Habronema microstoma and Habronema muscae. These lesions, also known as “summer sores’’, are often severe and disfiguring. Although Habronema-caused lesions at the coronary grooves have been described, cases of hoof cracks with secondary summer sores have never been reported. The present case describes clinic-pathological and surgical features of a quarter crack case complicated by cutaneous habronemosis at the dermal layers. A 15-year-old, Andalusian stallion was referred to the Veterinary Teaching Hospital of the University of Teramo because of a mass of the hoof and a severe lameness. The clinical examination revealed an exuberant granulation tissue protruding from a full thickness vertical quarter crack. The mass was surgically removed, and subjected to histopathological, microbiological, and parasitological analyses. A copromicroscopic examination was also performed. The feces scored PCR positive for H. muscae, while the skin for both H. microstoma and H. muscae, thus confirming the primary role of Habronema in causing the hoof mass. This is the first description of a hoof wall crack complicated by summer sores, with simultaneous gastric habronemosis. This case confirms that a prompt diagnosis during fly activity is imperative for an efficacious treatment and a timely prevention of disfiguring summer sores.Cutaneous habronemosis in horses is caused by larvae of the spirurid nematodes  Habronema microstoma, Habronema muscae and Draschia megastoma . These nematodes are transmitted by biting and secretophagous flies, e.g., Stomoxys calcitrans and Musca domestica [Habronema spp. is much more distributed than D. megastoma, thus gastric and (muco-)cutaneous habronemoses are the most important diseases caused by these parasites [Habronematidosis is a parasitic infection caused by adult and larval stages of omestica . When inomestica ,3,4,5. Tomestica . Wounds omestica ,3,5. Sumomestica ,8,9,10. Habronema-infected horses are known. Thus, the present report describes clinic-pathological and surgical features of a quarter crack case complicated by cutaneous habronemosis at the dermal layers.In horses, the so-called “crack” is the disruption of the hoof wall that occurs in parallel to the horn tubules and lamellae, which usually initiates at the level of the coronary band until the involvement of the full thickness of the hoof capsule ,12,13,14A 15-year-old Andalusian stallion living in Southern Italy was referred in April 2021 to the Veterinary Teaching Hospital (VTH) of the University of Teramo  due to the presence of a neoformation at the level of the hoof.The owner noticed a quarter crack of the right forelimb hoof two months before the referral, and a corrective shoe (three-quarter bar shoe) was applied. However, the horse worsened in the following 20 days, showing a 4/5 lameness of the injured limb and a neoformation on the hoof crack. The referring veterinarian then treated the horse with an anti-inflammatory and antimicrobial therapy for 10 days, which was stopped 15 days before the hospitalization Despite the treatment, the owner reported a continuous growth of the lesion and referred the animal to the VTH of Teramo.A bleeding full thickness vertical quarter crack on the lateral aspect of the right forelimb hoof, and an exuberant granulation tissue protruding from the hoof crack, were noticed at the clinical examination. The mass was brown in color and had a nut-shape of 2 cm in diameter with hard and rough consistency .A warm swelling on the coronary band and the lateral bulb of the heel of the injured leg was evident, and the horse showed pain at palpation and was 4/5 lame. No alterations of the third phalange (PIII) bone were evident at the radiographic examination.Streptococcus spp. Three days after surgery the antibiogram performed on the pure Streptococcus spp. culture revealed an infection susceptible to cefazoline. Thus, a loco-regional cephalic perfusion (right front leg) with 2 gr of cefazoline was performed. Furthermore, based on results of parasitological examinations  0.4 mg/kg per os.First, standing surgery was carried out, aiming at removing the mass and performing further examinations. The mass was excised after sedation with medetomidine (7 μg/kg IV) and butorphanol (0.02 mg/kg IV). The crack was explored and widened to allow drainage and application of antiseptics. Loco-regional cephalic perfusion (right front leg) with 1 gr of gentamicin and 10 mL of lidocaine 2% was also performed, and a foot bandage was applied and changed q24h for 2 days, then q48h. A sample of the excised mass (1 cm × 1 cm × 0.4 mm) was fixed in 10% neutral buffered formalin, embedded in paraffin, and then processed for histopathological examination . Skin scrapings obtained from the excised mass were placed in sterile tubes containing absolute ethanol for parasitological microscopic and molecular examinations. A fecal sample was also collected for copromicroscopic analysis. The swab carried out from the mass was inoculated onto blood agar and MacConkey agar and incubated at 37 °C for 24 h. Pure culture was observed on