Understanding the wear of mineral fillers is crucial for controllingindustrial processes, and in the present work, we examine the wearresistance and nanomechanical properties of bare calcite and stearicacid-modified calcite surfaces under dry and humid conditions at thenanoscale. Measurements under different loads allow us to probe thesituation in the absence and presence of abrasive wear. The slidingmotion is in general characterized by irregular stick-slip eventsthat at higher loads lead to abrasion of the brittle calcite surface.Bare calcite is hydrophilic, and under humid conditions, a thin waterlayer is present on the surface. This water layer does not affectthe friction force. However, it slightly decreases the wear depthand strongly influences the distribution of wear particles. In contrast,stearic acid-modified surfaces are hydrophobic. Nevertheless, humidityaffects the wear characteristics by decreasing the binding strengthof stearic acid at higher humidity. A complete monolayer coverageof calcite by stearic acid results in a significant reduction in wearbut only a moderate reduction in friction forces at low humidity andno reduction at 75% relative humidity (RH). Thus, our data suggestthat the wear reduction does not result from a lowering of the frictionforce but rather from an increased ductility of the surface regionas offered by the stearic acid layer. An incomplete monolayer of stearicacid on the calcite surface provides no reduction in wear regardlessof the RH investigated. Clearly, the wear properties of modified calcitesurfaces depend crucially on the packing density of the surface modifierand also on the air humidity. The most common crystalline formof CaCO3 used both in laboratory studies and in industryis calcite. Aragonite is less commonly used, and the third polymorph,vaterite, is unstable at normal temperature and pressure.5 There are two types of CaCO3 granulates widely used inindustry, ground calcium carbonate, GCC,6 and precipitatedcalcium carbonate, PCC, which usually is obtained by carbonation inwater of calcium hydroxide in the form of slaked burned lime.7 The filler market for this fine mineral is not only related to costreduction, but increasingly in functional products, covering a varietyof requirements in applications within packaging, plastics, pharmaceuticals,water treatment, and so on.6 However, depending onthe final goods’ requirements, untreated CaCO3 cannotalways be directly used, such as when mixed with low-surface-energyorganic materials.1 The hydrophilic CaCO3 surface is not compatible with hydrophobic polymers; therefore,surface modification of calcite must be performed.1Calcium is the fifth most common element of the earth’scrust8 The most frequently used coupling agents withcalcium carbonate surface are based on fatty acids,12 but silanes,14 zirconates,14 and titanates15 can also be considered. Stearic acid is one of themost commonly used fatty acid surface modifiers on CaCO3 surfaces,11 acting to decrease the surface energy of uncoatedCaCO3 mineral fillers from about 48–60 mJ/m218 down to 21–32 mJ/m2, dependingon the packing density of stearic acid,20 close to the value of many polymeric matrices.8 Such treatment influences not only the dispersibilitybut also mechanical properties .22 However, industrial surface modificationis not a straightforward operation. To optimize the balance betweendesired parameters and process costs, the amount of fatty acid shouldbe controlled. A too low amount reduces the surface resistance andexposes highly undesirable hygroscopic CaCO3 areas,23 whereas an excessive amount of organic materialcan lead to inferior mechanical properties, processing problems, andhigh costs.25 In addition to the complexityof the treatment, one should also consider degradation of the coatedsurface. Surface-modified calcite as well as final coatings, and primarilypolymer filler applications, are exposed to mechanical stresses andchemical changes leading to local wear, where the environment affectsthe severity of the wear process.26 In this work,we focus on the wear resistance of the modified calcite surface inhumid and dry air. This is of particular relevance to processing withhydrophobic polymers, such as poly(vinyl chloride) (PVC) and polyolefins,where the system is highly sensitive to the quality of surface treatmentand the presence of humidity.To achieve good adhesion and effective mixing between filler andpolymer matrix, their surface energies should be close to each other.28 To this end, we prepared andcharacterized unmodified and stearic acid surface-modified rhombohedralCaCO3 mineral surfaces using stearic acid vapor exposure.Both fully covered surfaces and surfaces coated with a patchy stearicacid layer were considered. The wear resistance and nanomechanicalproperties of calcite were measured and evaluated by the highly surface-sensitiveatomic force microscopy technique. Our study offers a deeper understandingabout local wear properties of unmodified and modified calcite surfaceunder air exposure and in contact with water vapor. To our knowledge,no similar study has been reported before, but we note a recent studyon calcite friction in aqueous solutions,29 where effects of different cations present in solution were highlighted.Surface forces between two rough calcite surfaces in aqueous solutionhave also been investigated by means of a surface force apparatus30 and AFM,31 and wehave previously investigated the effect of humidity on adhesive andlong-range forces between an AFM tip and calcite in air.23The local wear resistance of adsorbed fatty acid layers on calcite,and how it is affected by the environment, has not been clarified.So far, in particular, the initiation of wear on a local scale onmodified calcite surfaces has not been considered in detail, althoughthe atomic force microscopy (AFM)-based method itself for assessinglocal wear has been used for other organic systems and is consideredas well suited.23 following the natural crystalangles of the rhombohedron along the dominant {101̅4} cleavageplane. This highly hygroscopic and wettable surface33 was immediately purged with pressurized nitrogen . At least three samples for each study were analyzed, each beinga few millimeters thick with an approximate surface area of 50–100mm2, and with no evidence of microcracks or excessive surfacesteps. Epoxy glue  was used for sample attachmentto the magnetic disk used in the AFM studies. A sapphire calibrationsample (Bruker) was used for deflection sensitivity calibration ofthe AFM probes, and a titanium roughness sample  forthe determination of the probes’ tip end radii. Stearic acid(C18)  was used for modificationof the calcite surface.The calcite material analyzed was optical-qualityIceland spar cleaved to provide a uniform fresh surface with stainless steel chiseland hammer,3 surface wasmodified by exposure to saturated stearic acid vapor. Such modificationis possible only at a high enough vapor pressure, which strongly dependson temperature.34 For this reason, themodification has to be performed above the melting temperature (69.3°C for stearic acid35). The humidityinside the oven  was estimated to be well below 2.5%relative humidity (RH), and at such low RH, the calcite recrystallizationis retarded.23 The samples were kept inan unsealed 1.5 L glass box for one of the time periods: 10 min or4 h at a temperature of 105 °C. The box contained a glass beakerfilled with 20–25 g of stearic acid that was previously meltedat the same temperature for about 90 min.The CaCOkz, calculatedusing the thermal tune method within the Nanoscope program was determinedto be 18.6 ± 1.7 N/m. The torsional spring constant, kt, was calculated to be (8.5 ± 0.7) ×10–8 N m/rad following the method proposed by Álvarez-Asencioet al.36 The deflection sensitivity inthe normal direction was found to be 25.1 ± 1.1 nm/V with useof the sapphire calibration sample, while by utilizing a polycrystallinetitanium roughness sample, the tip radius of the AFM probe was determinedto be 6.1 ± 0.4 nm at a 0.5 nm indentation depth and 17.2 ±2.3 nm at a 5 nm indentation. The tip radius was found to change slightlyduring contact mode wear measurements with the calcite surface whenapplying high forces using long sliding distances. To minimize thiseffect to below a limit of 20% of the nominal radius at 0.5 nm indentationdepth (and 6% at 5 nm), a new tip was used for each sample.The wearresistance, topography, and nanomechanical properties of freshly cleaved,aged, and coated calcite surfaces were recorded by utilizing a NanoscopeV Controller connected to a Multimode 8 AFM with a standard scanner using hard diamond-like carbon-coated HQ:NSC35/Hard/AlBS probes (Mikromasch) with a nominal tip diameter of <20 nm,a resonance frequency of about 300 kHz, and a spring constant of 16N/m. The normal spring constant, The samples were studied under controlled relative humidity (RH)set at <5 and 75 ± 3%RH, using a gas flow rate of 0.8 ±0.1 L/min. The desired RH was achieved by connecting pressurized nitrogen(overpressure of 13.8 ± 3.5 kPa) to a P-50 membrane flow humidifier(Cellkraft AB). The outlet stream was fitted with a heated tube nearthe AFM probe holder, although without direct contact, allowing thestream to easily leave the holder. Ultrapure Milli-Q water  was used for creating the humid medium. The RH ofthe gas entering the AFM environment was measured with an externalsensor , placed near the calcite samples. The laboratoryroom temperature was 23 ± 0.5 °C, and the room humiditywas 24–30%RH.DSP, can be calculated fromFSP is theforce setpoint [nN]; kz is the normalspring constant [N/m]; and DS is the deflectionsensitivity [nm/V]. To achieve the right force setpoint (expectederror of maximum 0.02 V), it is crucial to set the deflection signalof the photodetector to exactly 0.00 V prior to measurements.The modified calcite surfaces were studied immediately after preparation.Each experiment was carried out in a closed cell under constant nitrogenpurging at controlled humidity following the cycle: dry nitrogen (<5%RH),followed by 30 min exposure to humid nitrogen vapor (∼75%RH),and next, by up to 60 min of dry nitrogen exposure (<5%RH), whereat each step, the same nominal deflection setpoint forces were applied:100, 200, and 300 nN. This allowed us to see the impact of the humiditycondition, the reversibility of the effect, and to consider possibleeffects of changes in the tip radius of the probe. The deflectionsetpoint [V], 2 recorded in PeakForce Quantitative Nano-scale Mechanical (PF QNM)mode. This allows simultaneous capturing of images showing height,tip–sample adhesion, and sample deformation. For wear measurementsin contact mode, the scan size was lowered to 1 × 0.67 μm2 (512 × 340 pixels) to minimize the damage of the tip.The load applied to the calcite surface during wear measurements wasincreased stepwise from 100, via 200, and through to 300 nN. The wornregion is located in the central area of the subsequent topographicaland nanomechanical images. The scanning frequency was kept constantat 1.0 Hz, and during imaging, the scanning angle was at times changedfrom standard 90–0° to check for possible image artifacts.Nanomechanical properties were determined using 512 × 512pixel images over a scanned area of 2 × 2 μmV/2, between the two scanning directions for each scan line (1 μm)was calculated. Here, 50 nm at each end was removed to exclude effectsresulting from the change in the scan direction. Next, the averagevalue of ΔV/2 was obtained from all lines ata given applied force as calculated using Ff, was quantified37heff isthe effective height of the probe (here determined to be 15 μm).The friction data were further analyzed using the NanoScope Analysisprogram and ForceIT software in Matlab. In all cases, no image enhancementwas performed apart from first-order plane fitting and flattening(with