Saccharum species such as sugarcane and energy cane are key players in the expanding bioeconomy for sugars, bioenergy, and production of high-value proteins. Genomic tools such as culm-regulated promoters would be of great value in terms of improving biomass characteristics through enhanced carbon metabolism for sugar accumulation and/or fiber content for biofuel feedstock. Unlike the situation in dicots, monocot promoters currently used are limited and mostly derived from highly expressed constitutive plant genes and viruses. In this study, a novel promoter region of Sugarcane bacilliform virus , SCBV21 was cloned and mapped by deletion analysis and functionally characterized transiently in monocot and dicot species and stably in sugarcane.SCBV21 [1816 base pair (bp)] identified two putative promoter regions (PPR1 and PPR2) with transcription start sites (TSS1 and TSS2) and two TATA-boxes (TATAAAT and ATATAA), and several vascular-specific and regulatory elements. Deletion analysis revealed that the 710 bp region spanning PPR2 (with TSS2 and ATATAA) at the 3′ end of SCBV21 retained the full promoter activity in both dicots and monocots, as shown by transient expression of the enhanced yellow fluorescent protein (EYFP) gene. In sugarcane young leaf segments, SCBV21 directed a 1.8- and 2.4-fold higher transient EYFP expression than the common maize ubiquitin 1 (Ubi1) and Cauliflower mosaic virus 35S promoters, respectively. In transgenic sugarcane, SCBV21 conferred a preferential expression of the β-glucuronidase (GUS) gene in leaves and culms and specifically in the culm storage parenchyma surrounding the vascular bundle and in vascular phloem cells. Among the transgenic events and tissues characterized in this study, the SCBV21 promoter frequently produced higher GUS activity than the Ubi1 or 35S promoters in a manner that was not obviously correlated with the transgene copy number.In silico analysis of SCBV21 promoter is distinct from the few existing SCBV promoters in its sequence and expression pattern. The potential of SCBV21 as a tissue-regulated promoter with a strong activity in the culm vascular bundle and its storage parenchyma makes it useful in sugarcane engineering for improved carbon metabolism, increased bioenergy production, and enhanced stress tolerance.The newly developed plant viral The online version of this article (doi:10.1186/s13068-017-0850-9) contains supplementary material, which is available to authorized users. Ubiquitin (Ubi) promoters, maize Ubi1  and CaMV 35S promoters  vector  dCTP using the Random Primers DNA Labeling kit . Pre-hybridization, hybridization, washing, and detection of DNA gel blots were conducted as described by Sambrook et al.  may not be required for SCBV21 activity. Similarly, previous studies have reported that RT/RNase H was not influenced by important promoter motifs, such as those identified in the SCBMOV-MOR or SCBIMV-QLD promoters  at its 3′ end retained the full promoter activity, suggesting that the RT/RNase H region and putative PPR1 may not be required for SCBV21 activity. Stable expression analyses demonstrated that SCBV21 conferred a preferential GUS gene expression in the storage parenchyma surrounding the vascular bundle and in vascular phloem and sclerenchyma of the sugarcane culm. It is further shown that SCBV21 exhibited significantly higher levels of GUS gene expression than the common maize Ubi1 and CaMV 35S promoters. The novel SCBV21 promoter expression pattern is distinct from that of the few existing SCBV promoters in its strong activity in the culm vascular bundle and its storage parenchyma, making it valuable for metabolic engineering to improve plant biomass characteristics through enhanced carbon metabolism for sugar accumulation or increased fiber content for biofuel feedstock.In this study, a novel plant viral promoter, Additional file 1: Table S1. List of primers used for cloning the SCBV21 promoter and its deletions.Additional file 2: Figure S1. Multiple alignment of nucleotide sequences of the SCBV21 promoter (KY031904) and the two published SCBIMV-QLD (NC_003031) and SCBMOV-MOR (NC_008017) promoter regions. Two potential promoter regions of SCBV21 are underlined in red, as identified with Neural Network Promoter Prediction . The putative transcription start sites TSS1 and TSS2 within the two regions are marked with an asterisk (*). The two TATA-boxes (TATAAAT and ATATAA) that were predicted by PlantCARE and PLACE databases are indicated in a red box. The partial RT/RNAse H region (782 nucleotides) is indicated in a green box. Nucleotides that are highlighted in black have the highest percentage identity.Additional file 3: Figure S2. Map of promoter:gene constructs used for sugarcane transformation. SCBV21: Sugarcane bacilliform virus promoter; Ubi1: Maize ubiquitin 1 promoter; Pr4: Ubi1 promoter without heat-shock elements (5′-TGGACCCCTCTCGAGAGTTCCGCTC-3′); E35S: Enhanced Cauliflower mosaic virus (CaMV) 35S (2×35S) promoter; EYFP: enhanced yellow fluorescent protein gene; GUS: β-glucuronidase gene; Nos: Agrobacterium tumefaciens nopaline synthase terminator. Ubi1:GUS (pAHC27 vector) [GUS (pBI221 vector)  are used. B, BamHI; Bb, BbsI; E, EcoRI; H, HindIII; N, NcoI; P, PstI; Sa, Sall; Sc, SacI; Sm, SmaI; Sp, SphI; X, XbaI; Xh, XhoI; and Xm, XmaI. Unique enzyme sites are indicated in red. Boxes are not drawn to scale. A triangle represents the deletion of heat-shock elements (25 bp) in the Ubi1 promoter.Additional file 4: Table S2. Identity (%) of nucleotide sequences of partial reverse transcriptase/ribonulcease H (RT/RNAse H) region (872 nt) of Sugarcane bacilliform virus (SCBV) promoter (SCBV21), and 12 SCBV and three Banana streak virus published isolates.Additional file 5: Figure S3. Transient expression of EYFP and GUS genes as directed by the SCBV21 promoter in monocot and dicot tissues. a–f fluorescent images and g–l color images were collected with a stereomicroscope  fitted with YFPHQ filters (excitation of 490–500 nm and emission of 515–560 nm) and a DP71 digital camera (Olympus)  with YFP filter and blank light, respectively at 48 h post-DNA bombardment with SCBV21:EYFP or SCBV21:GUS . a and g sugarcane leaf roll disc, b and h sugarcane culm, c and i sugarcane root, d and j sweet sorghum leaf, e and k tobacco leaf, f and l lima bean cotyledon.


A receptors. We discuss common ribs of inhibitory synapse dysfunction in the umbrella of NDD, highlighting alterations in the developmental switch to inhibitory GABA, dysregulation of neuronal activity patterns by parvalbumin-positive interneurons, and impaired tonic inhibition. Increasing our basic understanding of inhibitory synapses, and their role in NDDs is likely to produce significant therapeutic advances in behavioral symptom alleviation for interrelated NDDs.The term neurodevelopmental disorder (NDD) is an umbrella term used to group together a heterogeneous class of disorders characterized by disruption in cognition, emotion, and behavior, early in the developmental timescale. These disorders are heterogeneous, yet they share common behavioral symptomatology as well as overlapping genetic contributors, including proteins involved in the formation, specialization, and function of synaptic connections. Advances may arise from bridging the current knowledge on synapse related factors indicated from both human studies in NDD populations, and in animal models. Mounting evidence has shown a link to inhibitory synapse formation, specialization, and function among Autism, Angelman, Rett and Dravet syndromes. Inhibitory signaling is diverse, with numerous subtypes of inhibitory interneurons, phasic and tonic modes of inhibition, and the molecular and subcellular diversity of GABA Highlights:• Human studies and animal models need to be bridged in neurodevelopmental disorders• Inhibitory signaling emerges as a common contributor to neurodevelopmental disorders• Inhibitory signaling is diverse in mode, source, and target• Systematic evaluation of inhibitory diversity is lacking in neurodevelopment• Understanding of inhibitory signaling diversity will advance therapeutic strategiesTable 1) and common genetic factors (Table 2). Further, genetic studies are beginning to indicate converging biological and phenotypic traits in complex disorders such as NDDs  are a broad class of disorders involving disruption in one or more domains including cognitive, emotional, motor, and/or behavioral function. NDDs affect up to 36.8% of children in low- and middle-income earning countries as evaluated in 2015 . Althoug as NDDs .Autism spectrum disorder (ASD) is a prototypical NDD, and acts as a reference point for this class of disorders, with others often being described as possessing “autistic-like features.” Features of ASD include any range of impairments in social interaction, language development, and cognition, along with stereotyped behavior, and restricted interests . In addiTable 2). Specifically, RS is linked to the MECP2 gene  and Angelman Syndrome (AS), have a clear genetic cause . One example in the cortex is a type of bipolar cell that is positive for the neuropeptide vasoactive intestinal polypeptide (VIP), which have been shown to target the majority of PV-positive interneurons . A large body of work has documented and characterized 16 subtypes of interneurons in the hippocampus and/or cortex that possess unique morphologies, differential protein expression patterns, and specialized connectivity, physiological function, and functional impact within