United States between 2012 and 2017 6. Despite the apparent, widespread prevalence of this debilitating disorder, firm statistics that could support better public health attention continue to be studied7.As relatively tall mammals, humans have developed adaptations to manage the effects of gravity on the fluid balance in our tissues which, if unopposed, can add about 75 mmHg of pressure for each meter of height. Normally, interstitial fluid pressure (IFP), even in the human lower leg, is held at or just below atmospheric pressure by a tightly regulated balance between losses from the blood vessels and clearance by the lymphatic vessels10—which affects more than 36% in the general population over age 40 with increasing prevalence with age11.The rate at which interstitial fluid arises from the blood vessels can be increased by either increases in the pressure in the capillaries or increases in the permeability of the capillary walls. Normally, the capillary wall forms a relatively tight barrier to fluid loss, and the capillary bed is shielded from high pressure in the arteries by precapillary sphincters and by one-way valves in the veins that prevent the pooling of blood from above. But during angioedema or inflammation the capillary wall becomes more permeable to plasma, leading to an increased source of interstitial fluid even when the capillary pressure is normal. Alternatively, trans-wall flow from the capillaries can be increased by higher capillary pressure when the venous valves fail to prevent back flow—a condition known as venous insufficiency13. In the early stages of lymphedema, fluid accumulates in the subcutaneous tissues. As the lymphedema progresses into later stages, the fluid inundated tissue is replaced with adipose tissue14.Up to their limits, lymphatic vessels can respond to an increased source of interstitial fluid with stronger pumping. However, too little lymphatic clearance—called lymphedema may occur from surgical removal of lymph nodes, parasitic obstruction, congenital defects or other damage to lymphatic vesselsGravity imposes local challenges to maintaining tissue-fluid balance through its contributions to higher capillary pressure and the added difficulty of pumping lymph fluid uphill. In addition, gravity has a more global effect by directly pulling fluid downward through the interstitium. Here, we will consider both local and global effects of gravity on tissue-fluid balance.26. While known to be effective, mechanisms by which compression manages the fluid balance in the leg are only partially understood.When edema does arise, mechanical compression serves as one of the few clinical tools available to manage the swelling in the limbs33, injection sites39, under indentation probes42, near the edge of a compression wraps and in normal tissues47. The local models often include capillary permeability and lymphatic clearance, but over such relatively short distances, gravity has little opportunity to build significant hydrostatic pressures. Large-scale models of the entire limb in compression may include gravity, tissue elasticity, and sometimes even include large-scale percolation, but typically neglect local capillary and lymphatic source/sink effects56.Previous computational analyses have addressed many aspects of fluid management in the leg, but none have been sufficiently comprehensive to allow a full discussion of the competing processes and the use of compression in edema. Typically, the tissue is modeled as a saturated poroelastic material that is divided into solid and fluid subvolumes. At the local scale (about 1 cm) poroelastic methods have been employed in the contexts of solid tumors60. Guyton61 found that the volume of an excised dog leg increased rapidly when the IFP slightly exceeded atmospheric pressure. He also found that the easy dilation of the leg in this range of pressures was accompanied by an increase in tissue permeability of as much as five orders of magnitude due to expansion of available pore spaces in the interstitium57. More generally, the size of tissue pore spaces is determined by a balance between the IFP in the tissue pores which tends to dilate the tissue and compression of the solid subspace by external loading from gravity or wraps.Another key feature of the fluid balance in the leg is that the tissue permeability  depends strongly on the state of strain in the tissueOur present goal is to incorporate all of the aforementioned features, albeit each in simplified form, to better understand the relative contributions of each effect on lower leg edema.To that end, we represented the lower leg as a cylindrical bi-phasic porous medium Fig. . The idepi and by gravity based on Darcy’s law62pi in Eq. . Starling’s law is used to represent the capillary source63z) direction pc0 can include osmotic effects p0 is the hydrostatic pressure, 64 which if reduced has been demonstrated to contribute to edema65. While more sophisticated models of lymphatic pumping are available, this two-parameter  and the interstitium can persist before equilibrium is reestablished. Here we adopt29. The vascular absorption time scale corresponding to Lv isL is the distance over which fluid must transport. These parameters were first applied to solid tumors33 and injection sites39 where L was on the order of 1 cm. Whereas, here the distance that would be needed to force fluid from beneath a compression wrap would be considerably larger, perhaps approaching the height of the wrap on the limb—i.e., tens of centimeters. When we introduce estimated parameter values, we see that movement of fluid solely via percolation along a limb through the interstitium is much slower than vascular interactions unless local absorption by the vasculature is impeded.There are also two relevant characteristic time scalesThe model was implemented in COMSOL®5.5 using the CFD and structural mechanics modules. Poroelasticity is modeled with quasistatic, linear elastic solid mechanics that is linked to fluid modeled by a fully transient Darcy’s law by using the IFP as the pore pressure in the solid. Gravity is applied in the solid and fluid subvolumes. IFP dependent mass sources are added to Darcy’s law to model the capillary and lymphatic interactions. The compression wrap is