marked excluded area of wear) of the height channel. The frictionforce was extracted using the following procedure. First, half ofthe difference in lateral photodetector signal, Δ37The coefficient of friction, μ, was calculated from the slopeof the curve showing friction force as a function of load.38 and clear images of thesehave been reported and discussed in detail in our previous publications.23PeakForce QNM images, illustratingtopography, adhesion, and deformation of freshly cleaved calcite (watercontact angle below 10°), which were recorded before wear measurements,are shown in A freshly cleaved calcite surface was challenged by the AFM tipunder the combined action of load and shear. As the tip traversesthe calcite surface, the cantilever laterally bends due to frictionforces. The resulting lateral force was monitored by measuring thelateral photodetector response, and the data are shown in the toprow of The worn area can clearly be distinguished in the middle of theseimages showing typical surface features on calcite. At a load of 100nN, the wear is very small, but we note that the trenches, seen asdark regions in topography, adhesion, and deformation images outsidethe worn area, have disappeared. As the load is first increased to200 nN and then to 300 nN, clear wear scars due to abrasive wear areobserved, and the worn material has to a large extent accumulatedby deposition at the edges of the worn area. The loose wear materialextends from the surface and displays larger deformation and loweradhesion to the tip compared to unworn calcite. In the lateral forceimages 2a,one cBelow 5%RH, we notice that significant abrasive wear occurs atloads of 200 and 300 nN, and the material released is pushed to theside of the worn area. The worn area appears relatively homogeneouseven though the wear depths at the highest load approaches 4 nm. Thesituation is different at 75%RH. Some worn material is still pushedto the side, but clearly less so than in the dry state, and now manysmall wear particles are found inside the worn area. Further, thewear depth is smaller at 75%RH than in the dry state. When the sampleis dried again to below 5%RH, a similar wear pattern is observed asin the initial dry state. Thus, we reach the conclusion that adsorbedwater changes the wear characteristics of the calcite surface.40 or a continuous layer,42 where results from our previous study were consistent with the ideaof a continuous water layer.23 The waterlayer thickness under humid conditions has been reported to reachabout 1.5 nm.42 Thus, at 75%RH, we expect a water layer thickness of about 1 nm.This amount of water is not very large, but sufficient for allowingcapillary forces to develop between the hydrophilic wear particlesand between the wear particles and the hydrophilic calcite surface,as well as between the tip and the surface. The presence of capillaryforces between wear particles and between wear particles and the calcitesurface is the reason why at 75%RH the wear particles are more difficultto push to the side, and instead have a higher tendency to remainin the worn area. Since the wear particles tend to remain in the wornarea, they effectively reduce the average wear depth at higher humidity.Nonetheless, the worn area containing the wear particles remains locallysignificantly rougher than the virgin calcite surface. Based on therapid reaction of the calcite surface with water at high humidity,it seems plausible that the wear particles to a large extent are builtup from the hydrated form of calcite.One could expect that adsorbed water reduces the friction force,but as we will show below, the friction force at 75%RH is only marginallylower than that obtained at below 5%RH, so there must be another reasonfor the different wear characteristics found at the two humidities.It is known that water adsorbs increasingly to hydrophilic surfacessuch as calcite when the relative humidity is increased. Differentstudies of water adsorption on calcite suggest either a patchy wateradsorption layer23 we demonstratedthat the recrystallized form of calcite can be distinguished frompristine calcite by a lower adhesion force. In the adhesion imagesin In our previous work,–1, whichcorresponds to a sliding distance, d, of 20 nm andan oscillatory frequency of 50 Hz, i.e., the same as the AC frequencyin the electrical power lines. Thus, we assign this peak and its overtonesto electrical noise that carries no information. No other clear peaksare observed, which suggests that unlike for polymer surfaces, calcitedoes not undergo plastic deformations that give rise to characteristicstick-slip lengths.47 However, we note that in the humid state, the FT amplitude increasesmore at low values of 1/d compared to that in thedry state. Thus, in the humid state, there is a greater probabilityfor a larger stick-slip length, which is consistent with the largerstick-slip amplitude.We now take a closer look at the friction traces, and in At higher forces, we observe a few large peaks that appear in thelateral force trace. We suggest that these large stick-slip eventsgive rise to initiation of abrasive wear. They are more pronouncedat 5%RH than at 75%RH, which suggests that the presence of water reducesthe brittleness of the calcite surface and whereby counteracts abrasivewear. This is consistent with the topography images of wear shownin Thus, we conclude from the wear measurements that water adsorptionhas a significant effect on both wear of calcite and stick-slip betweencalcite and the tip. It appears that water makes the surface regionsomewhat softer and less brittle, resulting in larger stick-slip amplitude,stick-slip length, and reduced abrasive wear.As judged from the homogeneous AFMimage and a high water contact angle of ∼110°, calciteis completely covered by a stearic acid monolayer after 4 h exposureto its vapor at 105 °C. The presence of this layer affects thewear properties significantly. The wear scar on stearic acid-modifiedcalcite 4is moreS1 in the Supporting Information.In these images, we note clear differences depending on the humidity,which is somewhat surprising since the wear scar is similar at <5and 75%RH. At a low force, 100 nN, the stick-slip amplitude is clearlylarger in humid air than in dry air. Following the interpretationgiven for bare calcite in The lateral photodetector voltage variations and Fourier transformsof these are shown in At a high force, 300 nN, where some abrasive wear of the stearicacid layer occurs, larger stick-slip amplitudes are observed in dryair than in humid air. This finding can be rationalized as it is thestearic acid layer and not the calcite that wears. The larger stick-slipamplitude in dry state is thus due to the higher binding strengthunder dry conditions that requires a larger force for lateral displacementof the adsorbed molecules. In addition, the limited remaining wearscar suggests that once the tip has locally displaced some adsorbedstearic acid molecules, the layer partly reforms and heals the wearscar. Thus, it can be proposed that the stearic acid layer has a certainself-healing ability both under dry and humid conditions. More importantly,the presence of a stearic acid layer on the calcite surface makesthe calcite surface itself less vulnerable to wear, and in our experiments,we find no evidence for wear of the calcite substrate when coatedby a stearic acid monolayer.Figure S1), we notice two peaks due to electronic noise at 0.05 and 0.1 nm–1, and the stick-slip lengths move toward smaller valuesof 1/d (larger distance) with increasing load. Thisis similar to that observed for bare calcite, but unlike what hasbeen previously observed for polymer layers that deform plastically,47 there is no clear characteristic stick-slip length. Interestingly,two characteristic stick-slip lengths have been reported for adsorbedcatechol-containing polymers, whereas adsorbed polyelectrolytes withoutcatechol groups displayed one single characteristic stick-slip length.28From the Fourier analysis  to stearic acid vaporat 105 °C, where the initial water contact angle was found tobe ∼70°. The stearic acid patches can be distinguishedby lower adhesion and higher deformation than the unmodified calcite,as illustrated in At a low humidity, the wear scar is relatively smooth at forcesof 200 and 300 nN. In contrast, an inhomogeneous wear scar is foundat high forces at 75%RH, which was also observed for unmodified calcite.3 modified with a complete layer of stearic acid, is the largerstick-slip amplitude at a low force (100 nN) under dry conditions.It is under this condition that abrasive wear is very limited as seenin the topography image and, thus, here the stick-slip amplitude isrelated to the inhomogeneous surface structure. We note that the stick-slipamplitude is lower under humid conditions where the binding strengthof stearic acid to calcite is reduced. At higher forces, abrasivewear of the calcite substrate becomes important, and the stick-slippattern becomes similar to that observed for unmodified calcite.The effect of the patchy layer structure on the lateral force variationcan be observed in Ff, depends linearlyon the applied load, Fn, according toAmontons’ ruleIn many, but not all, cases,the friction force, 39 adsorbed waterlayer at 75%RH has no noticeable lubricating effect. However, at higherloads, the friction force is lower at 75%RH than under the dry condition.The reason for this is the more pronounced abrasive wear observedunder dry conditions . The data also demonstrate the importanceof packing density of the stearic acid layer and the air humidity.Abrasive wear of unmodified calcite was found to be negligibleat an applied force of 100 nN under both dry and humid conditions(≈75%RH). The exception is that recrystallized patches on thecalcite surface were removed 2. DuringA full coverage of stearic acid on the calcite surface was foundto significantly reduce the wear, and the wear depth did not exceed1 nm, i.e., less than the thickness of the monolayer, even at 300nN load under dry and humid conditions. Clearly, the stearic acidmodification is robust, and it protects the calcite surface from wearand the limited abrasive wear observed largely occurs within the adsorbedlayer. Nevertheless, the sliding at high forces was characterizedby large stick-slip amplitudes. The fact that the wear depth despitethis remains small suggests that the stearic acid layer has a certainself-healing ability.A patchy coverage of stearic acid did not have the same beneficialeffect on the wear, but in this case, the wear properties were largelysimilar to those of bare calcite. However, the inhomogeneous layerstructure remained after challenged by a load of 100 nN combined withshear under dry conditions, which gave rise to large stick-slip amplitudes.In contrast, the inhomogeneous stearic acid layer was worn away atthe same load at 75%RH. This implies stronger binding of the stearicacid layer under dry conditions.The friction force data show that the presence of a complete layerof stearic acid on the calcite surface slightly reduces the frictionforce at a low humidity, but it has no impact at 75%RH. An intriguingobservation is that a stearic acid-modified surface that was exposedto 75%RH and then redried displayed a significantly lower frictionforce at 100 nN (prior to abrasive wear) than a similar surface notexposed to high humidity. We speculate that this is due to water-inducedreactions where protonated stearic acid molecules are converted tocarboxylate form that subsequently reacts to form strongly bound calciumcarboxylates upon drying.To summarize, a complete stearic acid layer provides improved wearresistance, while a patchy one does not provide any significant benefit.Thus, it is important to achieve full stearic acid coverage not onlyfrom wetting and surface energy considerations but also from wearperspectives.