blood agar and gram stain, catalase and oxidase test were performed, yielding the identification of ions see  the horsTwo weeks after, a second surgery under general anesthesia, was carried out due the poor clinical improvement of the stallion and the presence of an exudate draining from the coronary band .The horse was premedicated with acepromazine (20 μg/kg IM) and sedated with medetomidine (7 μg/kg IV); general anesthesia was induced with ketamine (2.2 mg/kg IV) and diazepam (0.06 mg/kg IV) and maintained with isoflurane and a constant rate infusion of medetomidine (3.5 μg/kg/h IV). A cleansing and extensive debridement of the crack , with a per os), along with antimicrobial therapy (sulfadiazine (200 mg/mL) + trimethoprim (40 mg/mL) 20 mg/kg SID per os) for one-week hospitalization.The medical management after surgery included the administration of NSAID  containing diluted povidone-iodine every two days, starting 48 h after surgery, and a constant change of hoof bandage. The horse recovered uneventfully, the lameness improved (3/5) and the warm swelling on the lateral bulb of the heel of the injured leg was markedly reduced. The stallion was discharged from the hospital 7 days after the second surgery.Follow-up was obtained by telephone interviews with the owner and the referring veterinarian. The short-term follow-up reported improvement of the lameness and no recurrence of the exuberant granulomatous mass. A prosthetic hoof wall repair material was used to provide an appropriate stabilization for the quarter crack repair.The feces were undertaken to McMaster analysis and conventional flotation procedure using a ZnSO4 solution with a specific gravity of 1.35 . As prevSkin scrapings from the lesion obtained during the first surgery were divided in two equivalent aliquots for subsequent microscopic and molecular examinations . The firThe copromicroscopic examinations showed the presence of strongyle eggs with value < 50 eggs per gram of feces , while the skin material was microscopically negative for parasite elements.H. microstoma and H. muscae in horse feces and skin [H. muscae, while the skin for both H. microstoma and H. muscae.Stool supernatant and digested skin were subjected to DNA extraction and molecular examination as previously described using a two-step semi-nested PCR protocol able to discriminate target species-specific sequences within the rDNA ITS2 of and skin ,20,21. TAn intense inflammatory response was microscopically observed mainly at the dermal layer. More in detail, multiple foci of suppurative inflammation were seen, along with aggregates of spherical-shaped bacteria. Moreover, areas of necrosis and granulation tissue were present, along with scattered eosinophils within the dermis .In horses, the hoof capsule guarantees protection to the dermal and bone structures within the capsule . SeparatThe treatment for quarter cracks relies on corrective shoeing aimed at unloading the affected wall ,26. In mThe prognosis for successful treatment of a hoof crack varies from good up to guarded , and theThere is scarce information on healing process of foot wounds, though similarities exist between foot and skin . Foot inHabronema-infected horse. The secondary intention healing and the persistent bacterial and parasitic infections have contributed to the exuberant granulation tissue reaction. In fact, summer sores often evolve into non-healing cancer-like masses [Habronema in causing the exuberant mass was then confirmed by the PCR-positivity to both H. microstoma and H. muscae, although no larvae were found at the histological examination. In fact, larvae of Habronema live for less than one month in the cutaneous tissue because the local allergic immune response kills them and their death triggers the appearance of exuberant, necrotic, or calcified lesions [Habronema larvae can only be found in very early stages of infection, though their DNA may persist and detectable upon molecular amplification [This is the first report of hoof wall cracks in a e masses  due to te masses ,32. Moree masses ,5 are th lesions ,32,33, t lesions . Therefofication , as hereHabronema in causing the hoof lesions and confirm previous data which showed the correlation between animals suffering from summer sores and concomitant gastric habronemosis [The positivity of the horse at the molecular examination of feces  further supports the primary role of onemosis ,2.Habronema-caused lesions develop, sores usually are painful and itching, so that the infected animals bite and rub the lesions leading to auto-traumatisms. In this case it is likely that the crack attracted infected flies, which deposited the larvae which, consequently, triggered local immunity response and development of the local mass first, and the opportunistic bacterial infection, then.Before that In conclusion, this case illustrates the risk for horses affected by hoof crack to suffer from severe summer sores. The diagnosis, in this case, was untimely, as the horse was referred 20 days after that the mass appeared on the hoof crack. A molecular prompt diagnosis would have allowed a timely treatment and a faster recovery, minimizing the factual risk for a potentially disfiguring infection.The fly season is usually spring–summer, but the current global warming is extending its duration throughout much of the year ,35. Thus