modeled as an inward acting pressure on the lower portion the leg. Other boundary conditions are as described in Fig. The dimensions and estimated parameter values for a baseline case are available in Table 61 found that for IFP just above atmospheric pressure the volume of the dog hindlimb increased by about 10% for each mmHg increase in IFP under acute conditions  or the lymphatic clearance rate constant Conditions of lymphedema may be modeled by any combination of factors that limits the ability of the lymph vessels to match the supply of interstitial fluid from the capillaries. For example, while Fig. Another pathology of interest is venous insufficiency Fig. a where fg to Eq.  as it di91.The addition of a compression wrap on the lower leg Fig. b can comLg which lowers where on the leg the free fluid percolation through the interstitium begins.In Fig. Having shown that the ratio depends on each of the relevant parameters. Since density, gravitational acceleration, and the viscosity are effectively constant only the tissue permeability, tissue compliance and the capillary and lymphatic exchange rates matter. Figure Next, we consider the time course of the development of edema and its response to compression. First, we determine how much time is needed for dilation of the ankle area to occur when a hydrostatic gradient is initially added to the capillary pressure in the unwrapped leg as would occur in venous insufficiency Fig. a. Effectfrom Eq. ) constanL over which percolation must occur is on the order of 0.25 m. When vascular absorption is active, as it is in Fig. K. Only when local lymphatic clearance is significantly reduced does interstitial percolation occur fast enough to dominate.Removing the effects of gravity, but retaining the wrap, allows a direct comparison between vascular absorption and interstitial percolation rates. Figure In Fig. Management of lower limb edema is largely empirical in the clinic, with insufficient theoretical foundation to guide therapy. Here, we have presented a mathematical model that includes the key mechanical considerations and fluid dynamics that are responsible for maintaining tissue fluid homeostasis under normal physiological conditions—and that are disrupted in lower limb edema. The model shows that IFP is normally kept at a low level by a local balance between the blood and lymphatic vasculatures: fluid lost from the blood is quickly absorbed by nearby lymphatic vessels. Only under extreme conditions of tissue dilation do the tissue permeability and tissue compliance reach levels that allow interstitial fluid to move freely enough for hydrostatic effects to become significant within the interstitium itself. However, even when interstitial fluid movement is relatively local, gravity effects can initiate edema by increasing the pressure—and leakage from—the blood capillaries and/or by reducing lymphatic clearance.92 found that patients with primary lymphedema in one leg had subcutaneous IFP of 14.8 mmHg when supine and 17.9 mmHg when standing, while the IFP in the healthy leg remained just below atmospheric and did not change significantly with position. Similarly, they found that post-thrombotic syndrome (which impairs venous function) led to IFP in the affected leg of 2.8 mmHg when supine and 4.0 mmHg when standing with IFP remaining just below atmospheric in the healthy leg in both positions. Bates et al.73 also found that lymphedema in the arm following breast cancer surgery elevated the IFP by 3.5 mmHg above normal. Chronic kidney disease also produces edema and increased IFP in the leg to 5.5 mmHg above normal93. Our numerical predictions are consistent with these results—introduction of vascular and lymphatic pathologies raises the IFP and the imposition of gravity further exacerbates these effects.Comparisons with experimental reports in the literature support our findings. Christensen et al.One of the most common strategies for controlling edema is so-called RICE . Our results directly address the effects of a compression wrap and the gravitational effects of elevation. The imposed external compression can have multiple effects. Probably the most important effect is to improve the function of the one-way valves in the veins, which has the beneficial effect of decreasing backflow into the capillaries and the consequent increase in fluid pressure and leakage. In the present study, we did not model the mechanics of the veins and their valves explicitly, but rather assigned the capillary pressure according to whether the limb had functional valves (capillary pressure was constant) or whether there was venous insufficiency . Another important effect of compression is that it directly deforms and confines the interstitium. Immediately after application of compression the IFP may undergo a temporary spike before the fluid is either absorbed by the local vasculature or has time to percolate to sites not under the compression Fig. . In the Compression wraps are generally uncomfortable, and patient compliance is a challenge, so optimizing their effectiveness would be beneficial. Our analysis revealed important dynamics occurring at different time scales that can help guide this optimization. Key considerations are the time course of compression treatment and the eventual disposition of the fluid. For example, knowing how long the fluid takes to move in response to the compression wrap  would help determine whether it would be better to apply a stiff wrap that loosens as the leg dimensions diminish (and must be reapplied frequently) or to use a more adaptable/flexible/elastic wrap that holds a more nearly fixed pressure with respect to leg dimension. Optimization of intermittent/peristaltic and graded compression technologies will also benefit from a better understanding of the time course of fluid movement in the limb.94. In addition, locally cooled tissues became softer as measured by indentation even though there was little change in fluid volume95. Such reduced resistance to indentation might be explained by a favorable combination of changes in the interstitial fluid pressure, the interstitial fluid mobility and the solid tissue stiffness. Interstitial fluid pressure can be reduced by lowering the capillary blood pressure via