T cell activation, proliferation, and differentiation into effector and memory states involve massive remodeling of T cell size and molecular content and create a massive increase in demand for energy and amino acids. Protein synthesis is an energy- and resource-demanding process; as such, changes in T cell energy production are intrinsically linked to proteome remodeling. In this review, we discuss how protein synthesis and degradation change over the course of a T cell immune response and the crosstalk between these processes and T cell energy metabolism. We highlight how the use of high-resolution mass spectrometry to analyze T cell proteomes can improve our understanding of how these processes are regulated. Upon pathogen recognition, naive T cells rapidly increase energy production and produce a large number of new messenger RNA (mRNA) transcripts and proteins. Activated T cells also undergo massive growth, doubling to quadrupling in size over a 1–2 day period , followeThe protein production and degradation that drive T cell activation are energy- and resource-intensive processes that are shaped by the T cell environment and the availability of key nutrients –10. In tProtein synthesis involves linking amino acids into a polypeptide chain in the order specified by the nucleotide sequence of a mRNA transcript. A simplified diagram of protein synthesis is provided in Fig. Translation of an mRNA sequence into a protein is frequently described as involving three main steps: translation initiation, elongation and termination Fig.  11–13].–13.11–13The following sections will outline how protein synthesis changes as naive T cells respond to pathogens and differentiate into effector and memory cells. A schematic of the changes in nutrient uptake, protein synthesis, and cell metabolism is provided in Fig. In naive T cells, the rate of protein synthesis is low –18 but nOne estimate by Wolf et al. is that naive T cells translate ~60,000 proteins/min . These fIt has been known since the 1960s that stimulated leukocytes increase in size prior to entering the cell cycle . During Measurable T cell growth corresponds with the increased expression of ribosomal proteins and other translational machinery –7, 23. BSome proteome remodeling during T cell activation requires de novo mRNA production, including the expression of mRNA encoding transcription factors, cytokines, and chemokines, which are critical for immune cell activation and function , 30, 31.The magnitude of the increase in global protein synthesis during effector T cell differentiation is dependent on the system and time point studied, as outlined in Fig. When effector CD4 and CD8 T cells proliferate and differentiate in vitro in nutrient-rich media in the presence of inflammatory cytokines, they can sustain the high levels of expression of ribosomes and translational machinery. As a consequence, in vitro-generated effector T cells remain extremely translationally active with high levels of protein synthesis Fig.  2, 33, , 38. ThiEffector T cells produce cytokines and cytolytic molecules that drive pathogen clearance and express high levels of effector molecule mRNAs, including those encoding granzyme B and IFNγ. Granzyme B is one of the most highly expressed proteins in effector CD8 T cells , 37, 42.T cells undergo major transitions in protein synthesis activity when they are activated, proliferate and differentiate. The immunological drivers of the increased protein synthesis that supports T cell activation, proliferation, and effector function are signals generated by the T cell antigen receptor and inflammatory cytokines , 37, 38.Slc7a5 and Slc1a5 mRNA, and there is no substantial increase in any amino acid transporter protein upon T cell activation in the absence of MYC [T cells can obtain the amino acids needed to fuel protein synthesis by four main mechanisms: uptake from the environment via amino acid transporters , 51, dege of MYC . Accordie of MYC , 62. Stre of MYC . In effee of MYC , 63. More of MYC , 63. In e of MYC , 51, 63.It has been extensively demonstrated that environmental nutrient availability is key for T cell protein synthesis and growth. Depletion of a number of individual amino acids  in T cell culture media has been demonstrated to be sufficient to substantially inhibit T cell growth and protein synthesis during activation and differentiation , 64–68. Amino acids are needed by T cells as they are the biosynthetic building blocks of protein synthesis. However, other pathways of amino acid metabolism are equally important; for example, methionine is important for producing the methyl donor used for DNA, RNA, and protein methylation ; glutamiIt has, however, been shown that protein synthesis is the major user of amino acids in proliferating cell lines . In partIt is thought that low rates of protein synthesis in quiescent cells can be enforced by proteins known as translational repressors that modulate protein synthesis by inhibiting the function of eIF4F translation initiation complexes. The major translational repressor proteins that have been studied in T cells are eIF4EBP family proteins and PDCD4 Fig. . These pPdcd4 deletion caused a selective increase in the cytokines IFNγ, IL4, and IL10 [Another translation repressor that could be important in T cells is PDCD4, which binds eIF4A at a 1:2 ratio and prevents its association with eIF4G in the eIF4F translation initiation complex . Quantitand IL10 . In vivoand IL10 .The importance of matching T cell protein production to the demands of immune effector T cells has prompted a focus on the key signaling molecules that control T cell protein synthesis. In this context, there is a pervasive dogma that the serine/threonine kinase mTORC1, which is activated in T cells in response to antigens, cytokines, and nutrients , is a major regulator of protein synthesis in T cells. This dogma stems from work on mTORC1 as a regulator of protein synthesis in yeast and nonlymphoid cells –87. HoweHow does mTORC1 selectively control T cell protein synthesis? mTORC1 regulates the phosphorylation of numerous eukaryotic translation initiation factors, translational repressors, and ribosomal subunits in activated T cells. For example, mTORC1 controls the phosphorylation of the translational repressors eIF4EBPs and PDCD4 . HoweverMnk1/Mnk2 deletion, which ablated eIF4E Ser209 phosphorylation, failed to impact T cell activation, proliferation, or effector cell differentiation, with only specific effects on cytokine production in an in vivo setting [These studies highlight that it is important to look beyond mTORC1 signaling when trying to understand how antigen receptors and cytokines control protein synthesis during a T cell immune response. One hypothesis is that T cell protein synthesis is also regulated by the serine/threonine kinases MNK1/MNK2, which phosphorylate eIF4E at Ser209 within minutes of T cell activation ; this ph setting . These rHowden et al. analyzed how T cell activation shapes T cell proteomes and estimated that ~7–9% of T cell proteins are downregulated as T cells respond to antigen . ProteinProteins are targeted for proteasomal degradation when they are tagged with a polyubiquitin chain by ubiquitin ligases. Polyubiquitinated proteins are shuttled to the proteasome, where regulatory cap subunits recognize the protein, deubiquitinate it, and unfold it in preparation for proteolytic degradation by the protease activity of the proteasome core subunit proteins . This prListeria-OVA had high proteasomal activity if they had a memory precursor (CD62LhiCD25lo) phenotype and lower proteasomal activity if they had an effector precursor (CD62LloCD25hi) phenotype at 2.5 days post infection [T cells predominantly express a specialized form of the proteasome dubbed the “immunoproteasome”, which differs by several subunits from the proteasome in most nonimmune cell types , 99. T cnfection . How thinfection . The molnfection . It has nfection .Is there immune control of proteasomal protein degradation pathways in T cells? One obvious point of control is the phosphorylation of some proteins in immune-activated T cells  that targets these proteins for ubiquitination and degradation Fig.  6, 63, , 63, 83.The other major mechanism by which proteins are degraded is lysosomal proteolysis, wherein lysosomal fusion with other vesicles allows lysosomal hydrolases to destroy the contents of the vesicle. This process occurs either via endocytosis, whereby the plasma membrane and extracellular proteins are internalized, or via autophagy, whereby cytoplasmic proteins and organelles become encompassed by a double membrane structure called the autophagosome, which fuses with the lysosome –108. ForExaminations of T cell proteomes have shown a net increase in components of the autophagy pathway after 24 h of T cell activation . SeveralFollowing antigen receptor engagement, very fast changes occur in multiple cellular metabolism pathways. Activated T cells increase glycolysis and mitochondrial OXPHOS to produce ATP while decreasing mitochondrial fatty acid oxidation (FAO) , 43, 62.The increase in the T cell glycolytic rate is relatively rapid, detectable within a few minutes of TCR stimulation, and this rate continues to increase over hours of immune activation , 124. NaIncreased mitochondrial OXPHOS has also been measured within hours of T cell activation  and has Protein synthesis is a metabolically demanding process. It has been estimated that ~5 ATP per peptide bond or ~2300 ATP per typical protein synthesized are required . This reAutophagy can be triggered by nutrient deprivation in multiple cell types. Protein degradation via autophagy generates more resources and energy than are utilized in the process . Very liThe precise regulation of protein synthesis and degradation shapes the functional capabilities of T cell immune responses. Technological advances have revealed the constant dynamic turnover of proteins in both naive and effector T cells and the balance between protein synthesis and degradation that is key for controlling protein expression. There have also been major advances in identifying some crucial checkpoints for T cell protein synthesis, including the importance of the regulation of amino acid transporter expression and immune control of ATP production. There is, however, more to learn, particularly about how diet and competition with other cells shape nutrient availability to T cells and how signaling molecules and environmental nutrients selectively impact T cell protein expression. Understanding how T cells prioritize protein production and which proteins they will degrade under conditions of energy and nutrient stress will be of particular value for efforts to refine anticancer T cell therapies and develop T cell tissue-targeted therapies for inflammatory disease progression. However, there are now sensitive and quantitative technologies for measuring protein synthesis and T cell proteomes that will facilitate our understanding of these key issues.