neuronal networks . GABAARs are heteropentamers gating a chloride channel, composed of combinations of subunits from seven families: α(1-6), β(1-3), γ(1-3), δ, ε, θ, π, and ρ(1-3) . The most abundant subtypes of GABAARs are synaptic, sensitive to benzodiazepines, and composed of α1-3, β1-3, and γ2 subunits  . The gensubunits . In contsubunits .ARs are also the site of action for non-benzodiazepine Z-drugs . GABA released from the presynaptic neuron bind to GABAARs gating a channel permeable to chloride and bicarbonate ions at the postsynaptic membrane. Generally, this leads to a net inward flow of anions, and a hyperpolarizing postsynaptic response known as the inhibitory postsynaptic potential (IPSP). Defining features of this phasic mode of receptor activation are the rapid synchronous opening of a relatively small number of channels that are clustered at the synaptic junction and the short duration of the GABA transient to which the postsynaptic receptors are exposed. Phasic receptors enriched at synapses are most commonly characterized by two α(1-3), two β, and one γ subunit.GABAARs at synaptic sites relies upon the interaction of multiple receptor-associated, adhesion, and scaffolding proteins diagramed in Figure 2, including gephyrin . For example, receptors containing α1 subunits are enriched at inhibitory synapses on the dendrites, those containing α2 subunits are preferentially enriched at inhibitory synapses on the AIS, and both α1 and α2 subunits can be found clustered at somatic inhibitory synapses . GABAARs containing δ subunits tend to be purely extrasynaptic  receptor-mediated current and voltage responses. EGABA is controlled by the equilibrium potential for chloride (ECl) which in turn is regulated by the expression and function of cation-chloride cotransporters . Specific genes implicated within these categories are discussed in detail below: adhesion molecules from the neurexin, neuroligin, and contactin families; synaptic scaffolding proteins from the shank family; and GABAAR subunit genes within the 15q11–13 locus.Multiple genetic contributors to ASD are synaptic proteins, and in particular can be classified as trans-synaptic adhesion molecules, or synaptic scaffolding proteins, or synaptic signaling proteins (Table 2). In broad terms, CAMs are membrane proteins that extend into the extracellular space and facilitate physical interaction with other cells or with the extracellular matrix. NRXNs are enriched in the presynaptic compartment whereas NLGNs are enriched postsynaptically (The neurexin (NRXN/Nrxn), neuroligin (NLGN/Nlgn), and contactin (CNTN) families are cell adhesion molecules  signaling consistent with enrichment at excitatory synapses, along with repetitive behavior and impaired spatial memory  was shown to result in impaired social interactions paralleled by an increase in inhibitory synaptic transmission, leaving excitatory synapses unaffected . HoweverARs , CNTNAP proteins are members of the NRXN family based on structural similarity, and are also associated with ASD . CNTNAP Genetic analysis of CNTNAP2 in ASD has revealed deletions producing a frameshift mutations leading to early introduction of a stop codon . This cade novo and maternally inherited deletions have been detected in ASD populations in a related family member, CNTNAP4  family of proteins. CNTN proteins perform diverse functions in the CNS including myelination, synapse formation, and plasticity. This group of proteins, like the NRXNs and NLGNs, is also adhered to the cell membrane . DuplicaTable 2; Mutations in genes encoding scaffolding proteins, such as the SHANK family of proteins have also shown connection to the development of ASD Table 2; . Scaffolde novo mutations and deletions in SHANK2 . The 15q11-q13 region is subject to imprinting and recombination, and is regarded as one of the most complex regions in the genome , and three GABAe genome . The pare genome . The syne genome . Childree genome , and seve genome . Duplicae genome . 15q11-qARs leads to a prediction of excessive inhibitory neurotransmission due to gene dosage; however, an in vitro model of 15q duplication displayed reduced transcripts for GABRB3 (and other nearby genes) due to impaired homologous pairing  Ube3a gene  as a result of high-affinity binding to the α2-δ subunit of voltage-gated calcium channels (AR β3 subunit (GABRB3 gene product) displays ASD-like features and enhanced susceptibility to seizure , and with a relatively high rate of infantile spasms 42%; . In contchannels . Mouse mchannels . Further seizure .AR in Dup15q, postmortem studies from subjects with ASD with non-specified genetic background have shown reduced levels of [3H]-muscimol binding compared to control  is a NDD characterized by severe mental retardation, ataxic movements, speech impairment, and high incidence of seizures . In contromosome . The canTable 2; , althougAR genes, GABRB3, GABRA5, and GABRG3 genes (Table 2), encoding β3, α5, and γ3 subunits, respectively, are also within the 15q11-13 locus. The presence of this GABAAR subunit gene cluster in 15q11-13 has led to the hypothesis that GABA neurotransmission may also be involved in AS , but not paternal (Ube3am+/p-), deletion mice had increased spontaneous seizure activity and abnormal EEG, along with impairments in motor function, learning and memory, ultrasonic vocalizations, and an anxiety-like phenotype demonstrating the impact of GABAAR function on the severity of phenotype  has also been shown to result in alterations to the intrinsic properties of neurons , is a genetic disorder that leads to severe impairments, affecting nearly every aspect of a child’s life. ASD-like features, especially stereotyped behaviors, loss of language skills, learning and memory deficits, loss of social skills, and seizures are common characteristics of RS . RS may 308) produce a more moderate phenotype that shows the progressive pattern of symptom development  expression is enhanced, paralleled by enhanced responsiveness to THIP, which selectively activates extrasynaptic GABAAR subtypes , protocadherin 19 (PCDH 19), and Syntaxin Binding Protein-1  are characterized to have spontaneous seizures, including thermally evoked seizures  have been reported to result in NDDs with varied behavioral symptoms , which is a neuron specific RhoGEF. All collybistin isoforms possess three major functional domains: an N-terminal SH3 domain, a GEF domain, and a PH domain. Through these domains, collybistin has been shown to interact with gephyrin, neuroligin-2, and the GABAAR α2 subunit, playing an important role in clustering of GABAARs at inhibitory synapses , including ASD, seizures, and schizophrenia . A number of studies have indicated perturbations in PV-positive cells in NDD populations and mouse models , early in development and in NDDs. Studies have identified alterations in the expression of tonic GABAAR subunits in human populations  mutation in ASD, with the C-terminus being impacted . Recent e models . While se models . These se models . A thirdulations , as wellulations . Gaboxadx and AS .AR subunit alterations would provide insights about which modes of inhibition, and which subtypes of inhibitory synapses may be contributing to NDDs. Investigations comparing both phasic and tonic forms of inhibition across animal models of NDD would be illuminating, as tonic inhibition plays an important and powerful role in controlling brain activity but is understudied. In addition, subtypes of synaptic GABAARs, including somatic and AIS synapses, are known to have a powerful role in coordinating neuronal activity patterns, but we are just beginning to understand the roles of these synapses, how they are specified, how they develop, and their possible contributions to NDDs. In addition, there are many key adhesion, and scaffolding proteins required for the aforementioned processes at specific inhibitory synapse subtypes, including neuroligins, dystroglycan, dystrophin, gephyrin, and collybistin, among others. Increasing our understanding of these key proteins involved in the formation, maintenance, and function of specific inhibitory synapses will also advance our understanding of how subtypes impact neurodevelopment more globally.Despite these emerging ribs, our understanding of the details of inhibitory signaling lags behind that which is known about excitatory signaling. In particular, further investigation of regional or subtype specific deficits in GABAergic signaling is warranted in in NDD populations and animal models. More specific information about GABAFurther, assessments of specific interneuron function also seem promising, with evidence pointing toward PV-positive interneuron dysfunction as a key factor mentioned above. In particular, it may be important to examine the mechanisms causing changes in PV expression, as well as understand the functional implications of such changes to neuronal circuit activity. In addition, analysis of markers for other subtypes of interneurons in NDD and mouse models have not been described. In particular, analysis of interneuron targeting interneurons that have disinhibiting effects on network signaling is lacking in NDD populations and models. This is of particular relevance as a recent study has shown that Nav1.2 (SCN2A), which bears an ASD related mutation , is enriRAR contributed to the writing. CJ contributed to the writing and tables. RH conceived of the topic, wrote the manuscript, and prepared the figures.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.