thermoregulatory vasoconstriction or decreasing the permeability of the capillary walls as it might be with reduced inflammation96. Another possible softening mechanism that is little explored is a reduction in fibroblast tensioning within the tissue which may alter the pressure–volume relationship97. Offsetting these favorable effects are the possible deductions in lymphatic pumping98, increases in fluid viscosity and higher stiffness of the solid phase99, all of which tend to increase, rather decrease, the apparent stiffness of the tissue when cooled. Given that lower apparent stiffness is associated with positive clinical outcomes, further attention to the role of managing the interstitial fluid pressure and tissue stiffness with treatment would seem to be warranted. The present poroelastic model promises to be a useful tool for examining the tradeoffs among the various competing effects.Another component of RICE is icing, or cooling. The poroelastic perspective utilized here yields some insight into the effect of tissue cooling, even though temperature was not explicitly modeled. Cooler weather has been associated with less swelling in lymphedema patients108. Specifically, the lower leg of the giraffe has thick arterial walls, tight capillary walls, and relatively stiff skin and connective tissues all of which protect the giraffe from excess pooling of fluid in the leg. IFP in the giraffe leg is normally elevated (44 mmHg100) well above that in healthy humans, but swelling is seldom seen in giraffes due to the stiffer and probably less permeable soft tissues. The swelling in human pathologies comes from the breakdown of one or more of these adaptations—in particular the frequent presence of a thick adipose layer that is both highly compliant and highly permeable. The higher compliance and permeability of subcutaneous adipose tissue might explain the strong correlation between the risk of developing lymphedema after axillary lymph node dissection and body-mass index at the time of surgery109.Because it includes gravitational effects, it is interesting to use our model to analyze how animals of different sizes achieve tissue fluid homeostasis. Gravitational effects should be minimal in mice, where the largest vertical distance is on the order of a few centimeters (this must be carefully considered when extrapolating conclusions regarding edema and tissue drainage obtained using murine models). Giraffes, on the other hand, are tall enough to generate hydrostatic pressures over several meters. Consequently, they have multiple adaptations to manage vascular and interstitial pressures14. The conversion of the fluid phase of lymphedema to the fatty, fibrotic phase of advanced lymphedema is not well understood.It is important to note that chronic edema can lead to tissue stiffening as fibrosis occurs and the tissue interstitial matrix is modified. Likewise, there are age-related changes in the stiffness and thickness of the tissues that could have important implications for tissue mechanics. Stiffer, less permeable tissues would be less susceptible to large-scale pooling of fluid and would probably react more quickly to fluid transport when compression is applied. Unfortunately, last stage disease is often less treatable with compression because the fluid has been largely replaced with fibrotic adipose tissue that contains less fluid110. While beyond the scope of the current study, surface tension effects in an open wound may warrant further study.Late stage disease may also lead to wet, open wounds in which interstitial fluid may come into contact with the air. Under these circumstances, surface tension effects may become significant113.As with any model, ours includes several assumptions and has notable limitations. First, the model does not explicitly include the blood vessels or lymphatic vessels. An interesting extension would consider how compressive stress in the tissue might alter venous valve function or impede blood or lymph flow. We also did not explicitly include the regulation of capillary pressure by precapillary sphincters. Failure of this mechanism could also lead to high capillary pressures and add to the source of interstitial fluid from the capillaries. This would produce increases in capillary pressure similar to those seen in venous insufficiency. In addition, it would likely be more realistic to use highly nonlinear mechanical properties for the tissues. The bulk modulus of the subcutaneous tissue has at least three distinct ranges: a stiff compressive range, a highly compliant range with pressure just above atmospheric, and a stiffening range for large levels of tissue dilation. In the current implementation such dramatic changes in properties led to numerical instabilities. Even with the linearized properties we employed, we were able to examine the behaviors of the system within each stiffness range that could then be compared between ranges. Finally, the assumed model of lymphatic pumping has the benefit of having only two simple parameters but does not capture the full range of behaviors that have been observed in vivo and ex vivoThe accumulation and removal of fluid from tissues is a complex process, and additional investigations can extend from our analysis. For example, the active effects of skeletal muscle contraction, especially during walking should dynamically change tissue mechanics and fluid pressures, and would be an interesting extension of the present study. Also, water in tissues can quickly change between mobile and immobile states as it is associated with or released by tissue hydrogels such as hyaluronic acid. This could also affect edema, and could be considered in future studies.We have shown that a simple set of length and time scales can clarify the relative importance of competing processes in fluid transport in the leg. The combination of high tissue permeability, high tissue compliance and low lymphatic clearance can lead to a positive feedback loop in which fluid can percolate so freely through the interstitial space that further buildup of fluid can occur in the lower leg due to gravitational pooling. Our model also shows that compression therapy can break this cycle by reducing large distance percolation of interstitial fluid.