Mammalian target of rapamycin (mTOR) is crucial for endothelial function. This study is aimed at assessing whether the glucagon-like peptide-1 (GLP-1) analogue liraglutide has a protective effect on endothelial function via the mTOR signaling pathway.  Human umbilical vein endothelial cells (HUVECs) were administered liraglutide (100 nM) for 0, 10, 30, 60, 720, and 1440 minutes, respectively. Then, the expression and phosphorylation levels of mTOR, mTOR-Raptor complex (mTORC1), and mTOR-Rictor complex (mTORC2) were determined by Western blot and immunoprecipitation, while mTORC1 and mTORC2 expression was blocked by siRNA-Raptor and siRNA-Rictor, respectively. Akt phosphorylation was detected by Western blot. HUVECs were then incubated with liraglutide in the absence or presence of Akt inhibitor IV. Nitric oxide (NO) release was assessed by the nitrate reductase method. Phosphorylated endothelial nitric oxide synthase (eNOS), human telomerase reverse transcriptase (hTERT), and apoptosis-related effectors were assessed for protein levels by Western blot. Telomerase activity was evaluated by ELISA.  Sustained mTOR phosphorylation, mTORC2 formation, and mTORC2-dependent Akt phosphorylation were induced by liraglutide. In addition, eNOS phosphorylation, NO production, nuclear hTERT accumulation, and nuclear telomerase activity were enhanced by mTORC2-mediated Akt activation. Liraglutide also showed an antiapoptotic effect by upregulating antiapoptotic proteins and downregulating proapoptotic proteins in an mTORC2-Akt activation-dependent manner.  Liraglutide significantly improves endothelial function, at least partially via the mTORC2/Akt signaling pathway. Accelerated atherosclerosis characterizes type 2 diabetes (T2DM). Endothelial cell dysfunction (ECD) in premature atherosclerosis found in T2DM is considered the causal factor in atherogenesis. ECD is characterized by decreased endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) release, reduced telomerase activity, and elevated cytokine expression, resulting in impaired vasodilation and increased sensitivity to apoptotic stimuli, which could worsen the deficits and promote the pathogenesis and progression of atherosclerosis. Therefore, exploring the potential mechanism and identifying a strategy for the improvement of endothelial cell function are critical for preventing and treating macrovascular complications in T2DM.Liraglutide represents a glucagon-like peptide-1 (GLP-1) analogue employed in T2DM treatment. Similar to endogenous GLP-1, liraglutide induces postprandial insulin secretion, reduces glucagon release, and enhances satiety. Mounting evidence suggests GLP-1 analogues might equally decrease the risk of cardiovascular diseases in individuals with T2DM . In partPrevious findings have demonstrated that the mammalian target of rapamycin (mTOR) is closely associated with endothelial cell function. Recent studies suggested GLP-1 has protective effects on pancreatic beta cells , cardiomThe purpose of the present work was to assess whether the protective effects of the GLP-1 analogue liraglutide on endothelial function involve the mTOR signaling pathway.2 and used at passages 3–5.Human umbilical vein endothelial cell  isolation was performed from human umbilical cord vein specimens by collagenase treatment as previously described . HUVECs These assays were carried out according to previous descriptions .5 cells per well of 6-well plates) were cultured in ECM medium (2 ml) to 60% confluency. This was followed by 7 h of incubation in the presence of respective siRNAs. After transfection, HUVECs were further cultured for 24 h. Finally, mTOR, Raptor, and Rictor expression levels were analyzed by Western blot.For gene silencing, mTOR, Raptor, Rictor, and control siRNAs, respectively, were transfected into HUVECs following a previous report . Briefly5 cells per well) and incubated with liraglutide in the absence or presence of Akt inhibitor IV. Following PBS washes, the HUVECs were lysed as previously described [μg of protein with a TeloTAGGG Telomerase PCR ELISA Plus Kit (Roche).Cells were seeded into 6-well plates  by the nitrate reductase method.t-test was employed to compare group pairs. One-way analysis of variance (ANOVA) with subsequent Tukey post hoc test was employed for multiple group comparisons. p < 0.05 indicated statistical significance.Data are mean ± SEM from 3 or more experiments performed independently. Student's HUVECs were administered liraglutide (100 nM) for 0, 10, 30, 60, 720, and 1440 minutes, respectively, and immunoblot was carried out to assess the expression and phosphorylation of mTOR . As showGLP-1 activates protein kinase B (Akt), which activates eNOS via direct phosphorylation. Therefore, the effects of liraglutide on activated phosphor-Akt expression were