Aircraft-produced contrail cirrus clouds contribute to anthropogenic climate change. Observational data sets and modelling approaches have become available that clarify formation pathways close to the source aircraft and lead to estimates of the global distribution of their microphysical and optical properties. While contrail cirrus enhance the impact of natural clouds on climate, uncertainties remain regarding their properties and lifecycle. Progress in representing aircraft emissions, contrail cirrus and natural cirrus in global climate models together with tighter constraints on the sensitivity of the climate system will help judge efficiencies of and trade-offs between mitigation options. Condensation trails produced by jet aircraft contribute to anthropogenic climate change, yet the magnitude of their impact remains unclear. Here, the author reviews progress in understanding contrails since the last IPCC report (1999), assesses the remaining uncertainty, and identifies potential mitigating options and pathways forward. Depending on whether or not they retain their linear shape, they have been referred to as persistent contrails and contrail cirrus, respectively, or together as aircraft-induced clouds (AIC). A change in global cloudiness due to AIC creates an imbalance between incident radiation from the Sun and upwelling radiation from the Earth’s surface and atmosphere, resulting in a radiative forcing (RF) of climate that induces a tendency to change the temperature structure in the lower atmosphere.Condensation trails (contrails) are line-shaped ice clouds generated by jet aircraft cruising in the upper troposphere at 8–13 km altitude. Depending on surrounding atmospheric conditions, contrails can be short- or long-lived. Long-lived contrails are those that remain for at least 10 min—defined by the World Meteorological Organization as Cirrus homogenitus3. Prior to 1990, quantitative interpretation was hampered by a lack of knowledge of the properties of contrail cirrus, processes involved in their formation and tools and technologies to observe and represent them in models. A special report by the Intergovernmental Panel on Climate Change (IPCC) summarised and assessed in 1999 the scientific progress made regarding the climate impact of aviation4. While studies had begun to explore extensively the physical characteristics of persistent contrails, observational data on contrail cirrus were, and still are, very scarce, since it is difficult to distinguish them visually from naturally occurring high ice clouds (cirrus) owing to their irregular shape5, especially if their history is not known. Therefore, the IPCC special report did not provide a best central estimate of contrail cirrus RF. The resulting scientific uncertainty limited projections of the climate impact of aviation as a whole and prevented the formulation of options for mitigation. Since that report, climate model simulations have shown that RF due to contrail cirrus greatly exceeds that from persistent contrails6, making contrail cirrus the most important contribution to AIC and to RF associated with aviation. However, knowledge gaps still exist regarding the transition of contrails into contrail cirrus, optical properties determining their radiative response and the sensitivity of the climate system.Widespread recognition of a possible large-scale climatic influence of contrails dates back to several decades2), and aerosol particles. In addition to CO2 emissions, civil aviation affects the climate through a number of non-CO2 climate forcing agents that are unique to this sector of transportation. Taken together, in the year 2011, these agents caused 4% of the total global RF from all human activities  gases, most prominently carbon dioxide —has led to concerns about the growing impact of aviation on climate. Depending on the economic projections and aircraft emission scenarios, aviation fuel usage and therefore CO2 emissions may double by 2050 (ref. 11). Some scenarios predict 3–4-fold increases12. When scaled to fuel usage, AIC RF relative to that of aviation CO2 emissions stays constant. However, such a scaling disregards non-linearity in atmospheric response to cloud changes. Exactly how this contribution will evolve also depends on changes in regional air traffic patterns and climate13. To eliminate AIC from the aviation climate equation, the research community needs to address knowledge gaps by advancing the scientific understanding of contrail cirrus and providing an assessment of options to mitigate the climate impact of AIC14. If the objective is to minimise the aviation contribution to climate change on long time scales (relating to centuries), then reductions in CO2 emissions are much more important than mitigating non-CO2 effects. However, persistent contrails and contrail cirrus have much shorter life times than CO2 emissions (hours versus centuries) and are therefore amenable to rapid mitigation.Despite significant improvements in aviation technology and operation efficiencyThis review brings together and draws upon knowledge on understanding their lifecycle and effects on Earth’s radiation budget since IPCC 1999. Determining conditions with predominant meteorological or microphysical control of contrail cirrus, connecting their radiative effects with global temperature change and exploring mitigation solutions will help develop appropriate technological and operational responses to reduce the climate impact of aviation as a whole and inform decision-making.15. Ice supersaturation denotes sufficiently cold and moist atmospheric conditions—relative to saturation with respect to the ice phase—that influence radiative properties, extent and lifetimes of all cirrus cloud types and is required for contrails to be long-lived and evolve into contrail cirrus . Through a combination of models, laboratory and in situ measurements, the last two decades have seen significant advances in identifying and understanding thermo-, fluid-dynamical and microphysical processes in the formation stage. Understanding these processes is essential to model and predict initial AIC properties.The formation stage unfolds behind engines of cruising aircraft and lasts for about 10 min  provide a threshold below which either short-lived or long-lived contrails appear behind jet aircraft cruising above ≈8 km (formation constraint). Contrail occurrence is predicted with confidence, if ambient pressure and relative humidity, water vapour and heat emissions, and propulsive characteristics of aircraft engines are known. The underlying definition of threshold temperatures rests on the assumption that exhaust plumes surpass water saturated conditions (water saturation constraint). The thermodynamic approach is not capable of predicting formation (nucleation) pathways and microphysical properties of ice crystals in contrails.Contrails begin to form when jet engine exhaust plumes expand and their constituents mix with surrounding ambient air Fig. . In line21. Laboratory studies24 demonstrate that only after water droplets have formed does substantial ice nucleation occur, with rates of homogeneous (pure liquid) droplet freezing increasing dramatically with decreasing temperature (phase constraint). The presence of ice crystals in contrails is supported by ample observational evidence25. An analysis of aircraft observations shows that many  small (<1 µm) ice crystals need to form within a wingspan behind cruising aircraft to make contrails visible already at formation26 (visibility constraint). This is possible since plume cooling rates driven by turbulent mixing27 are high and plume particles acting as water condensation nuclei are abundant28.Aerosol particles types present in aircraft exhaust plumes comprise emitted soot particles, nanometre-sized (ultrafine) aqueous aerosol particles formed within the plumes and particles mixed (entrained) into the plumes from the ambient air Box . In the 33 and remote sensing34 and aircraft36 measurements.Figure 128 and an electrically neutral mode too small to contribute significantly to ice nucleation. Fuel combustion produces condensable vapours including water vapour, sulphuric acid, nitric acid, and low-volatile hydrocarbons. Sulphuric acid is produced by oxidation of emitted sulphur oxides and is highly water-soluble. Nitric acid is produced by oxidation of emitted nitrogen oxides and is only taken up by UAPs that are sufficiently diluted (water rich)129. The chemical nature of organic compounds from emissions of unburned hydrocarbons in aircraft exhaust is poorly characterised. The number of UAPs in the chemi-ion mode, exceeding 1017 per kg of fuel burnt41, is insensitive to variations of, and UAP sizes (1–10 nm) increase with, the sulphur content in the fuel131.As mixing and associated cooling of jet plumes with surrounding air progresses, ambient aerosol particles are gradually mixed into them and exposed to moister and warmer plume air. Ultrafine aqueous particles (UAPs) are generated from gaseous emissions before ice crystals form. UAPs partition into a larger mode that formed on ionised molecules (chemi-ions)14–1015 per kg-fuel93. The fractal morphology of soot particles affects the relationship between total soot particle mass and number emissions. Their number and chemical state as well as the size gap between soot and UAP modes vary depending on engine type, engine power setting and type of fuel132.Incomplete combustion of carbonaceous phases present in aviation fuel produces aromatic compounds that merge to form elementary spherules. These solid black carbon (soot) aerosol particles grow further into fractal clusters mostly in the size range 10–100 nm and with numbers 10−1) generate high supersaturation capable of activating the rather insoluble soot particles28. Modern jet engines extract more heat for propulsion133, meaning that plumes stay cooler and therefore develop higher supersaturation for similar water vapour emissions. Activation of plume particles ceases when the newly formed droplets start to quench the supersaturation due to condensational growth. This happens along with homogeneous water droplet freezing at plume temperatures 229–232 K (ref. 28).Aerosol particles attract water molecules depending on their solubility and on relative humidity. Hygroscopic properties of liquid and mixed (liquid–solid) particles are determined by the chemical nature of the solutes. Insufficient knowledge of the size distribution and the surface-to-vapour interfacial energy (surface tension) renders model predictions of UAP activation into nearly pure water droplets uncertain. Hygroscopic properties of soot particles are mainly determined by chemical oxidants, condensation and particle scavenging processes in the jet exhaust plume producing thin aqueous coatings affecting their droplet formation (water activation) behaviour. In liquid water-supersaturated conditions, the largest and most soluble aerosol particles preferably activate into water droplets. Below contrail formation threshold temperatures, vigorous plume cooling rates (>10 K s12–1013 (kg-fuel)−1) ambient particles. UAPs contribute to the pool of water droplets only if plume supersaturation is high enough to overcome the size (Kelvin) barrier for activation, i.e., if soot particles are removed from the exhaust to an extent that they no longer act as the primary condensational sink for water vapour and if temperatures fall significantly below formation threshold temperatures37. Desulphurising jet fuel decreases the size of UAPs and turns them into less potent droplet forming agents.The first particles to freeze are the water droplets originating from the largest soot particles and the more water soluble but much less abundant , consistent with small amounts (1% by volume) of soluble coatings as found in other measurements24. Together, these findings support the notion of homogeneous droplet freezing as the predominant ice nucleation mode in fresh contrails.Laboratory studies suggest that exhaust water vapour by itself will neither form water droplets nor ice crystals4), refined models have become available only in recent years39.Robust predictions of changes in climate that result from AIC rely to a large degree on the ability to transfer the process understanding outlined above into models that are not capable of treating the formation stage explicitly due to a lack of spatial or temporal resolution. This goal can be reached by allying findings from observations with local