The purpose of this study is to evaluate the influence of anti-vascular endothelial growth factor (VEGF) in the appearance or progression of epiretinal membranes (ERMs) in age-related macular degeneration (ARMD) and investigate confounding factors causing ERMs.Seventy-six eyes that were treated for more than 36 months from the first anti-VEGF injection were assessed. Binary logistic regression analysis was performed between smoking, lens status, subretinal hemorrhage, posterior vitreous detachment (PVD) status, peripheral retinal degeneration, type of AMD, conditions of contralateral eye, and the number of injections as independent variables and appearance or progression of ERMs during 36 months as dependent variables.p = 0.005) with the appearance or progression of ERMs. Moreover, peripheral retinal degeneration was significantly associated . Injection number of anti-VEGF was not significantly associated .The presence of vitreomacular adhesion (VMA) or development of PVD during the observation period was significantly associated (Odds ratio [OR]: 5.77; 95% confidence interval [CI], 1.72–19.4; This study suggests possibilities that anti-VEGF injections alone are unable to cause the development of ERMs, that VMA or developing PVD has a prior impact on the developing ERMs in ARMD similar to that of idiopathic ERMs, and that peripheral retinal degenerations and vitreomacular adhesion were both related to ERMs development and pathogenesis of ARMD. Epiretinal membranes (ERMs) are a common type of fibro-cellular proliferations that form a fibrous sheet along the inner retinal surface. The Beaver Dam Study revealed the prevalence in older adults as 11.8% .Recently, several articles reported the occurrence of ERMs in patients with age-related macular degeneration (ARMD) –5. Eyes A previous study discussed the possible progression of ERM after intravitreal ranibizumab injection in branch retinal vein occlusion (BRVO). Marticorena et al. reported that 4/25 16%) eyes injected with anti-VEGF drugs progressed ERMs 6–7 weeks after injection nevertheless had been diagnosed 5.5–12.5 months earlier % eyes in. A similClinicians would not concentrate on ERM appearance or progression in patients with ARMD because those rarely affect the visual outcome. However, the progression of ERMs proliferation is gradual over several years. The purpose of this study was to investigate the relationship between intravitreal injection and progression of ERMs taking into account the confounding factors.The study design was approved by the Ethics Committee of Sakura Medical Center, Toho University (No. S18032). The Institutional Review Board (IRB) of Sakura Medical Center, Toho University and the study design adhered to the tenets of Declaration of Helsinki. All patients provided written informed consent for treatments; all private patient information was excluded from the database. The use of anonymous information was approved by the IRB without the need to seek further consent. In a retrospective manner, medical records of all consecutive patients who had visited the ARMD clinic of our hospital between April and August 2019 were evaluated. The durations between the time point when they had first undergone the intravitreal anti-VEGF injection and the time point following 36 months were examined as observation periods. These periods included the time period between July 2012 and August 2019. The inclusion criteria were as follows: those who had continuous treatment of more than 36 months from the first intravitreal injection and evaluation of spectral-domain (SD) optical coherence tomography . The exclusion criteria were (1) previous vitrectomy; (2) gas injection; (3) photodynamic therapy; (4) previous laser photocoagulation; (5) presence of retinal break; (6) eyes with other conditions that are known to affect the ERMs, such as retinal vascular disease and uveitis; (7) subjects with only time-domain OCT in the early period. All the patients underwent a complete ophthalmological examination. Classic CNV or occult CNV were diagnosed with well-demarcated choroidal fluorescence in the early phase or irregular elevation of the retinal pigment epithelium without an intensely bright area in the early phase and stippled or granular hyperfluorescence on fluorecenin angiography (FA) . On the We investigated confounding factors causing ERMs (1) smoking , 14; (2)The classification of ERM was according to previous reports. Kleins et al. classified ERMs into two groups, cellophane macular reflex (CMR) that did not distort the macula, and preretinal macular fibrosis (PMF) that contracted with the appearance of superficial retinal folds, graded by fundus photographs . WilkinsMoreover, ERMs were divided as either idiopathic or secondary, based on etiology following cataract surgery, retinal breaks, laser photocoagulation, retinal cryopexy, ocular inflammation, trauma, vascular abnormalities, and vitreous hemorrhage . Cho et Progression of membranes was defined as per previous reports. Based on fundus photographs, BMESII defined the area of ERM increase more than 25% as an increase and the area of ERM decrease more than 25% as a decrease. Moreover, the proportion of CMR to PMF and disappearance of membranes were evaluated . Byon etThe PVD status was examined throughout the 36 months period evaluated by SD-OCT, slit-lamp examination, and ultrasonography. The classification was according to previous reports that investigated the PVD status in patients with ARMD –23. We cThe injection numbers contained both aflibercept  and ranibizumab . Moreover, we further built another binary logistic regression analysis, containing all referred determining factors, dealing with each injection number of each medicine as different independent variables.p-value of < 0.05 was considered significant.Data analysis was carried out using SPSS software version 25. Relationships between the appearance or progression of ERMs and considerable determining factors were analyzed by binary logistic regression analysis, referred previously. A Pearson correlation was used for the correlation between the injection number of aflibercept and ranibizumab. A A total of 222 ARMD patients visited our ARMD clinic during the study period. The inclusion criteria were met by 76 eyes . The characteristics of these patients were shown in Table At baseline, 30 eyes (39%) had no ERM, 33 eyes (43%) had GA, and 13 eyes (17%) had PA. After 3 years, 17 eyes 22%) had no ERM, 35 eyes (46%) had GA, and 24 eyes (32%) had PA. The incidence and appearance or progression during the 3 years were shown in Table 2% had noThe representative cases are shown in Fig. Table r = − 0.49, [95% confidence interval (CI): − 0.64 to − 0.29], p<0.0001).Table Coexistence of ERMs was reported to be 15–38% of ARMD eyes , 24, 25.The reported incidence of VMA in ARMD eyes in literature ranged from 12 to 41% , 25, 27,Cho et al. stated no spontaneous resolution of ERMs in ARMD patients in their study  and therRegarding the reports , 29, 30,p = 0.005]. Moreover, the presence of peripheral retinal degeneration was significantly associated . The other factors examined were not significantly associated.The results of the logistic regression analysis, among considerable determining factors, revealed that presence of VMA or progression of PVD during 36 months against no PVD or complete PVD was significantly associated with ERM appearance or progression [OR, 5.77; 95% CI, 1.72–19.4; As referred previously, incomplete PVD had been considered to progress ERMs with chronic traction . We consWe carefully excluded eyes with a retinal break to prevent contamination of ERMs secondary to the break, and most of the peripheral retinal degeneration prevalence was not due to lattice degeneration (1 eye), but due to cobblestone, naevus, or CHRPE, etc. We could search only one article that reported the prevalence of peripheral retinal degenerations of idiopathic ERMs was similar to that of normal eyes . HoweverWe hypothesized that these peripheral retinal degenerations and vitreoretinal adhesions would co-exist and, vary on the stage or prevalence to some extent, and that both of them are related to the development of ERMs and pathogenesis of ARMD.There would be possibility that the appearance and progression were affected by both similar factor and different factor. Perhaps, appearance would be evoked by any dynamic change inside the eye ball while progression would reflect a secondary or continuous condition. However, we were unable to conduct an analysis distinguishing appearance and progression owing to the small sample size in this study.Regarding the present results, there seemed to be no significant influence of the type of AMD, PCV against other groups, on appearance or progression. However, tAMD was reported to have more subjects with incomplete PVD than PCV . If examExposure to cigarette smoke extracts had been reported to provoke activation of the TGF-β pathway and up-regulate genes related to fibrosis, that was known to play a critical role in the pathogenesis of ERMs , 14. HowMoreover, there was a limited relationship between the history of subretinal hemorrhage and the development of ERMs. We had started this study with the impression that there might be a relationship between developing ERMs in ARMD patients and subretinal hemorrhage treated with aflibercept. However, this hypothesis seemed to be contradicted by the observations. Subretinal hemorrhage inside the retina itself would be unable to cause the development of ERMs, whereas ERMs following vitreous hemorrhage had been distinguished as secondary ERMs .The BMES II study revealed the rate of new appearance of ERMs to be 9.1% in eyes with history of cataract surgery during 5 years from BMES I, whereas it was 4.9% in non-surgical group . In the In the BMESIIstudy, moreover, 13.5% of another eye of the first eye with ERMs at baseline had reported developing new membranes, while 5.3% of another eye of the first eye without ERMs . In our The observation that the number of injections did not have an impact on the appearance or progression of ERMs could suggest the absence of side effect of anti- VEGF injection in the development of ERMs whereas we had no controls of ARMD eyes that had not received any injections. The fact that the proportion of GA to PA was 24% during 36 months and had no priority to 33% during 24 months of idiopathic ERMs  also supWe hypothesize that NO would decrease due to anti-VEGF injection and PDGF-A, TNF-α, TGF-β and some other cytokines would be up-regulated –9, howevThis study had several limitations. First, there was no control of eyes with ARMD and without any injection, previously referred. Second, this was a retrospective study and invitation spans had variety. Moreover, appearance and progression of the ERMs were not differentiated. A further prospective study differentiating these parameters is necessary.The number of anti-VEGF injections in ARMD had little relationship with developing secondary ERMs with neither aflibercept nor ranibizumab and this related possibility that injections alone were unable to develop ERMs. Though their side effects of hypertension or thrombosis following reduction of NO, endophthalmitis, retinal detachment, or iatrogenic cataract are sometimes critical, and clinicians must be aware of these risks. VMA or developing PVD and peripheral retinal degenerations had an association with developing ERMs, and these conditions could be related to the pathogenesis of ARMD.


Asthma is one of the commonest respiratory illnesses among elderly patients undergoing surgery. Detailed preoperative assessment, pharmacotherapy and safe anaesthetic measures throughout perioperative period are the keys to decrease complications. Resistance to expiratory airflow results in positive alveolar pressures at the end of expiration, which causes air-trapping and hyperinflation of the lungs and thorax, increased work of breathing, and alteration of respiratory muscle function. This systematic review was conducted according to the Preferred Reporting Items for systematic review and metanalysis (PRISMA) statement. Search engines like PubMed through HINARI, Cochrane database and Google Scholars were used to find evidences. Low-dose IV ketamine, midazolam, IV lidocaine or combined with salbutamol are recommended to be used as premedication before induction. Propofol, ketamine, halothane, isoflurane and sevoflurane are best induction agents and maintenance for asthmatic surgical patients respectively. Among the muscle relaxants, vecuronium is safe for use in asthmatics. In addition, Succinylcholine and pancronium which releases low levels of histamine has been used safely in asthmatics with little morbidity. •Asthma is one of the commonest respiratory illnesses among patients undergoing surgery.•This systematic review was conducted according to PRISMA protocol.•Propofol, ketamine, halothane, isoflurane and sevoflurane are best pharmacologic agents for asthmatic surgical patients.•Vecuronium is safe for use in asthmatic surgical patients. StudiesRegardless of the techniques of anesthesia, perioperative respiratory adverse events like bronchospasm, laryngospasm, desaturation, coughing and excessive secretion may occur at any stages of the anaesthetic course ,5.Surgical patients with history of uncontrolled asthma three months before surgery had nearly double risk of postoperative mortality and three times risk of developing post-operative pneumonia as compared to surgical patients with controlled asthma .To minimize the risk of perioperative respiratory adverse events in asthmatic surgical patients, adequate preoperative assessment and optimization that includes detailed history, pulmonary function test, and medications are imperative. Adequate depth of Anesthesia, using less histamine releasing agents during intraoperative period and close follow up of post op respirator system is a prerequisites to minimize morbidity and mortality among the surgical patients .In many asthmatic patients, treatment with systemic corticosteroids and bronchodilators is indicated to prevent the inflammation and bronchoconstriction associated with endotracheal intubation .1.1. These changes in the extracellular matrix, smooth muscle, and mucous glands causes a decrease in forced expiratory volume in one second (FEV1) and bronchial hyper-responsiveness [The pathophysiological hallmark of asthma is a reduction in airway diameter due to the contraction of smooth muscle, vascular congestion, edema of the bronchial wall, and tenacious secretions . The airsiveness .Resistance to expiratory airflow results in positive alveolar pressures at the end of expiration, which causes air-trapping and hyperinflation of the lungs and thorax, increased work of breathing, and alteration of respiratory muscle function. In addition, airflow obstruction is not uniform, and the mismatching of ventilation to perfusion occurs, leading to changes in arterial blood gases ,9.1.2Asthma is a common disorder with increasing prevalence rates and severity worldwide. Asthmatic patients often present for surgery and anesthesia and can pose challenges for the anesthetist, especially when General anesthesia with endotracheal intubation is required. A rational choice of anaesthetic agents and airway management based on the available resources in limited resource settings are crucial to minimize perioperative respiratory adverse effects.This evidence based guideline systematic review focused on comprehensive assessment, risk stratification and identification of uncontrolled asthma, optimization during preoperative period with available drugs. This review suggested also the best possible pharmacologic and anaesthetic techniques. In addition, it provides comprehensive intraoperative and post-operative management algorithms.2Asthma OR Bronchial asthma) AND (Surgery OR Elective surgery) AND  AND (Respiratory drugs OR Bronchodilators) AND perioperative respiratory adverse events (PRAEs). In this review, studies on patients with younger than 18 were not included. Publication dates were not used as inclusion or exclusion criteria and only those articles written in English language were considered for this review. Furthermore, after comprehensive and in-depth appraisal of literature, evaluation of quality was conducted according to the According to WHO 2011 level of evidence and degree of recommendation  2020 statement  Fig. 1)Fig. 1). endation .Fig. 1Fl2.1Papers fulfilling the following criteria were included in the study: studies presented as original articles, comparative studies on airway intervention for a patient with asthma undergoing surgery, premedication for a patient with asthma undergoing elective surgery, incidence and management of perioperative respiratory adverse events in patient with asthma undergoing surgery, studies written in English.Studies done on patient with asthma at emergency department, surgery undergoing under local infiltration, child with asthma, studies where full articles were no longer available online were not selected to be included in the current review. All of the research articles that were identified from searches of the electronic databases were imported into the ENDNOTE software version X6  and duplicates were removed. Before findings had begun, full length articles of the selected studies were read to confirm for fulfilling the inclusion criteria.3The chronic inflammatory process leads to tissue injury and subsequent remodeling of the airway structure. Bronchospasm and mucous plugging obstruct both inspiratory and expiratory airflow. Optimization of patients with asthma at preoperative, intraoperative and postoperative period play a pivotal role for a good out come of the victims.3.13.1.1Most well-controlled asthmatics tolerate anesthesia and surgery well. The incidence of perioperative bronchospasm in asthmatic patients undergoing routine surgery is less than 2%, especially if routine medication is continued. However, perioperative respiratory adverse events are increased in patients over 50 years undergoing major surgery and in those patients with unstable disease.1b.Poorly controlled asthmatics patients with current symptoms, history of frequent exacerbations or hospital admissions are at risk of perioperative respiratory problems; bronchospasm, sputum retention, atelectasis, infection and respiratory failure. Hence, to minimize perioperative respiratory adverse events elective surgery should take place when the patient's asthma is optimally controlled 1b.1b. Preoperative assessment should focus and include: Activities of daily living (ADL) and physical status, presence of infectious symptoms, amount and purulence of sputum, presence of allergies, Factors known to trigger attacks or exacerbations, use and effectiveness of medications, previous history of surgery and anesthesia, coexisting medical disorders and obesity or sleep apnea syndrome [The purpose of preoperative evaluation in asthmatic patients is used to determine the respiratory dysfunction and its magnitude, the effectiveness of current therapy and prepare suited anaesthetic plan 1b. Preopsyndrome .2a , which can be assessed by listening over the trachea while the patient exhales forcibly and fully. An FET >6 s correlates with a substantially lowered FEV1/FVC ratio and should initiate further investigation 1b. Preop1.Pulmonary function test/Spirometry: this investigation helps for chronic and uncontrolled asthmatic patients to determine whether airflow obstruction is at least partially reversible after use of bronchodilators in patients of all ages, reversibility is indicated by an increase of at least 12% in FEV1 from baseline. In adults, an increase in FEV1 of greater than 200 mL from baseline also constitutes reversibility [2a and 1b respectively.sibility 2a and 1b2.Electrocardiogram (ECG): asthmatic patients may show right atrial or ventricular hypertrophy, acute strain, right axis deviation, and right bundle branch block, so we should routinely obtain an ECG during and after the acute attack for comparison.3.Chest x-rays: is useful to rule out pneumonia or heart failure, Hyperinflation and increased lung markings, bronchial thickening are common radiographic findings of asthma but has little value in formulating changes in therapy [ therapy 1a.Even though, investigation for asthmatic patients is not routine done in low resource setting areas, the below mention laboratory investigation may help the perioperative team for decision on the severity of asthma and to predict post-operative respiratory adverse events.3.1.21b, is recommended to maintain preoperative disease control in asthmatics scheduled to undergoing surgery under general anesthesia [1b  is the cornerstones to stabilize persistent asthma and decrease morbidity and mortality in asthmatic surgical patients 1b. in AdCombined treatment with corticosteroids and a β2-adrenergic agonist  and salbutamol respectively can improve preoperative lung function and decrease the incidence of wheezing following endotracheal intubation 1a.1b and 1a respectively.Patients who are on systemic corticosteroids for >2 weeks during the prior 6 months should be considered at risk for adrenal suppression needs intra operative supplementation of 1–2 mg/kg of hydrocortisone iv every 8 h and more on the day of surgery followed by return to previous dosage by gradual tapering off 1b and 1aIV TID), Infections should be eradicated and Fluid and electrolyte imbalances should be corrected [1a.According to Enright preoperative management of acute exacerbated asthma should treated with steroids and inhaled beta 2-agonists  in addition, patients with a preoperative FEV1<80% of predicted should receive steroids  with that of (2 mcg/kg IV) fentanyl effectively provide analgesia and prevents fentanyl induced cough 1a  is based on the type of surgery, patient condition and other clinical parameters. However, there is evidence that tracheal intubation causes reversible increases in airway resistance not observed with placement of LMA ,322a. De2a.Inadequate depth of anesthesia at any point can allow bronchospasm to be precipitated. Anaesthetic maintenance with a volatile agent such as isoflurane or sevoflurane confers protective bronchodilation. In selecting a ventilatory mode, attention should be given to providing an adequately long expiratory time to avoid the build-up of intrinsic or auto-PEEP. This can be facilitated by using smaller tidal volumes than usual 2a.3.61b.Intraoperative course is a major determinant for post-operative management, if the surgery was uneventful and pain, nausea, and respiratory status are well-controlled, asthmatics may safely be discharged to the appropriate inpatient unit without further intervention, but in significant intraoperative complications with severe bronchospasm, special care must be taken to ensure patient safety during the postoperative period  1b.1c. Respiratory rehabilitation should be performed by a team of physicians, nurses, physiotherapists together with the patient's family, if necessary [1a.Postoperative control of the amount of sputum, recovery and maintenance of ventilator gas exchange become possible with early respiratory rehabilitation, leading to prevention of complications and early discharge from hospital 1c. Respiecessary 1a.1b. Ensure patient's usual medications are prescribed in an appropriate formulation after surgery [1b. So that, prescribe Salbutamol regular, Review dose and route of administration of steroid daily and Avoid NSAID in poorly controlled asthmatics [1b is paramount during the post-operative period.It is prudent to re administer β-agonists prior to emergence and throughout the postoperative recovery period as needed for recurrent bronchospasm and maintaining a head of the bed up position is preferable for prevention of atelectasis 1b. Ensur surgery 1b. So ththmatics 1b is par4In many asthmatic patients, treatment with systemic corticosteroids and bronchodilators is indicated to prevent the inflammation and bronchoconstriction associated with endotracheal intubation.Low-dose ketamine , Intravenous lidocaine (1–1.5 mg/kg) or combined with salbutamol, Standard dose of Anti muscarinic are safe drugs used as premedication before induction.Propofol, ketamine, halothane, isoflurane, sevoflurane are best induction agents and maintenance for asthmatic surgical patients. In addition, vecuronium is safe for use in asthmatics during induction and maintenance, Succinylcholine and pancronium which releases low levels of histamine, has been used safely in asthmatics with little morbidity, while atracurium and mivacurium should avoid as much as possible.Regional < Facemask < LMA < ETT.Even if some literatures are in controversial most of them agreed on deep level of intubation and extubation for asthmatic patients. According to high level controlled studies the perioperative complication from low to high in asthmatic patients Reversals such as neostigmine (40 μg/kg) with atropine (10 μg/kg) mixture could safely be used for patients with airway hyperactivity. The Post-operative management mainly depends up on the Intraoperative course, if surgery was uneventful, and pain, nausea, and respiratory status are well-controlled, asthmatics may safely be discharged either to home or to an appropriate inpatient unit without further intervention, but in significant intraoperative complications with severe bronchospasm, special care must be taken to ensure patient safety during the postoperative period .Fig. 2Fl