scale, process-oriented models. While first attempts have been made to devise simulations addressing formation stage processes prior to IPCC 1999  below which molecular diffusion removes spatial gradients of water vapour and temperature. Water droplet formation followed by homogeneous freezing defines a minimal microphysical framework. By invoking a heterogeneous (solid surface assisted) ice nucleation mode involving bare exhaust soot particles, it is possible to tune these nucleation rates to trigger rapid formation of copious ice crystals preventing water supersaturation from occurring. However, this violates the water saturation constraint.Together with observations, process models point to exhaust soot particles as the most important source of contrail ice crystals for current propulsion technology37  codes, they have been employed to study effects of aircraft wake development on ice crystals. In this regard, LES have provided valuable insights into systematic dependencies of ice crystal losses on aircraft-dependent and atmospheric variables.43, in which initial conditions of ice supersaturation and nucleated ice crystal properties in jet exhaust plumes have been prescribed. The more ice crystals nucleate, the larger the sublimation losses which decrease with increasing supersaturation. Observations confirm a reduction in ice crystal numbers in descending aircraft wakes42. First steps towards a unified description of these trends across different LES models have been taken43, but sublimation loss factors and ice crystal number concentrations remain uncertain until measurements become available providing tight links between soot emissions and ice crystal numbers across the entire formation stage.Figure Advances in research in the last two decades do not abrogate the need to measure more accurately physical and chemical properties of aerosol particles and ice crystals in the size range 1–1000 nm and to re-examine model predictions of ice crystal size distributions in and vertical extensions of contrails with better informed models at the end of the formation stage. These variables serve as initial conditions of AIC in the spreading stage and have important repercussions regarding the transition of contrails into contrail cirrus. It is important to ascertain model predictions by which sublimation decreases differences in nucleated ice crystal numbers induced by changes in soot emissions.40; little progress has been made since IPCC 1999 (ref. 4) in quantifying lifecycles of contrail cirrus and in identifying and understanding factors controlling them.Microphysical and optical properties of ice crystals in contrails change as they spread or transition into contrail cirrus, depending on the meteorological situation and microphysical processes Fig. . Over ti19, the fraction of ice supersaturated areas in which aircraft actually fly is relatively small (10–15%) (ref. 44). Projected changes of extent and frequency of occurrence of ice supersaturated regions in the upper troposphere have implications for contrail formation45. Persistent contrails may merge with or form in natural cirrus49. AIC can be transported considerable distances (many 100 km) away from their source regions, therefore contrails and contrail cirrus may be found in areas where formation conditions are not met50. Over congested airspace, they manifest as ice cloud layers (‘contrail outbreaks’), which can extend over as much as 100,000 km2 and are readily apparent in satellite imagery16.While formation conditions including those for short-lived contrails are frequently met at cruise altitudesOptical properties of ice crystals influence AIC RF. Cloud optical depth (OD) measures the attenuation of radiation passing through cloud Box  and is o51. Changes in the diurnal cycle of coverage and outgoing radiation in the North Atlantic region were estimated in correlation with air traffic52. Another study determined cloud properties formed during several contrail outbreaks that occurred over North America53. Both studies reported persistent contrails embedded in cirrus or sharing an atmospheric column with extant cirrus and suggested enhancements in OD and effective ice crystal size over the values found for contrails alone. A study quantified over the eastern North Pacific statistically the effect of contrails on OD of already existing cirrus, resulting in enhanced cirrus OD inside selected flight tracks, mostly in vertically thinner ice clouds49.Satellite remote sensing reveals slight seasonal differences in contrail mean OD  and particle effective diameter (36 µm) without diurnal variations54 or an automated tracking algorithm able to follow for some time a fraction of spreading contrails that are no longer line-shaped55. These studies have yielded values of mean geometrical thickness of 670 m, average contrail top altitude (temperature) of 10.9–11.7 km (≈219 K) and OD of 0.19–0.34, showing that contrails are mostly optically thin (OD < 0.3) in line with other studies56. Differences in the results of these studies reflect variability in microphysical and optical properties and meteorological conditions, differences in observation regions and air traffic activity, and inherent differences in detection methods.Some space borne observations include lidar measurements with increased temporal and spatial resolution of, and sensitivity to, optically thin contrails, assisted by manual detection57. The optically thinnest ice clouds escape detection when using satellite-based, passive remote sensing methods that rely on upwelling radiance measurements, because detection efficiencies decline rapidly at low (<0.1) OD59. Meaningful comparisons of observed AIC properties, acquired via aircraft data sets25, with models are often precluded due to: missing information on contrail source aircraft and local formation conditions; sparse sampling of AIC that exhibit large spatial variability; poorly constrained dilution factors reducing ice crystal number concentrations; counting and chemically analysing small (sizes <0.01 µm) aerosol particles; counting and sizing errors in ice crystal size distributions taken with optical spectrometers; inability to directly measure with imaging probes the shapes of ice crystals with maximum dimensions <40 µm; and substantial uncertainties in measurements of supersaturation and ice water content—the amount of cloud ice per unit volume of air. Few attempts have been made30 to relate ice crystal size distributions of AIC from in situ measurements60 to microphysical simulations of the formation stage. While AIC might be detected in situ through a combination of measured aircraft co-emissions of NOx and model-based trajectory analyses17, all types of observations remain inconclusive regarding the ability to separate contrail cirrus from natural cirrus due to the large variability in geometrical and microphysical properties of the latter. Moreover, it is difficult in dense traffic areas to determine whether natural cirrus have already been perturbed by aircraft emissions.Detectability of AIC from space depends on a large number of factors related to OD61.Space borne and aircraft measurements provide a fairly detailed picture of contrail properties, albeit with a considerable spread of observation results. While first steps resolving the transition of contrails into contrail cirrus have been taken, cloud system resolving models linking the regional scale evolution of AIC to specific meteorological situations and simulating their microphysical interactions with natural cirrus are lacking. To inform our incomplete understanding of the spreading stage, such models are needed along with collocated observations of mesoscale (1–100 km) vertical air motion and associated ice supersaturation variability as well as properties of ice nucleating ambient atmospheric particles135. An initial perturbation of the radiation balance defines RF, a first-order indicator of the importance of different climate change mechanisms. RF refers to initial imbalances over a specified region and period of time; those provided by the IPCC7 are global averages taken relative to a pre-industrial time (1750). Global RF cannot be observed directly and is therefore obtained with the help of models or by extrapolation of regional values inferred from satellite observations.At the top of the atmosphere, annually and globally averaged, incoming shortwave  radiation and the outgoing longwave  radiation cancel out unless a climate forcing, such as changes in properties of clouds, perturbs this balanceλ, passes through it—is a crucial determinant of RF. OD and RF are determined by a number of radiative transfer processes. Ice in high level clouds, i.e., thin cirrus and long-lived contrails, acts similar to cloud-free air (a): it decreases the energy lost to outer space, enhancing the temperature in the atmosphere below the cloud and at the Earth’s surface (cloud greenhouse forcing). Cloud ice crystals affect RF by reducing thermal emission (radiant energy with λ > 4 µm) to space via absorption and re-emission of LW radiation at cold temperatures. Contrary to low clouds composed mainly of liquid water droplets (b), high ice clouds are often optically thin , i.e., partially transparent to solar radiation (λ ≈ 0.2–4 µm), meaning their ability to scatter SW radiation back to space  is small. The greenhouse forcing is stronger for higher and colder clouds, in which ice crystals are increasingly abundant. The net average diurnal RF due to thin cirrus and contrails is a small positive residual from the wavelength-integrated SW and LW forcings. RF contributions due to thickening of natural cirrus by embedded contrails and new ice crystal formation in contrail areas with low ice crystal concentrations, blurring the distinction between natural cirrus and contrail cirrus, have not yet been determined.OD—measuring the opacity of a medium when electromagnetic radiation of a given wavelength, 137, but exactly which habits prevail and how individual habits vary with contrail age and ice crystal size is not well known.Beside the power received from the Sun (solar irradiance) and Earth’s atmosphere, Earth surface conditions and vertical distribution (overlap) of clouds, wavelength dependent scattering and absorption properties of single ice crystals are needed to estimate OD and RF. Those properties depend on shapes (habits), surface roughness and orientation against incident light of cloud ice crystals across their size distribution, all of which are difficult to quantify in airborne measurements. Emissivity  and reflectance  of high ice clouds are determined by OD and physical properties of ice crystals. Reflectance is sensitive to the habits of small ice crystals. Optical contrail models assume mixtures of various habits with fixed fractional weightsT, is estimated by the climate sensitivity parameter138, RF/ΔT. Values of this parameter and the associated equilibration times depend on the nature, spatial pattern and temporal evolution of RF and on the heat capacity of the climate system.Positive (negative) RF induces a warming (cooling) tendency that is subsequently communicated throughout the whole atmosphere. The strength of the resulting response in globally averaged, equilibrium near surface air temperature, Δ62. Global climate models can augment such observations by providing OD and RF estimates associated with AIC of all ages including contrail cirrus to various degrees, depending on how they are represented in their cloud schemes. Crude estimates of changes in the global mean surface air temperature as a result of RF are only available for persistent contrails, not for contrail cirrus. Regarding changes of RF due to aircraft-induced modification of natural clouds, model studies have not led to robust estimates.Uncertainty in the understanding of the processes central to AIC formation and evolution is, to an extent, reflected in the tools used to estimate the associated RF Box . Satelli64. In addition to satellite observations, high-resolution lidar47 and cloud models65 show marked variability in properties and RF of ageing persistent contrails.Variability in cloud properties—conveniently illustrated with the help of probability distribution functions (PDFs)—arises from variations in meteorological conditions and from cloud heterogeneity. Both sources of variability affect OD and hence RF and are not fully captured by global models. Large variability in microphysical and optical properties was observed in aircraft measurements of young (age <10–30 min) contrails65 simulating in ice supersaturated conditions the dilution and spreading of contrails along with depositional growth and sedimentation of ice crystals within them. Figure 56. The model PDF derives from an analysis of satellite observations66 and contains many optically thin and even invisible portions within contrails. This indicates that contrails that are non-detectable by passive remote sensing and invisible by ground-based observers may exert a non-negligible RF despite their low OD, since they are likely associated with large coverage67.Figure −2 (refs. 56). In such rare cases their development may be affected by radiative heating68 enhancing vertical mixing. Regional contrail RF can be close to or above 1 W m−2 (refs. 71). Globally averaged, annual mean RF values and uncertainty ranges for persistent contrails alone (≈0.01 (0.005–0.03) W m−2) and together with contrail cirrus (≈0.05 (0.02–0.15) W m−2) are compiled in Fig. Local radiative flux changes due to contrails can be as high as 100 W m−2 refs. 1. Global73. The first RF estimate for persistent contrails and contrail cirrus was based on a global climate model that represents AIC as a separate cloud class74. An offsetting feedback of AIC on natural clouds due to humidity and (to a lesser degree) temperature changes was estimated with large uncertainty to reduce the direct AIC RF by almost 20% (ref. 6). The RF values compiled in Fig. 69 reports much lower AIC RF for 2006 aircraft operations than others, possibly because in the model contrails, while evolving consistently with the hydrological cycle, are undifferentiated from other clouds. The same model predicts an increase in AIC RF from 2006 to 2050 by a factor of seven due to a non-uniform regional increase in air traffic and different sensitivities for the forcing in different regions75. While model-based central estimates of AIC RF converged over time, results for current traffic levels are not available and the level of scientific uncertainty remains large.Until 2011, a best central estimate of RF due to contrail cirrus including RF due to persistent contrails was not available. This caused large uncertainty in aviation RF as a whole. After IPCC 1999, AIC RF estimates have been based on associating air traffic with regional trends in cirrus coverage without proof of causality and by assuming equal radiative efficiencies of contrails and contrail cirrus−2)−1 (ref. 76) and 0.3 K (W m−2)−1 (ref. 77), meaning that contrails induce a smaller global temperature change per unit RF than CO2 emissions. In combination with the most recent IPCC uncertainty range in contrail RF of 5–30 mW m−2 −1 ref. 6 and 0.37, 0.4–1.2 K (W m−2)−1. Combining the latter range with the most recent (year 2011) uncertainty range for AIC RF, 20–150 mW m−2, leads to a highly uncertain equilibrium global warming contribution of 0.008–0.18 K due to AIC. This may be compared to the range 0.014–0.042 K due to accumulated aviation CO2 emissions alone inferred from the associated RF of 35 mW m−2 .The contrail climate sensitivities mentioned above are at the low end of the overall climate sensitivity uncertainty across climate models61 is also needed to simulate AIC RF and its dependence on future air traffic patterns and climate change with greater confidence. Understanding forcing-response relationships in the climate system to better quantify surface air temperature changes warrants further scientific scrutiny81. In particular, time series of AIC RF and climate sensitivity estimates for contrail cirrus are needed to better constrain the contribution of AIC to global warming.More effort is required to understand the causes of spread in AIC RF estimates across different global climate models and methodologies. Total uncertainty estimates should account for correlations between uncertainties due to different processes. Besides a more complete representation (parameterisation) of AIC, an improvement of the representation of natural cirrus in global models82. They also suggest that aircraft emissions increase the number concentrations of fine (sizes < 100 nm) aerosol particles significantly in some areas83. Soot particles, regardless of their source, do not appear to contribute much to natural cirrus formation85. In contrast, sufficiently large sulphate particles tend to be efficient liquid cloud nucleating agents; several climate models predict significant modification of low level liquid phase clouds by emissions of small aircraft sulphate particles86. A global model study of the influence of soot particles emitted by aircraft and other sources on natural cirrus found RF estimates that would dominate the total aviation RF, but the sign of the simulated forcing could not be ascertained87. A contrail model run within a climate model predicted reductions in the total water column and high- and low-level cloud coverage88. Based on the assumption that aircraft soot particles—once preconditioned in contrails—act as highly efficient ice forming agents, one climate model predicted substantial effects on natural cirrus89. This relied, in part, on assumptions of how ice forms in background cirrus—a matter of current debate61.Full quantification of aircraft-induced cloudiness changes is hampered by a lack of knowledge on whether and how particle emissions affect natural liquid phase and ice phase clouds. Climate models indicate that increases in upper tropospheric sulphate and soot particle mass concentrations due to aviation emissions are at least tenfold smaller compared to other sourcesGiven the lack of observational evidence for effects of aircraft particle emissions on natural clouds and the poor model treatment of underlying processes, reducing scientific uncertainty and building confidence in these effects remain challenging.90 limiting global warming to 2 °C (preferably 1.5 °C) above pre-industrial levels by the end of this century, requires a dramatic pace for decarbonisation. If greenhouse gas emissions continue for just 15 more years, global surface temperature could rise as much as 3 °C (ref. 91). International aviation can help achieve the COP21 goals by mitigating the contribution of AIC to aviation RF on short time scales and thereby buy time to more substantially reduce aircraft CO2 emissions. Figure The achievement of the 2015 Paris (COP21) agreement9. Replacement fuels are limited to those that pass both technical and safety requirements. Alternatives to kerosene from crude oil include synthetic fuels  and biofuels 92. To meet technical and safety standards, those can be implemented rapidly only when blended with kerosene. Alternative fuels need to be assessed in terms of their environmental footprint including lifecycle analyses to ensure an overall carbon offset.Understanding how changes in fuel composition affect engine reliability and safety presents engineering and scientific challenges93 and exerts a rather small impact on ice crystal numbers (−35%)42, since lower nucleated ice numbers due to reduced soot particle emissions , and thereby causes contrails to form more frequently, this does not lead to a significant increase in ice crystal numbers at the end of the formation stage (<5%)42. The effect of alternative fuels on contrail ice crystal formation is currently being investigated in airborne measurements93.As pure synthetic fuels and biofuels contain next to no sulphur and aromatic species, their use would lead to a substantial reduction in ice forming aqueous plume and soot particles Box . The use94). Other technical options to reduce contrail ice formation have been proposed95; their viability has not yet been demonstrated and they may become operative only in the long term.Pure biofuels cannot satisfy current supply demands and research into emissions arising from the use of pure synthetic fuels is still in the demonstration stage. It is therefore meaningful to explore the capability of modifications to propulsion technology to reduce in-flight soot emissions. Lean combustion technology (employing high air to fuel ratios during fuel combustion) produces low soot particle emissions by number in ground-based measurements  showed that AIC RF increases by 50% (40%) compared to conventional future technology due to changed contrail formation conditions99. While the effect of reduced soot emissions has not been investigated in detail, the study suggested that a reduction of AIC RF might be achieved when employing blended wing body technology together with soot-poor fuels.Liquid hydrogen (LH2) and liquefied natural gas  have zero soot and sulphur emissions. In a climate model study, RF due to a hypothetical fleet of LH2-propelled aircraft (cryoplanes) with increased water vapour emissions was smaller than for kerosene aircraftx emissions) due to longer lifetimes of emissions. The lower stratosphere is commonly too dry to support AIC, but those gaseous emissions affect polar stratospheric cloud formation with negative repercussions for the ozone layer101. Evaluating the climate impact of a hypothetical civil supersonic fleet without accounting for effects on stratospheric cloudiness, one study predicts a total RF of 22 (9–29) mW m−2 in 2050 (ref. 102).The long-term transition to a fleet of supersonic aircraft operating in the lower stratosphere  will increase levels of water vapour and nitric acid  plays a key role in enabling safe and energy efficient operation. Studies have begun to explore the mitigation potential of changes in ATM with regard to wind-optimised improvement in fuel consumption8. The chemical impact of contrail avoidance measures is considered small107. While entirely avoiding the formation of persistent contrails by re-routing is arguably difficult, it is desirable to identify and avoid meteorological conditions that cause the potentially largest climate impact: long lived, large scale, ice supersaturated areas108. Simply re-scheduling global air traffic to more daytime flights to promote the compensation of SW and LW AIC RF . Adopting the two principal stages of AIC evolution discussed here as a conceptual framework in mitigation studies offers the possibility to systematically explore the implications for mitigation by analyses of subcomponents within each stage, i.e., the jet and vortex regimes within the formation stageage Fig.  focus on2 emissions need to be placed on a common scale by the use of suitable metrics beyond RF. To compare the associated climate responses on various time horizons, climate change metrics other than RF are more suitable for AIC113. Choosing a time horizon depends on the specific climate policy the metric should serve and helps evaluate the relative importance of short-lived and long-lived climate perturbations.The climate impacts resulting from RF due to AIC and CO114 are neither expected to be efficient liquid cloud-nucleating particles nor potent heterogeneous ice nuclei. This casts doubt on their capability to facilitate cloud water droplet or ice crystal formation in atmospheric conditions. Pre-activation refers to an increase in an aerosol particle’s freezing temperature after ice nucleation occurred. While pre-activation has been observed in laboratory settings, it is not known whether it occurs on aircraft-emitted, small (<100 nm) soot particles in the atmosphere or in contrails.Robust RF estimates of this potential contribution to AIC RF are missing due to lack of observational evidence and uncertainties in underlying mechanisms and their parameterisation. Mechanisms include modification of ultrafine aqueous plume aerosols during transport from cruise altitudes to lower cloud levels and pre-activation of emitted soot particles in contrails. Given the small size and the substantial fraction of organic matter in aqueous plume particles and the largely insoluble nature of exhaust soot Box , particl2) emission index in kerosene varies widely around a mean value ≈1 g (kg-fuel)−1 depending on the origin of the crude oil4; most jet fuel has lower emissions. The fraction of SO2 converted to condensable sulphuric acid at emission (few %) and the mass and chemical nature of emissions of condensable organic vapours are not well known. With regard to aircraft-emitted soot particles, emission inventories only provide mass-based soot emissions117—often as a single, fleet-averaged surface-level value—from which particle numbers at cruise are inferred using empirical correlations. This seriously underestimates variability in soot particle numbers in contrails