The significant mortality rate and prolonged ventilator days associated with invasive mechanical ventilation (IMV) in patients with severe COVID-19 have incited a debate surrounding the use of noninvasive respiratory support (NIRS)  as a potential treatment strategy. Central to this debate is the role of NIRS in preventing intubation in patients with mild respiratory disease and the potential beneficial effects on both patient outcome and resource utilization. However, there remains valid concern that use of NIRS may prolong time to intubation and lung protective ventilation in patients with more advanced disease, thereby worsening respiratory mechanics via self-inflicted lung injury. In addition, the risk of aerosolization with the use of NIRS has the potential to increase healthcare worker (HCW) exposure to the virus. We review the existing literature with a focus on rationale, patient selection and outcomes associated with the use of NIRS in COVID-19 and prior pandemics, as well as in patients with acute respiratory failure due to different etiologies  to understand the potential role of NIRS in COVID-19 patients. Based on this analysis we suggest an algorithm for NIRS in COVID-19 patients which includes indications and contraindications for use, monitoring recommendations, systems-based practices to reduce HCW exposure, and predictors of NIRS failure. We also discuss future research priorities for addressing unanswered questions regarding NIRS use in COVID-19 with the goal of improving patient outcomes.The online version contains supplementary material available at 10.1186/s40560-021-00593-1. The high mortality rate and prolonged ventilator days associated with invasive mechanical ventilation (IMV) reported in COVID-19 patients have renewed the discussion surrounding the utility of noninvasive respiratory support (NIRS), an umbrella term encompassing high flow nasal cannula (HFNC), continuous positive airway pressure (CPAP), and noninvasive ventilation (NIV). NIV has been shown to be efficacious in critically ill patients with conditions, such as chronic obstructive pulmonary disease (COPD), cardiogenic pulmonary edema, obstructive sleep apnea (OSA), and hypercapneic respiratory failure, whereas its utility is less clear in the management of patients with pneumonia, acute respiratory distress syndrome (ARDS), and particularly, COVID-19. Knowledge gained from the use of NIRS in prior viral pandemics such as SARS, MERS, and H1N1 may provide insight into appropriate use during the COVID-19 pandemic. Here, we outline the three most commonly utilized forms of NIRS, discuss the evolving literature pertaining to NIRS with and without proning, outline the aerosolization risks with different forms of NIRS, and review available evidence for the use of NIRS in patients with COVID-19 as well as in viral pneumonia from prior pandemics, ARDS, and community acquired pneumonia (CAP). Based on this analysis we suggest an algorithm for NIRS in COVID-19 patients which includes indications and contraindications for use, monitoring recommendations, practices to reduce HCW exposure, and predictors of NIRS failure. Finally, we discuss research priorities for addressing unanswered questions regarding the use of NIRS in COVID-19.Additional file COVID-19 has various modes of transmission. While contact and fomite transmission do occur, the primary modes of transmission appear to be droplet and airborne. Airborne transmission is a direct result of inhalation of aerosolized viral particles. The primary differentiating factor between droplets and aerosols is size. According to the World Health Organization, respiratory droplets are greater than 5–10 µm in diameter, whereas aerosols are less than 5 µm in diameter. Droplet transmission occurs within minutes of exposure to an infected individual and is typically the result of droplet formation from talking, singing, coughing, sneezing, or laughing. Due to the size and weight of droplet particles, they do not stay suspended in air for very long, and transmission typically occurs within six feet of the infected individual. In contrast, airborne transmission is a result of aerosolized particles remaining suspended in the air for a prolonged period of time, which makes this mode of transmission particularly concerning in large crowds, indoors, or in areas with poor ventilation. Of particular concern to healthcare workers is the role of aerosol generating procedures in the spread of COVID-19. Aerosol generating procedures such as laryngoscopy and bronchoscopy are thought to produce high concentrations of aerosolized particles which would increase the risk of airborne spread. While evidence remains lacking as to the precise proportion of spread that is due to airborne transmission from aerosolized particles, healthcare workers must remain vigilant when performing aerosol generating procedures to prevent inadvertent spread of COVID-19.Much of our understanding of aerosolization associated with different ventilatory support strategies arose from research catalyzed by the previous pandemics of the twenty-first century. Yu et al. found six risk factors responsible for nosocomial spread of SARS which included minimum distance between beds ≤ 1 m), access to washing/changing facilities for staff, resuscitation administered on the ward, symptomatic staff continuing to work, and whether the patient required supplemental oxygen or NIV  m, acces. A retroOne of the feared complications of NIV in patients with transmissible respiratory infections is air leakage from an imperfect seal resulting in aerosolization of infective pathogens and a resultant superspreading event –7. This When utilizing HFNC, higher flows are likely to increase risk of aerosolization. The dispersion distance associated with HFNC use increased from 6.5 ± 1.5 cm at a flow of 10 L/min to 17.2 ± 3.3 cm at a flow of 60 L/min . When usIn pandemic medicine, the use of a dual limb circuit for CPAP and NIV is far superior to a single limb circuit as the dual limb circuit is a closed system. If a single limb circuit is to be utilized, a viral filter should be placed over the leak port to further reduce the risk of aerosol dispersion. The lowest possible pressures should be used with CPAP and NIV, and the lowest possible flows with HFNC. While limiting the risk of aerosol dispersion is of clinical importance, in a review of HFNC use in COVID-19, Lyons and Callaghan highlight the lack of current evidence regarding any clinically meaningful relationship between increased aerosolization and increased risk of spread to HCW . On the In Additional file ventilation-induced lung injury, to underline the fact that it is not the ventilator itself injuring the lung, but rather the unprotective ventilation. Along the same line, the concept of patient-self-inflicted lung injury (P-SILI) has been developed to describe the potential injurious ventilation in spontaneously breathing patients.It is well known that the clinical presentation of COVID-19 varies drastically, from asymptomatic to severe ARDS, multiorgan system failure and death. While it has been hypothesized that there may be different COVID-19 phenotypes that could explain the variance in clinical presentation, more evidence is needed to support those claims. Nevertheless, the heterogeneity witnessed in disease severity and presentation can make initial treatment challenging. It is known from studies in mechanically ventilated patients with ARDS that ventilation with high tidal volumes and elevated driving pressures may induce ventilator induced lung injury. In spontaneously breathing patients we may expect similar consequences if patients are breathing with large driving pressures and large tidal volumes without being appropriately monitored. Based on this concept, the traditional term ventilator-induced lung injury has been modified by some authors into A recent study showed that the beneficial effect of lowering the tidal volume on mortality in mechanically ventilated patients with ARDS varies according to elastance, suggesting that lung protective ventilation strategies should target driving pressure rather than tidal volumes . WhetherIdeally, to minimize the risk of injurious ventilation during spontaneous breathing, both tidal volume and driving pressure should be monitored. The use of esophageal manometry can help in determining the patient’s work of breathing and estimate the driving pressure, although it requires placement of an esophageal balloon catheter in awake patients with respiratory distress and it is not yet considered a standard of care. While HFNC does not allow monitoring of tidal volume, NIV would provide some tidal volume measurement, although leaks at the NIV interface might affect measurements. Patients who are breathing with tidal volumes larger than 9 mg/kg should be considered high risk for NIRS failure, and IMV should be strongly considered . However2/FiO2 > 200 mmHg and 63% in those with PaO2/FiO2 ≤ 200 mmHg [2/FiO2 significantly improved after 1–2 h of NIV; however, 29% of those patients ultimately required IMV [There remains limited data pertaining to the efficacy and safety of NIRS in the management of COVID-19. A prospective cohort study out of New York found that 22% of patients admitted with COVID-19 were critically ill and 79% of those critically ill patients required IMV . Of note200 mmHg . In addi200 mmHg . Of thosired IMV . This su2/FiO2 increase of approximately 100) and respiratory distress (RR decrease from 28 to 24) [Coppadoro et al. conducted an observational study to evaluate the efficacy of helmet CPAP on COVID-19 patients (study included both full code and DNI patients) who had failed standard oxygen therapy. Helmet CPAP was successful in treating 69% of full code patients and 28% of DNI patients . Helmet 8 to 24) . Ing et 8 to 24) . The HEN8 to 24) . Duan et2/FiO2 ratio after one hour of NIRS was not predictive of NIRS success, whereas independent predictors of NIRS failure included SAPS II greater than 33 and LDH greater than 405 [2/FiO2 ratio in COVID-19 patients on a trial of NIRS is not consistently predictive of NIRS success [More recent literature pertaining to acute respiratory failure secondary to COVID-19 has the added benefit of providing clinicians with evidence-based outcomes that can help guide clinical decision-making. Liu et al. conducted a retrospective, multicenter observational study in an effort to develop a nomogram that would predict likelihood of NIRS failure in COVID-19 patients and found that predictors of NIRS failure included age, number of comorbidities, GCS score, ROX index, and vasopressor use on day one of NIRS . Menga ethan 405 . Hill anthan 405 . This ma success . The uni success .One of the strategies utilized in the fight against COVID-19 that has potential widespread implications for future management is awake prone positioning, both with and without concurrent use of NIRS, as reviewed in Additional file The World Health Organization conducted a systematic review of ventilation strategies for coronavirus  and concluded that NIV may reduce mortality and need for intubation, but that it also has the potential to increase spread to HCW . Societa2 < 92–94% on room air (RA) or declining SpO2, with escalation to NIRS in patients with mild to moderate respiratory distress, increased work of breathing, PaO2/FiO2 > 150 but < 300, or SpO2 < 90–94% on non-rebreather [2/FiO2 < 150, or SpO2/FiO2 < 196 [2 > 75 (in hypercapnic ARF) after two hours of NIV, lack of improvement in blood gas, lower bicarbonate, lower PaCO2 (in hypoxemic ARF), higher lactate, and failure to maintain a PaO2 of 60 mmHg on FiO2 of 0.6 [2/FiO2 < 150–200, a tidal volume of > 9.0–9.5 ml/kg of predicted body weight, and high peak pressure requirement [Based on the literature review provided above, we posit that NIRS, when utilized in the appropriate setting, is an appropriate alternative to early IMV for patients presenting to the hospital with suspected or confirmed COVID-19. However, stringent observation is necessary with NIRS to allow for early detection of clinical deterioration as NIRS failure is associated with increased risk of hospital mortality, ICU stay and hospital stay . A recenbreather . They prO2 < 196 . There iO2 < 196 , 36–42. 2 of 0.6 , 36–42. uirement –41, 43. uirement , 38–41.2 of < 80%, while on supplemental oxygen. Table Table 2, PaCO2, and PaO2/FiO2 ratio. If at any point the patient begins to decompensate, develops a contraindication to NIRS  of patients with severe COVID-19 have incited a debate surrounding the use of noninvasive respiratory support (NIRS)  as a potential treatment strategy. We review the existing literature with a focus on rationale, patient selection and outcomes associated with the use of NIRS in COVID-19 and prior pandemics, as well as in patients with acute respiratory failure due to different etiologies  to understand the potential role of NIRS in COVID-19 patients. Based on this analysis we suggest an algorithm for NIRS in COVID-19 patients which includes indications and contraindications for use, monitoring recommendations, systems-based practices to reduce healthcare worker (HCW) exposure, and predictors of NIRS failure.Additional file 1: Text S1. Noninvasive respiratory support strategies.Additional file 2: Text S2. Historical use and efficacy of noninvasive respiratory support.Additional file 3: Text S3. Prone positioning.