at formation.Liquid aerosol particles produced from gaseous aircraft engine emissions are influenced by fuel composition Box  but are To parameterise aqueous plume particle formation and contrail ice nucleation properly in climate models, it is necessary to inform global aircraft emission inventories with additional sulphate/organics mass and soot particle number emissions. Such improved inventories better represent aviation activity and allow for more realistic simulations of the formation stage, AIC RF and effects on natural clouds by way of parameterisation.118. As neither geometric optics nor Mie theory accurately describe such cases, other optical models might be employed to provide more realistic parameterisations of ice crystal scattering properties in contrails and contrail cirrus. Shapes of small ice crystals are largely unconstrained by direct observations  SW radiation at the Earth’s surface in cloudless scenes has been observed at several locations across the continental U.S.A. in the period 1996–2007 (ref. 120). The observed trend was almost entirely due to an increase in the diffuse irradiance, while the direct component remained virtually unchanged, ruling out a concomitant decrease in atmospheric aerosol OD as the sole cause of the increase. Recent analyses using diffuse SW spectral measurements121 confirmed this trend coined as ‘clear-sky whitening’. Ref. 121 hypothesised that while a decreased aerosol load did increase the total SW irradiance, an increase in subvisible contrail-generated ice crystal ‘haze’ repartitioned this increase from the direct into the diffuse component. Formation and spreading stage processes including radiative effects of small ice crystals must be understood well to explore this plausible hypothesis.Solar radiation fluxes declined from the 1950s to 1980s and partially recovered since mid-1980sOn the one hand, instrumental challenges pose significant limitations in measurements of microphysical and optical properties of contrail cirrus. This heightens the role of models Table  in provi122 to improve their representation of AIC supporting areas50. They also use parameterisations of formation stage processes to initialise AIC properties and rely on progress in simulating the formation of natural cirrus more realistically61.Local scale process and fluid-dynamical models systematically explore how plume turbulence and wake dynamics affects nucleation and properties of contrail ice crystals Fig.  and how 123 is very promising. More aircraft travelling in the same regions may not necessarily lead to more AIC coverage due to overlap effects and merging with natural cirrus, thus limiting increases in RF for higher air traffic volume. Moreover, AIC and natural cirrus do not evolve independently49. A new generation of high-resolution global models explores such effects in the spreading stage with increased confidence.Global models represent much of the system complexity, but account for processes and their variability affecting RF only to a degree, depending on the model’s representation of clouds. They track atmospheric motion and clouds with ≈10 min time steps and represent them with coarse spatial resolution. This means that formation and effects of clouds must be parameterised. Cloud microphysical and optical variables evolve on unresolved temporal and spatial scales. The connection of unresolved processes to resolved  model variables is not always obvious. With higher resolution, fewer parameterisations are required. The prospect to perform in the near-term global studies that resolve explicitly the regional scales crucial for AIC development31, reminiscent of the ‘Goldilocks principle’. A suitable framework combines aircraft and active remote sensing observations and local and regional models to study characteristics of and linkages between individual processes (bottom-up) with passive remote sensing observations, and global models to explore large-scale constraints arising from the coupled dynamics-aerosol-cloud-radiation system (top–down).Improving and validating AIC in global models requires an integrative analysis framework that unravels crucial cloud-controlling factors and conditions. Propitious weather conditions leading to large radiative impact of contrail outbreaks are tied to synoptic-scale, enhanced moisture transport and uplift (cooling), in which moderately high ice supersaturation at the same time minimises sedimentation losses and maximises OD by generating ice crystals within an optimal size range62. Adding or elaborating on processes may be required to improve on specific problems. However, this would expand the space of underlying model parameters, possibly compromising the robustness of the models’ predictive capability and the goal of reducing overall model uncertainty124. Parameterisation schemes used in such models should therefore be informed by theory, whereby it is important to capture the most influential processes governing a specific application, and document the physical soundness and plausibility of the scheme. Understanding how well global climate models work for a specific purpose  is important, but in itself is not a straightforward task. At times the disagreement of a model with observations motivates model improvements.Methodologies combining estimates of contrail coverage from meteorological reanalysis data and mean contrail OD in off-line radiative transfer models and used to diagnose global contrail RF in climate models have been replaced by simulations of AIC within global modelsAnother important task in support of model improvement is ascertaining its “truth” , e.g., by comparison with suitable measurements. In models with many compensating uncertainties, several pathways of process representation conforming to the same set of underlying physical laws may lead to similar plausible agreement with observations. Just because models agree with some measurements does not imply reduced uncertainty in their RF predictions. In view of the case study nature of many measurements, observationalists and modellers should aspire to clarify if and exactly how measurements can be used to validate models and constrain uncertainty. Introducing microphysical mechanisms in cloud parameterisations without critical analysis, theoretical underpinning or support by empirical evidence increases the likelihood of model fallacies. Unexamined process representation carries the danger of proliferating unsubstantiated conclusions. The resulting enhanced uncertainty levels are difficult to judge and may be irreducible.Lack of rigorous model evaluation and validation has serious repercussions for the interpretation of model results. A way out could be to devise models based on a minimal set of processes with only a few open physical parameters and expounding meaningful ways of how model behaviour could be confirmed or falsified. Complexity would be added only when needed, fulfilling a methodological principle known as ‘Ockham’s razor’. To minimise AIC model uncertainty, ice cloud parameterisations should conform to a number of tenets: full inclusion of AIC into the model’s hydrological cycle, as cloud-induced changes in the moisture and heat budget cause feedbacks on natural cloudiness; consideration of vertical overlap between AIC and natural clouds, as cloud overlap statistic has a profound impact on overall fractional cloud coverage within a model grid box and on net RF; realistic representation of AIC within the model’s radiation scheme, as optical properties differ from those of natural cirrus; and abandonment of instantaneous conversion of water vapour above ice saturation into cloud ice (and the reverse process below ice saturation), as ice crystal growth and sublimation can be kinetically hindered in thin or aged contrail cirrus; moreover, the kinetic treatment of water vapour uptake involving size-dispersed populations of disparate ice nuclei is a perquisite for the proper treatment of ice nucleation in models. Implementing these improvements will also lead to better simulations of natural cirrus.Commercial aircraft are in service for 20–30 years meaning that adoption of new technologies from research to integration takes many decades. Therefore, in addition to scientific challenges, the implementation of AIC mitigation options also faces engineering and economical hurdles and will only be realised when disruptive change in the air transportation system can be avoided. Accomodating the expected growth in air travel while reducing the impact of AIC RF may require a combination of options.125, but does not explicitly address AIC in their environmental protection activities. Integrating AIC in these activities would constitute an important stepping stone in support of widespread implementation of AIC mitigation options. The use of biofuels has already been initiated by the aviation industry in the necessary transition away from fossil-based fuels. Besides implementing a standard for carbon neutral growth, ICAO has also developed certified testing procedures applying from 2020 for particulate emissions from aircraft engines based on particle mass and number125. This action facilitates the realisation of mitigation solutions addressing formation stage (emission-related) processes and holds promise to reduce AIC effects on climate on short time scales96. With regard to the spreading stage, while an interim strategy has been conceived126, ATM is considered to offer a potential long-term solution as meteorological forecast skills mature and if ATM proves to be flexible enough to allocate climate-compatible flight routes quickly and safely.The International Civil Aviation Organization (ICAO) agreed upon a carbon offsetting and reduction scheme for global aviation127. Studying climate change in response to AIC involves basic scientific challenges and is important to address in the larger context of how anthropogenic activities affect natural cirrus clouds and climate61. Better understanding the radiative response of contrail cirrus as it emerges from coupled meteorological and microphysical controls requires an integrative approach aiming at understanding on the process level at small scales and at the same time be cognisant of interactions that operate on large scales.Compelling evidence has been presented as to the urgency to prevent human activities from causing unacceptable environmental change


Blastococcus sp./Propionibacterium, Cryobacterium-related., Flavobacterium sp., Pedobacter sp., and Polaromas sp. that are frequently found in the six tested ice layers from 1990 to 2000. Live and total microbial density patterns were examined and related to the dynamics of physical-chemical parameters, mineral particle concentrations, and stable isotopic ratios in the precipitations collected from both Muztagata and Dunde ice cores. The Muztagata ice core revealed seasonal response patterns for both live and total cell density, with high cell density occurring in the warming spring and summer months indicated by the proxy value of the stable isotopic ratios. Seasonal analysis of live cell density for the Dunde ice core was not successful due to the limitations of sampling resolution. Both ice cores showed that the cell density peaks were frequently associated with high concentrations of particles. A comparison of microbial communities in the Dunde and Muztagata glaciers showed that similar taxonomic members exist in the related ice cores, but the composition of the prevalent genus-related groups is largely different between the two geographically different glaciers. This indicates that the micro-biogeography associated with geographic differences was mainly influenced by a few dominant taxonomic groups.In this study, six bacterial community structures were analyzed from the Dunde ice core (9.5-m-long) using 16S rRNA gene cloning library technology. Compared to the Muztagata mountain ice core (37-m-long), the Dunde ice core has different dominant community structures, with five genus-related groups  A variety of microorganisms including bacteria, archaea, fungi, protozoa, algae, and viruses, and even invertebrates, have been found in glaciers and ice sheets in the Arctic, Antarctic, Greenland, and in other mountains across the world . MicroorComamonadaceae and Flavisolibacter sp. are common in both the Kuytun 51 and Qiangyong glaciers but only Rhodoferax (Betaproteobacteria) is dominant in the Kuytun 51 glacier  and the Dunde ice cap . Six structures of bacterial communities were established from the Dunde ice core columns (at field depth 0.8–5.3 m) using bacterial 16S rRNA gene clone library technology. Additionally, live bacteria were examined and related to the physical-chemical parameters from the Muztagata and Dunde ice cores.Figure 1; Table 1). The Muztagata Glacier is located in the most western margin of the Tibetan Plateau, where precipitation is mainly controlled by westerly circulation originating in the arid and semi-arid regions, including the deserts Sary-Ishykotrau, Muyun Kum, Kyzyl Kum, Kara Kum, Taklimakan, and Gurbantunggut , the Dunde ice cap , and the Puruogangri ice cap  where precipitation patterns were mainly controlled by two different circulations- westerly and monsoon  was extracted at 7010 m ASL (above sea level) from the Muztagata Glacier in the summer of 2003 . The DunA 10 ml aliquot of melt-water from the Muztagata and Dunde ice cores was used for the analysis of the mineral particles. Total micro-particle concentrations were measured by using a Coulter counter Multisizer 3 (Beckman). A total of 44 ice samples were analyzed from the Muztagata ice core taken at a depth of 2.5–12.5 m, and 74 samples were analyzed from the Dunde ice core taken at a depth of 0.50–9.8 m.18O/16O (δ18O) in the precipitation. A Finnegan MAT-252 mass-spectrometer was used to determine δ18O values within ± 0.05%. The Dunde ice core was dated by using seasonal δ18O variations and annual visible dust layers and confirmed by the previous data . Microbial analyses were carried out on 156 and 37 samples from Muztagata and Dunde, respectively. The ice samples were cut between the visible dust layers, and ice layers were collected separately. The improved procedures were used for the decontamination of the outer surfaces of ice core samples. The snow and firn-ice columns  were decontaminated by cutting away the 10-mm annulus with an autoclaved sterile sawtooth knife. The knife was sterilized over an alcohol flame following each ice slice cut. A total of three sterile sawtooth knives were used for each ice sample. The decontaminated samples were then completely melted in clean and sterile glass beakers at 4°C. These handling procedures were undertaken at temperatures below 20°C within a sterile, positive pressure laminar flow hood as described before . The freHaeIII-based ARDRA  out of the 406 clones from the Dunde ice core. Each sequence was named using the initial of Dunde ice cap  and aligned with the Blast references (2 (Methanosaeta harundinacea strain 8Ac (accession no. AY817738) and Methanosaeta concilii strain GP6 (accession no. NR102903) were used as outgroup references on all trees. All the obtained sequences from the glaciers were identified by the recognized species and were related to the ecological clusters . Sequences obtained displaying similarities of >97% with known species were identified as the reported species. Most of the obtained clones were related to known cultivated genera or genus clones . A few clones had <97% similarity with reported species, and thus were designated separately.All 137 sequences from the Dunde ice cap were checked by DECIPHER . An apparent seasonal temperature change was indicated by the proxy value of the stable isotopic ratios, 18O/16O (δ18O), with a low value in winter and a high value in summer . The live cell density was greatly variable and ranged from 6.5 × 102 to 2.1 × 104 cells/ml between 1964 and 2000 . The total cell density varied from 4.4 × 104 to 8.7 × 105 cells/ml . Several live cell density peaks were formed during the summer seasons in 1969, 1970, 1973, 1979, 1982, 1983, 1988, 1990, and 1993 for a total of nine events, a1 to a9 , while cell density peaks were found in spring . This ice core also had an increased density of the total number of microorganisms in the summer of 1978, 1988, and 1993 , and in the spring of 1995 and 2000 , which was consistent with the live cell density patterns . The microbial cell density correlated with the concentrations of mineral particles and possessed a high R2 value of 0.68 only from 1994 to 2000 , but did not correlate with mineral particle concentrations from 1990 to 1993 .There was an obvious seasonal effect on temperature and biological parameters along the ice core extracted at 7010 m ASL of the Muztagata Glacier . Oxygen isotope ratios of the melt-water samples from the Dunde ice core showed a change range from –10.78‰ to –8.24‰ , while microbial cell density varied from 1.2 × 103 to 9.1 × 104 cells/ml  and 1.3 × 105 to 1.9 × 106 cells/ml  for live and total cell density, respectively. Three peaks c2, c3, and c4 of the total cell density were found in the spring of 1988–1989, 1992, and 2000, only one peak, c1, was found in the summer of 1985 . The live cell density response pattern was consistent with the total cell density tendency . An abundance of microbial cells frequently occurred at the dirty ice layers , but were rarely found at the clean ice layer .Seasonal analysis of the Dunde ice core was not successful due to the limitations of sample resolution . Three genus groups Cryobacterium-related, Salinibacterium/Frigoribacterium sp., and Propionibacterium/Blastococcus sp. were clustered with 65–76% similarity to the known species but grouped with genus Knoellia sp. with 95% similarity in the family members of Actinobacteria . Only one clone DD1D107-100 was 100% similar to the uncultured Bacteroidetes clone AKYG1686 . All tested bacterial clones in the ice fell into members of bacteria phyla Alpha, Beta, Gamma, and Deltaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Verrucomicrobia, and TM7 candidates.The dominant bacteria in six ice layers of the Dunde ice core were investigated using 16S rRNA gene clone library, sequencing techniques, BLAST and phylogenetic tools. A total of 24 bacterial genera were identified in the Dunde ice core. They belonged to genera Figures 8A1–A6). The bacterial clones were comprised of five dominant genus groups, Polaromonas sp., Pedobacter sp., Flavobacterium sp., Propionibacterium/Blastococcus sp., and Cryobacterium-related, which accounted for more than 55% of the total 406 clones and frequently appeared in the six tested ice layers from 1990 to 2000 . Nine genus groups such as Rhodoferax sp., Variovorax sp., Burkholderiales, Flectobacillus sp., Cytophagales, Sphingobacteriaceae, Knoellia sp., Cyanobacteria rarely occurred in the ice. Other opportunistic bacterial clones occasionally appeared in the ice.There was a large difference in the proportion of the main phylogenetic groups along the Dunde glacier depth profile, which indicated the seasonal changes of microbial communities in the glacier  used in this study is unlikely because the outer surfaces of ice core and reagents for DNA analysis were cautiously decontaminated, and all the procedures were performed within a sterile, positive pressure laminar flow hood. Only small DNA fragments (<100 bp) were detected from the ice column control of (autoclaved sterile water), which were not considered for further sequence analysis in this study. It should be noted that  samples . Howevern-fixing  and non-n-fixing . They wen-fixing .Various molecular techniques, CFM with cell stains cFDA, PI, and SYTOX have been used to investigate viable bacteria . These tFigures 8A1–A6). This indicated that the identified clones were based on the dominant bacterial taxa.For the phylogenetic analysis of bacteria, more than 600 clones were picked and sequenced. A total of 406 valid bacterial clones were obtained from the Dunde ice core after vector, and chimeric checking. The rarefaction curves of six clone libraries from the ice core were approaching asymptotes (dada not shown). Data also showed the prevalence of a few dominant genus-related groups at the different ice core depths . This was also consistent with another independent microbial investigation on the Muztagata glacier at 6300 m above sea level . The strong association of microorganisms with dust was also found in previous data from the Antarctic glacier  revealed a high correlation between dust and microbial abundance from 1994 to 2000, which indicated a strong influence of aeolian activities on the microbial deposition in the glacier snow . The Dunde ice core data also showed one small cell density peak c2 appearing at the clean ice layer a2 in Figure 4. These results indicate that microbial deposition in the glacier snow does not always associate with the dust deposits or “dirty” wind and may in fact be transported by “clean” wind or snow, which implies influences of the processes such as aerosol and precipitation deposition, along with other factors , which indicated positive temperature effects on the microbial density patterns. This was consistent with another independent microbial investigation on the Muztagata glacier at 6300 m ASL , revealed clear seasonal patterns with high microbial cell density occurring in the warming summer months , there were also many density peaks in the spring from 1963 to 2000 . The seasonal pattern of bacterial density was generally consistent with the dynamic mineral particle deposition with frequently dust outbreaks in spring and summer . The phenomena of Polaromonas sp. from the same location readily grouping together was also found in the Muztagata and Puruogangri glaciers . Bacterial genus groups Cryobacterium-related, Flavobacterium sp., Pedobacter sp., Polaromonas sp., and Propionibacterium/Blastococcus sp. were frequently found at the six tested ice layers of Dunde glacier from 1990 to 2000 . Another example of similar group members sharing the related ice core layers can be found in the recently reported Dunde ice. Genera Polaromonas sp. and Flavobacterium sp. commonly found between 1990 to 2000 were also identified from the Dunde ice column AD 1780–1830 . Seven genus groups Polaromonas sp., Enterobacter sp., Acinetobacter sp., Flexibacter sp., Thermus sp., Propionibacteria/Luteococcus sp., and Flavisolibacter sp. were frequently identified in the four tested ice layers of Muztagata glacier from 1970–1988 , while Polaromonas sp. and Flexibacter sp. were found at all three tested ice columns of Puruogangri glacier from 1600 to 1920 . Precipitation over the Muztagata glacier is mostly influenced by the westerly depressions, while precipitation over the Dunde ice cap and Puruogangri ice cap is mainly driven by the westerly depressions in winter and Indian monsoon in summer  and thus its dust components are more likely strongly affected by local dust storms and dominated by mineral particles from the two deserts Qaidam Basin and Tarim Taklimakan (The geographic pattern of bacterial taxonomic groups could be attributed to the influence of the moisture and dust source area, which might vary across the mountain glaciers on the Tibetan Plateau (n summer . Dust inklimakan , 2010. TPolaromonas, Pedobacter sp., Flavobacterium sp., Propionibacterium/Blastococcus sp., and Cryobacterium-related frequently appeared at the six tested ice layers, constituting the dominant species endemic to the Dunde ice cap, while seven genus groups Polaromonas sp., Enterobacter sp., Acinetobacter sp., Flexibacter sp., Thermus sp., Propionibacteria/Luteococcus sp., and Flavisolibacter sp. were frequently found at the four tested ice depths of Muztagata glacier. The results demonstrate that the spatial differences in microbial communities between the two ice cores are more significant than the temporal differences. This study also showed a seasonal pattern of microbial cell density with high cell density occurring in the warming spring-summer.The members of bacterial genus-related groups were found to be similar in the related ice cores at a historical scale but largely different between the two glaciers Muztagata and Dunde, even if microbial communities fluctuated along the two ice core depth profiles. Compared to the Muztagata glaciers, the Dunde ice core presented distinct members of the taxonomic groups. The five bacterial genus groups YC: Design of the laboratory experiment outline, data collection, analysis, and interpretation, and draft of the manuscript. X-KL: Sequence data analysis, and interpretation. JS: Sequence data collection, analysis, and interpretation. G-JW: Mineral particle concentration examination of ice core, data analysis, and interpretation. L-DT: Oxgen isotope ratio analysis, and interpretation. S-RX: Design of the research outline, data analysis and interpretation, and revision of the manuscript.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.