Sophora flavescens, is a traditional Chinese medicine that is a key ingredient in several promising cancer treatments. This activity is attributed in part to two quinolizidine alkaloids (QAs), oxymatrine and matrine, that have a variety of therapeutic activities in vitro. Genetic selection is needed to adapt S. flavescens for cultivation and to improve productivity and product quality. Genetic diversity of S. flavescens was investigated using genotyping-by-sequencing (GBS) on 85 plants grown from seeds collected from 9 provinces of China. DArTSeq provided over 10,000 single nucleotide polymorphism (SNP) markers, 1636 of which were used in phylogenetic analysis to reveal clear regional differences for S. flavescens. One accession from each region was selected for PCR-sequencing to identify gene-specific SNPs in the first two QA pathway genes, lysine decarboxylase (LDC) and copper amine oxidase (CAO). To obtain SfCAO sequence for primer design we used a targeted transcript capture and assembly strategy using publicly available RNA sequencing data. Partial gene sequence analysis of SfCAO revealed two recently duplicated genes, SfCAO1 and SfCAO2, in contrast to the single gene found in the QA-producing legume Lupinus angustifolius. We demonstrate high efficiency converting SNPs to Kompetitive Allele Specific PCR (KASP) markers developing 27 new KASP markers, 17 from DArTSeq data, 7 for SfLDC, and 3 for SfCAO1. To complement this genetic diversity analysis a field trial site has been established in South Australia, providing access to diverse S. flavescens material for morphological, transcriptomic, and QA metabolite analysis. Analysis of dissected flower buds revealed that anthesis occurs before buds fully open suggesting a potential for S. flavescens to be an inbreeding species, however this is not supported by the relatively high level of heterozygosity observed. Two plants from the field trial site were analysed by quantitative real-time PCR and levels of matrine and oxymatrine were assessed in a variety of tissues. We are now in a strong position to select diverse plants for crosses to accelerate the process of genetic selection needed to adapt kushen to cultivation and improve productivity and product quality.Kushen root, from the woody legume  Sophora flavescens Ait.) have long been used in traditional Chinese medicine. The medicinal use of kushen root preparations was first documented in 200 A.D. in the Chinese book Shen Nong Ben Cao Jing1 and continues today for treatment of a variety of conditions, including cancers, hepatitis, and heart disease (Smilax glabra in compound kushen injection (CKI). CKI has been used in Chinese hospitals to treat cancer for over 20 years (Extracts from roots of the woody deciduous legume kushen ( disease . Of part20 years  and has 20 years .Lupinus angustifolius), and they are responsible for the bitter taste of some lupin varieties  and CAO (LaCAO) have been isolated and found to be expressed in leaves and stems , both cadaverine and cadaverine-derived alkaloids were produced  has been isolated and reported to be most abundantly expressed in leaf and stem tissues, with little or no expression detected in inflorescence tissues  or in callus , n = 18) and observed statistically significant variation in biochemical composition  and 11 morphological traits among field-grown plants.Traditionally, kushen roots were harvested from wild populations in China, but with increasing demand for the product, kushen began to be cultivated, especially in the northwestern provinces Shanxi, Hubei, Henan, and Hebei . AgriculS. flavescens obtained from nine provinces of China. Further, to extend understanding of QA synthesis in kushen, a full-length SfCAO coding sequence was isolated from publicly available transcriptome data. A kushen field nursery was established in southern Australia and the expression of SfLDC and SfCAO was investigated in seed-grown plants from that nursery.In the research reported here, genetic diversity of kushen was investigated by applying GBS to 85 accessions of S. flavescens were obtained from a collection maintained at Shanxi Agricultural University. They were collected from non-cultivated  habitats in nine different provinces in China (counting inner Mongolia as a province), representing the natural distribution of the species. Seed were collected from a single region per province, ranging from Meihekou City, Jilin province in the northeast of China to Anshun City, Guizhou province in the southwest of China , in a glasshouse under 12 h daylength. Young leaf tissue was harvested from up to 10 seedlings from each region for DNA analysis. Additional seedlings were transferred to a field trial site at the Waite Campus of the University of Adelaide, with five to seven plants per row. An initial planting was done in October 2017, and a second planting was done in October 2018, to replace plants that had died. Plants were drip irrigated daily in summer. Tissues for RNA extraction were harvested on 26 November 2018, except for seed and seedpod tissues, that were harvested on 12 February 2019.Seeds of of China . In each3 Markers scores were provided as single-nucleotide polymorphisms (SNPs) (pairs of sequence tags differing only by 1 nt) or presence/absence polymorphisms detected in some accessions but not others. Two rounds of DArTseq were performed, a preliminary round on four samples and a full round on 89 samples.Genomic DNA was extracted from freeze dried young leaf tissue using a modified cetyltrimethylammonium bromide (CTAB) protocol , that waSfCAO, mirabait transcript capture (S. flavescens (project PRJDB3906) (SfLDC (AB561138.1) and LaCAO (MF152953) . The datasets were trimmed and error corrected using Quorum, version 1.1.1 , was used as a positive control Sophora for the mirabait short-read capture method. The L. angustifolius bait sequences [full coding sequence plus up to 20 base pairs (bp) of the 5′ and 3′ untranslated regions (UTRs) (if available)] were as follows: LaCAO (MF152953), tubulin (XM_019596948.1), cyclophilin (XM_019595391), elongation faction-α (XM_019597475.1), and ubiquitin-60S ribosomal protein (XM_019577360.1). The first round of mirabait (mirabait1) used low stringency parameters {-k 21 [k = k-mer-length (default = 31)]; and –n 2 [n = minimum number of k-mers needed for a sequence to be selected (default = 1)]}. Sequences were assembled in Geneious and contigs evaluated by BLASTn to identify the best sequences. A second round of mirabait was performed (mirabait2) at high stringency (default parameters) using the best contigs from mirabait1, short reads assembled in Geneious, evaluated by BLASTn and the best contigs used for further analysis.To obtain a full length cDNA sequence for  capture  was usedJDB3906)  as they on 1.1.1  or Trim on 1.1.1 . HousekePhylogenetic analysis was performed using the DArTSeq SNP data generated on the 89 DNA samples using MrBayes v3.2.6  with thePhylogenetic analysis of CAO proteins was performed using a maximum likelihood method with MEGA 10.0.5 . SequencSfLDC and SfCAO PCR products were amplified from genomic DNA using PhusionTM High-Fidelity DNA Polymerase . SfLDC (full length) gene sequences were amplified using SfLDC_F_5pr and SfLDC_R1. Two partial gene fragments were amplified for SfCAO: a 5′ fragment using SfCAO_5p_Fd and SfCAO_e1_R, and a 3′ fragment SfCAO_e10_F and SfCAO_R1. Primer information is listed in To identify SNPs in the two QA pathway genes, 4 PCR products were purified using the NucleoSpin gel kit from Macherey-Nagel , or by gel isolation when two or more PCR products were produced (see section “Results”), as per manufacturer’s instructions. PCR products were sequenced using both forward and reverse primers. Sequencing chromatograms were analysed using Geneious 8.1.9. The following additional primers were used to sequence the genomic fragments: SfLDC_F314, SfLDC_F752, SfLDC_R596, SfLDC_R1026 and in some cases SfCAO_e11_F and SfCAO_e11_R . The KASP markers for GBS-derived SNPs were assayed on the four DNA samples that were used in the preliminary round of GBS analysis. The KASP markers for SNPs in SfLDC and SfCAO1 were assayed on 86 of the DNA samples used in the second round of GBS analysis. KASP genotyping was conducted using an automated SNPLine system .Kompetitive Allele Specific PCR (KASP) uniplex assays (hereafter KASP markers)  were dev® Works (Asia) Sdn Bhd, Selangor, Malaysia]. Total RNA was extracted from 100 mg of frozen ground tissue using the SpectrumTM Plant Total RNA Kit according to manufacturer’s instructions. Total RNA was treated with DNase I , then 750 ng of RNA was used to prepare cDNA using SuperscriptTM III reverse transcriptase (ThermoFisher) using oligodT18 as primer (50°C for 1 h).Tissues for RNA were harvested, immediately frozen in liquid nitrogen and stored at −80°C until required. Tissues  were sampled from two field grown plants, 1_LC1  and 9_AC1  in triplicate, without destroying the plants , 2. SeedSfLDC and SfCAO was carried out with all cDNA samples using quantitative real-time PCR (qPCR) methods as described  was used with primers SfCAO_5p_Fd and SfCAO_R1. PCR products were sequenced (Sanger) with the PCR primers and additional internal primers: SfCAO_e5_F, SfCAO_ex8_F, SfCAO_ex7_R, and SfCAO_ex4_R (Full length AO_ex4_R .S. flavescens tissues and a reversed-phase high performance liquid chromatography (HPLC) quantification method were based on the Hong Kong Chinese Materia Medica Standards (HKCMMS) (g for 5 min), 500 μL of supernatant was transferred to a fresh tube and placed on ice. The pellet was re-extracted (twice) with 600 μL 30% ethanol (v/v) + 20 μL ammonium hydroxide solution  as before, and the supernatants combined. An additional 500 μL of 30% ethanol (v/v) was added to the pooled supernatants . Insoluble material was removed from the soluble extracts by centrifugation  and 100 μL of supernatant was removed and fully dried in a SpeediVac (Savant). The remaining supernatant was removed and stored at −20°C, and subsequently used to prepare concentrated extracts from some samples by drying down 1 mL of soluble extract. Dried samples were resuspended in 30–50 μL 30% ethanol (v/v) and clarified by centrifugation  prior to transferring to HPLC vials for use and/or short-term storage at −20°C. QAs were extracted from additional aliquots of tissues used for qPCR and two control samples .Protocols for extraction of oxymatrine and matrine from (HKCMMS) , with moQuinolizidine alkaloids were analysed using an Agilent 1200 HPLC unit, with a diode array detector (set to record at 220 nm) and a Zorbax Eclipse Plus C18 rapid resolution column . HPLC was performed using a mobile phase composed of A  and B  with a gradient elution from 4% A to 10% B over 10 min, flow rate 0.4 mL/min, column temperature 35°C. The injection volume was 2 μL. Sigma standards were used to establish identity of oxymatrine (O0891) and matrine (M5319) based on their relative retention times and published UV spectra . A standAn initial round of DArTseq using four DNA samples  from Hardy–Weinberg expectations, according to Chi-square tests conducted with one degree of freedom. For 1053 of these markers (98.4%), the observed frequency of heterozygotes was lower than the expected frequency  and consisted of 104 bp of 5′ UTR, 2343 bp of coding sequence, and 169 bp of 3′ UTR (Cicer arietinum (chickpea) shows that the protein encoded by the SfCAO cDNA contains the important catalytic residues and a C-terminal peroxisomal targeting signal as expected (SfCAO is the orthologue of LaCAO involved in QA biosynthesis (To develop gene-specific SNP markers for mirabait  at low savescens . After aRR031281 . It was f 3′ UTR . Alignmeexpected . Phylogeynthesis .SfLDC and different variants (1 and 2) were obtained from the two datasets, DRR031281 and DRR031283, respectively. The variants were greater than 98% identical to the SfLDC coding sequence at NCBI (AB561138.1) and differ from each other only at 4 positions (data not shown).Full length transcripts were also obtained by mirabait for SfLDC, one from each regional clade and one commercial sample  . For plants 4_15 and 9_8, standard cycling at a reduced annealing temperature of 55°C resulted in multiple bands (not shown) whereas touchdown PCR (Ten DNA samples were selected for SNP discovery