Introduction: The introduction of the internal lengthening nail (ILN) has changed the treatment of complex malalignment and shortening about the knee. Acute correction of the deformity and gradual lengthening through this osteotomy site has greatly simplified postoperative recovery. This manuscript is a review of the techniques that are currently being used in surgery.Methods: The article is broken into two sections: distal femur osteotomy and tibia osteotomy. Each is addressed separately since they have different personalities. Also included are topics of particular interest that surface in ongoing conferences regarding the ILN. This work is a mix of expert opinion and best practice supported by peer reviewed publications on the topic.Results: Most published series demonstrate excellent results with the ILN. Certain precautions are reiterated including avoiding mechanical failure, need for a percutaneous osteotomy, need for over-reaming, and the need for blocking screws.Discussion: Current controversies will be brought to light and discussed. The reader should find this aspect particularly helpful in navigating this rapidly evolving field. The introduction of the internal lengthening nail (ILN) has revolutionized how deformity surgeons approach the treatment of complex malalignment and shortening about the knee. Where once external fixation was required to manage simultaneous deformity with lengthening, internal fixation has utilized the tactic of acutely correcting the malalignment and gradually lengthening through this osteotomy site . Some stThe retrograde approach to the femur offers excellent control over distal femoral deformity. The distal femur is an ideal location for osteotomy due to its reliable bone formation during distraction osteogenesis. The challenge of the distal femur lies in its width which allows for the reamer and intramedullary (IM) nail to move in many possible directions. In order to secure a path for the nail to pass, blocking screws have been implemented. Well-placed blocking screws ensure that the reamer and IM nail will head in the proper direction both correcting the acute deformity and preventing lengthening-induced deformity. This concept of lengthening-induced deformity is well known to deformity surgeons with experience using external fixators but may not be so obvious to other orthopedic surgeons attempting deformity correction and lengthening with the IM nail. For retrograde nailing with a distal femoral osteotomy the distal bone fragment can be expected to flex during lengthening. This requires that a blocking screw be placed posterior to the nail at the time of the index surgery in anticipation of the impending deformity . The mecThe location of the deformity in the distal femur is important to determine. This technique is best suited for meta-diaphyseal deformities , 7. A faPreoperative planning: There are many ways to plan for the deformity correction and lengthening , 8. WhatOsteotomy site: The osteotomy site that was selected on the preoperative plan is located on the femur under fluoroscopy. A 10 mm incision is made in the lateral thigh at this level and taken through the iliotibial (IT) band. The incision can be extended for an IT band release. We release the IT band routinely for correction of valgus and lengthening and for large lengthenings (over 4 cm). A drill is then inserted with a protective sleeve, and multiple drill holes are created in one plane. This creates vent holes during reaming and marks the osteotomy site to help with blocking screw placement.Blocking screws: When correcting coronal plane deformity blocking screws are inserted. The position of the screw needs to be decided as a part of the preoperative planning and replicated in the OR. For valgus deformity a blocking screw is placed lateral to the proposed path of the ILN . For varHalf pins: A half pin is inserted into the distal femur using cannulated technique. A K-wire is inserted posterior to the path of the IM nail and checked using C-arm. The wire is then over-drilled with a cannulated drill bit through a small incision. The solid half pin (5–6 mm) is then inserted . A seconDistal fragment: The knee is flexed, and the distal femur is prepared through an arthrotomy either through or medial to the patellar tendon. A 2.4 mm K-wire is drilled in the center of the notch under AP and lateral fluoroscopy until it reaches the osteotomy site. The ACL 12 mm reamer is then inserted over the guide wire up to the osteotomy site taking care not to damage the patellar cartilage.Osteotomy: The osteotomy is then performed with an osteotome through the previous drill hole incision under fluoroscopy. The reduction is performed with translation and rotational correction. The external fixator can then be placed to hold this reduction .Reaming: Sequential reaming is then performed 1.5–2 mm over the nail diameter. There are different teachings on the correct way to prepare the canal. This will be further discussed.ILN insertion: The external fixator is removed. The nail should slide into the canal without too much resistance. A mallet can be used, but if aggressive tapping is needed then we suggest removing the nail and reaming an additional 0.5–1.0 mm. The distal locking is then performed with the targeting device . The rotFollow-up: Lengthening begins after a latency period and proceeds at approximately 1 mm per day. The patient is followed at regular intervals with radiographs checking for regenerate quality and joint contractures  and 7b.Surgical steps:An acute correction of the femur coronal plane deformity  will not impact the ability of regenerate bone to form through a distal femoral osteotomy , 7, 9–11As a general rule G.A. Ilizarov taught the world that bony distraction achieved optimal osteogenesis when performed at a rate of 1 mm per day split into at least four separate adjustments of 0.25 mm per day. With the precision interlocking (IL) nails available, rate can be strictly controlled. In our practice, distraction needs to be started four days after surgery at the accelerated rate of 1.32 mm per day (0.33 mm four times daily) for four days and then slowed to a rate of 0.99 mm per day (0.33 mm three times daily). Rate is then further fine-tuned based on regenerate formation observed during the postop period. We have not experienced any implant-related premature consolidation or nonunion in the femur using this formula (one premature consolidation occurred due to a non-compliant patient) . Singh eThe role of minimally invasive osteotomy is believed to be paramount to the success of acute deformity correction followed by distraction. The creation of drill holes at the osteotomy site followed by reaming leads to the deposition of endosteal autograft at the osteotomy site. This pre-grafting is noticeable on the intraop radiographs . OsteotoThe United States is exiting an era of heavy narcotic prescribing by launching an initiative to drastically cut down on opioids. The field of limb deformity surgery in the US has relied greatly on prolonged narcotic use for the duration of the lengthening process and often the entire time that external fixator is on the patient. Based on promising results in pediatric patients , adult sThe use of external fixation during this surgery is helpful for maintaining the deformity correction during the reaming process. Since the ILN will follow the path of the reamer we believe it is best to create that path with the bony deformity reduced and stabilized. The half pins also offer rigid control of the bone fragments. This helps to rotate the osteotomy to ensure it is complete, translate the bone fragments to achieve the desired reduction, and control the final rotation prior to interlocking. Some surgeons feel that the half pins and the external fixator are not necessary , howeverThe decision of whether to ream the femoral canal using rigid reamers or flexible reamers has been debated extensively. The advantage of rigid reaming is that the sagittal curve of the femur is minimized allowing for less over-reaming of the canal. This in turn gives the ILN a tighter fit with less potential for malalignment at the time of insertion or during the lengthening process , 11. If The ILN comes with both curved and straight distal options. There has been little discussion as to the advantages of each. There may be an increased tendency to create a procurvatum deformity when using the curved nail . In caseAcute deformity correction of the tibia followed by distraction with an ILN is gaining popularity. Tibial nailing is more challenging than retrograde femoral nailing with regard to alignment control. The entry point of the tibial nail is difficult to pinpoint due to the need to flex the knee with subsequent poor AP fluoroscopic imaging. The suprapatellar approach to tibial preparation may help with alignment precision  althoughPreoperative planning: The tibia does not deviate from the mechanical axis during lengthening making planning straightforward. An osteotomy site is picked, translation is accounted for, blocking screw position is planned, and nail length and width are selected  and 10b.Osteotomy site: The osteotomy site that was selected on the preoperative plan is located on the tibia under fluoroscopy. A 10 mm incision is made in proximal leg. A drill is then inserted with a protective sleeve, and multiple drill holes are created in one plane. This creates vent holes during reaming and marks the osteotomy site to help with blocking screw placement.Blocking screws: When correcting coronal plane deformity, blocking screws are inserted. The position of the screw needs to be decided as a part of the preoperative planning. The distance of the screws from the osteotomy site and cortex should be replicated in the OR. For valgus deformity a blocking screw is placed lateral to the proposed path of the ILN . For varHalf pins: A half pin is inserted into the proximal tibia using cannulated technique. A K-wire is inserted posterior to the path of the IM nail usually superior and anterior to the fibular head and checked using C-arm. The wire is then over-drilled with a cannulated drill bit through a small incision. The solid half pin (5–6 mm) is then inserted. A second half pin is inserted in the distal tibia, distal to the tip of the ILN. Rotation should be accounted for in this step for later correction .Distal syndesmotic screw: The distal syndesmotic screw is placed to protect the ankle during distraction. We recommend a 4.5 mm fully threaded solid cortical screw.Fibular osteotomy: The fibula is cut percutaneously by pre-drilling with a K-wire and then using an osteotome .Proximal fragment: The knee is flexed, and the proximal tibia is prepared through an arthrotomy either through or near the patellar tendon. A suprapatellar approach can also be used. A 2.4 mm K-wire is drilled under AP and Lateral fluoroscopy until it reaches the osteotomy site. The ACL 12 mm reamer is then inserted over the guide wire up to the osteotomy site.Osteoplasty: The osteotomy is then performed with an osteotome through the previous drill hole incision under fluoroscopy. The reduction is performed with translation and rotational correction. The external fixator can then be secured to hold this reduction.Reaming: Sequential reaming is then performed over-reaming the canal by 1.5–2 mm.ILN insertion. The external fixator is loosened. The nail should slide into the canal without too much resistance. A mallet can be used, but if aggressive tapping is needed then we suggest removing the nail and reaming an additional 0.5–1.0 mm. The proximal locking is then performed with the targeting device. The rotation and alignment are checked and adjusted with the half pins. The distal interlock screws are inserted with a free hand technique.Proximal syndesmotic screw: The proximal screw is placed using a cannulated technique. A guide wire is placed through the fibular head from lateral to medial. It is over-drilled and then a solid screw is inserted to prevent migration of the fibula during lengthening .Fasciotomy: It is routine to perform a percutaneous fasciotomy of the anterior compartment when acutely correcting a tibial deformity to prevent compartment syndrome .Follow-up: Lengthening begins after a latency period and proceeds at approximately 0.75 mm per day. The patient is followed at regular intervals with radiographs checking for regenerate quality and joint contractures  and 15b.Surgical steps:It is clear that tibial lengthening places tension on the common peroneal nerve and can cause nerve damage , 25. SpeThis topic is not as well studied as in the femoral counterpart. Acute deformity correction of in the tibia may affect the quality of the regenerate, as measured by the consolidation index and circumferential healing, greater than the effect seen in the femur. The consolidation index (CI) for femoral lengthening has been excellent, averaging 24–32 days/cm , 14, 16 The tibial consolidation rate is significantly slower than that of the femur using ILN . The sitThe ILN is a great advancement in the area of limb deformity and lengthening, particularly in the femur. Surgeons are still learning the capabilities and shortcomings of these devices. The quality of the available implants continues to improve. The tibia continues to challenge surgeons’ abilities to control deformity and bone formation during lengthening and remains an area where external fixation is incredibly useful.Author ATF received no funding or financial benefit related to this work. Author ATF does function as a consultant for NuVasive, Smith & Nephew, and Synthes. Author SRR received no funding or financial benefit related to this work. Author SRR does function as a consultant for NuVasive, Smith & Nephew, and Stryker.