in l sample . For SfL38928.1) . The revdown PCR  providedSfLDC by Sanger sequencing are detailed in SfLDC genomic DNA sequences of the 10 samples are reported in The SNPs identified in CAO, is expected to have 12 exons (11 introns), based on the 6520 bp lupin CAO gene sequence (Lup000530), with one very long intron   . For thiCAO genes, hereafter called SfCAO1 and SfCAO2, that share the same primer binding sites. Most samples produced a lower band of the expected size ≈ 720 bp  and all samples produced an upper band (≈1200 bp). Sample 4_15 did not produce the 720 bp band and sample 9_8 produced a smaller PCR product, ≈550 bp)  than for SfCAO1 and intron 11 is 399 bp longer (at 546 bp) (A 5′ product of the expected size (247 bp) was obtained and sequenced, revealing two polymorphic positions . Surpris≈550 bp) . Sequenc 546 bp) .SfCAO1 in sample 9_8  and quantification of secondary metabolites (see section “Tissue-Specific Expression of Two QA Pathway Genes and Accumulation of Oxymatrine”). One of these plants, 9_AC1 was grown from seeds collected in region 9, and therefore potentially contained the deletion variant of SfCAO1 missing exon 11 , with the exception of expression of SfCAO in leaves of both 1_LC1 and 9_AC1 , and root  9_AC1 (110 ± 28 units).To determine where in 03 units . SurprisS. flavescens seeds (0.26 ± 0.02%)  and at higher levels in immature seeds (0.32% fresh weight) . Matrine± 0.02%) . No oxymS. flavescens plants from a large part of its observed range within China. A combination of techniques has provided resources for the future, with over 10,000 SNPs identified and 27 of these validated through the development of high throughput KASP markers. These and the other results are discussed below in the context of the existing literature and future plans to achieve more control of secondary metabolite production in S. flavescens through the selection of diverse and genetically defined germplasm.Our research provides much needed information on the molecular diversity of SfCAO, the second gene in the QA pathway, with its cDNA sequence obtained using targeted capture and assembly from publicly available RNASeq data using the lupin LaCAO sequence  that groups with the LaCAO gene QA pathway  , comparison of the metabolites produced by plants from region 9, with and without the deletion  would be warranted. The similar levels of oxymatrine in the roots of 1_LC1 and 9_AC1, demonstrate that the QA pathway is not compromised by the 181 bp deletion in SfCAO1, indicating that SfCAO2 is functional in these plants.Gene duplication is a key process in the evolution and diversification of secondary metabolite production by providing opportunities for either sub-functionalization (greater substrate specificity or more restricted range of substrates) and/or neo-functionalisation (new activities) . Most ofquences)  which enquences) . This dele 9_AC1 . If the SfCAO2 gene from plant 1_LC1 . The coding sequences obtained in this work, part of exons 1 and 10 and all of exons 11 and 12, were identical between the two genes in most varieties, noting that in some samples not all of exon 11 could be sequenced due to frameshifts created by SSRs in the introns (SfCAO1 and SfCAO2 and (2) whether there are sequence differences between the two full length coding sequences that could lead to functional or regulatory differences between the encoded proteins. The second question in particular could have important implications for QA metabolite production.Full genome sequencing of a variety of accessions would have obvious advantages including obtaining full length genomics sequences for  introns , 10. Ful introns  are actuSfLDC and SfCAO genic sequences we were able to identify 14 SNPs in SfLDC and even more (16) in SfCAO1 by sequencing just 4 of the 11 exons. Fewer SNPs (8) were identified in the corresponding region of SfCAO2 despite its longer introns.The utility of the DArTSeq GBS approach was clearly demonstrated with the finding of over SNPs  providing robust data for phylogenetic analysis . By seleS. flavescens based on anecdotal evidence that it was an outbreeding species. However, in attempting to make crosses, we noted that anthesis can occur before flowers open  and they observed about twofold higher SfLDC levels in leaves compared to stems, which was consistent with their RNAseq data. In contrast, we observed higher levels in stems than in leaves. For a plant from region 1 (1_LC1) we observed over 2 × 105 normalised transcript levels for both SfLDC and SfCAO in stem tissue, whereas for the sample from region 9 (9_AC1) there were high levels of SfCAO expression but almost non-detectable levels of SfLDC expression. With the exception of the low SfLDC transcript expression in the stems of 9_AC1 all of the other data is as expected and consistent with the expectation that LDC and CAO are expressed in green tissues, predominantly leaves, and stems with generally lower expression in seedpods, with very low levels in roots, flowers, and seeds  have identified a growing number of candidate genes involved in QA accumulation in as yet unknown ways. The candidate genes include transcription factors, transporters, and a range of potential genes likely involved in metabolite modification  even after multiple freeze thaw cycles (−20°C) and we had good recovery of matrine in a mock extraction control with the QA extraction and storage protocols (data not shown). Our preliminary results showed little difference in oxymatrine concentration between big tuberous roots and small roots . If thisWe are now well positioned to perform more detailed phenotyping of field grown plants in future research. While evaluation of plant growth, metabolite profiling and transcript analysis can be time consuming and expensive, they have the potential to provide vital information to underpin successful production of pharmacologically active compounds, whether from roots or from seeds.S. flavescens, it will be possible to investigate the genetic control of important traits. We are now in a strong position to make informed decisions about the most informative plants to select as parental combinations to maximise diversity. GBS could be applied to mapping populations and SNPs can readily be converted to KASP markers for high-throughput application in selective breeding. It would be highly desirable to have a complete genome sequence, as well as isoform sequencing. All of this is achievable with current technology, indicating that rapid progress is possible despite the constraints of working with a species that takes years to reach harvest potential.With the development of mapping populations for The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/SfCAO cDNA sequences  and, performed and analysed the Sanger sequencing of SfLDC and SfCAO gene fragments. CS performed the purification and quantification of oxymatrine and matrine with significant protocol development assistance from JLa. CS, DM, and KC wrote the manuscript, with input from all authors. All authors have read and approved the final manuscript.KC and JLi planned and executed the collection of seeds in China. KC established and maintained the field trial site. KC and DM developed the strategy for DArTSeq and KASP marker design and supervised its implementation. SNG prepared the DNA samples for DArTSeq, designed and assayed the KASP markers (designed from DArTSeq SNPs), and conducted the SNP based phylogenetic analysis. KC, RB, TB-M, and CS designed the strategy for analysis of QA genes, qPCR and QA metabolite analysis. KL performed the microscopy on the developing flower buds. CS obtained the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Our results support the importance of red blood cell distribution width, electrocardiographic ratios and collagenopathies for assessing pulmonary hypertension prognosis.Pulmonary hypertension is a rare condition that impairs patients’ quality of life and life expectancy. The development of noninvasive instruments may help elucidate the prognosis of this cardiorespiratory disease. We aimed to evaluate the utility of routinely performed noninvasive test results as prognostic markers in patients with pulmonary hypertension. We enrolled 198 patients with mean pulmonary artery pressure >25 mmHg measured at cardiac catheterisation or echocardiographic pulmonary artery systolic pressure > 40 mmHg and tricuspid regurgitation Vmax >2.9 m/s, and clinical information regarding management and follow-up studies from the date of diagnosis. Multivariate analysis revealed that female sex [HR: 0.21, (95% CI: 0.07–0.64);  Pulmonary hypertension (PH) is a disabling and deadly haemodynamic condition characterised by the excessive proliferation of pulmonary artery smooth muscle cells, which results in decreased quality of life and impaired survival. PH is defined as an increase in mean pulmonary arterial pressure (mPAP) > 25 mmHg at rest and a mean pulmonary capillary wedge pressure < 15 mmHg, as assessed by right heart catheterisation (RHC) . PH is gOver time, various functional parameters have been reported as reliable mortality predictors for primary cardiovascular diseases ,5,6. ForThe lack of accessible and less invasive diagnostic tools has hindered the elucidation of clinical risk factors and limited the ability to obtain reliable epidemiological data, particularly in developing countries ,20. Given = 198), and a review of their medical records was performed. For this study, patients with PH were grouped into mild (40–49 mmHg), moderate (50–59 mmHg) and severe (≥60 mmHg). Baseline data were collected from a total of 198 consecutive patients during their clinical assessment. Anthropometric, demographic and clinical information available at the time of diagnosis were collected and analysed to predict severity and to assess mortality throughout the study. PH diagnosis was established at the department according to the current guidelines , a higher P°/T° ratio  and a higher RDW%  were severity-associated factors ; p < 0.001], a lower FEV1  and lower haematocrit (HTO) levels  were mortality-associated factors  and red blood cell distribution width (RDW%) were included in the multivariate analyses as quantitative and qualitative variables for the severity and mortality analyses. Quantitative multivariate analysis of PH-associated factors revealed that female sex .Although there have been studies attempting to explain the role of the electrocardiographic axis, none of them has examined the potential clinical implications of the relationship between the P, T and QRS axes in PH. Individually, the P, T and QRS axes have shown significant differences in PH and have been associated with a worse prognosis. Elevated P or T axis deviations are linked to the presence of increased pulmonary pressures ,42, and p < 0.05), and prolonged QRS duration is an independent predictor of mortality and is associated with a 2.5-fold increased risk of death from idiopathic PH (p = 0.024) [Prolongation of the QRS wave is associated with shorter 6 min walk distances and higher serum uric acid when compared with the same parameters in patients with an expected QRS duration (= 0.024) ,45.A QRS right axis deviation ≥110 degrees has been shown to have the highest positive predictive value for discriminating severe PH, which is similar to the findings of Zhang et al. , who detIn addition, we found common abnormalities in P and T waves that should not be ignored. A P°/T° ratio in the lowest percentile is the strongest independent predictor of severity, overcoming the classic ECG risk indicators, and the QRS°/T° ratio is a robust independent predictor of death in patients with PH. The determination of the electrocardiographic axis ratios is an accessible tool for primary care physicians, and the proper interpretation of electrocardiographic axis ratios may contribute to identifying subjects susceptible to worsening and should be considered part of clinical practice to individualise follow-up strategies for patients with PH. Further studies to validate these findings in other cohorts should be carried out.Our results support the importance of clinical markers (red blood cell distribution width and haematocrit) and routinely performed tests (electrocardiographic ratios) as prognostic assessment tools in patients with pulmonary hypertension.