assessed. Akt phosphorylation at Ser473 (primary phosphorylation site) was increased in HUVECs treated with liraglutide for 60 min, whereas total Akt levels remained unchanged . It is kEndogenous NO has a critical function in endothelial cells. Therefore, we assessed the effects of liraglutide on NO output and the activity of eNOS, the main NO synthetase in endothelial cells. Treatment of HUVECs with liraglutide at 100 nM for 60 min resulted in significantly increased amounts of bioactive NO in cell supernatants compared with control values . In addiCell senescence is controlled by telomerase activity modulated at the hTERT expression, phosphorylation, and nuclear translocation levels. To explore mechanisms downstream the mTORC2/Akt pathway, whether liraglutide regulates hTERT protein expression in HUVECs was assessed by Western blot. As shown in Apoptotic-related proteins, including Akt, B cell lymphoma- (Bcl-) 2, and Bcl-2-associated death promoter (BAD), were quantitated by Western blot Figures .Liraglutide promoted Akt phosphorylation at Ser473 with a subsequent increase of Bcl-2 protein expression levels ; meanwhiThis work showed liraglutide protected endothelial function via the mTOR signaling pathway by inducing mTOR phosphorylation, mTOR-Rictor complex formation, and mTORC2-dependent Akt phosphorylation. In addition, mTORC2-associated Akt activation enhanced the downstream NO production, hTERT translocation into the nucleus, and nuclear telomerase activity, upregulating and inhibiting antiapoptosis and proapoptosis proteins, respectively, in HUVECs. The above findings provide critical mechanistic insights into the effects of liraglutide on HUVECs.The mTOR protein regulates cell growth, proliferation, viability, and functions . We obseNO represents the main endothelial vasodilator, with powerful antiatherosclerotic effects due to its antioxidative, anti-inflammatory, and anticoagulatory properties , 15. PatIn addition to regulating eNOS phosphorylation and endothelial NO production, Akt also controls multiple cell cycle modulators and cell survival pathways. We assessed whether liraglutide inhibits HUVEC senescence. As shown above, liraglutide increased nuclear hTERT accumulation, markedly enhancing nuclear telomerase activity in HUVECs. Such effects were blunted by Akt inhibitor IV and Rictor knockdown, indicating that liraglutide enhances nuclear telomerase activity in HUVECs via mTORC2 and Akt activation.β cells, cholangiocytes, neurons, cardiomyocytes, and vascular endothelial cells [β cells [Multiple reports have demonstrated GLP-1 is antiapoptotic in various cell types, e.g., pancreatic al cells . It is wal cells . BAD is [β cells . Further[β cells . ConsistThe mTOR signaling pathway is closely related to the regulation of cell metabolism. Therefore, we will further study the effects of liraglutide on cell metabolism regulation and mitochondrial function under pathological conditions (such as high-glucose and high-fat condition) and whether it regulates cell homeostasis through autophagy. That will be beneficial to further clarify the cardiovascular protective mechanism of liraglutide. In summary, liraglutide stimulates mTOR phosphorylation, mTORC2 formation, and Akt activation in HUVECs, with protective effects via mTORC2/Akt-dependent increase of nuclear telomerase activity and NO biosynthesis. The current findings reveal new mechanisms underlying liraglutide-associated protection of HUVEC function and suggest mTORC2 to be a critical modulator of HUVEC function.


Drosophila is one of the most fascinating observations in biology and serves as a paradigm for gradient formation, yet its mechanism is still not fully understood. Two distinct models were proposed in the past, the SDD and the ARTS model.The formation of the Bicoid (Bcd) gradient in the early cis- and trans-acting factors that are indispensable for gradient formation. The first one is the poly A tail length of the bcd mRNA where we demonstrate that it changes not only in time, but also in space. We show that posterior bcd mRNAs possess a longer poly tail than anterior ones and this elongation is likely mediated by wispy (wisp), a poly A polymerase. Consequently, modulating the activity of Wisp results in changes of the Bcd gradient, in controlling downstream targets such as the gap and pair-rule genes, and also in influencing the cuticular pattern. Attempts to modulate the Bcd gradient by subjecting the egg to an extra nuclear cycle, i.e. a 15th nuclear cycle by means of the maternal haploid (mh) mutation showed no effect, neither on the appearance of the gradient nor on the control of downstream target. This suggests that the segmental anlagen are determined during the first 14 nuclear cycles. Finally, we identify the Cyclin B (CycB) gene as a trans-acting factor that modulates the movement of Bcd such that Bcd movement is allowed to move through the interior of the egg.We define novel Our analysis demonstrates that Bcd gradient formation is far more complex than previously thought requiring a revision of the models of how the gradient is formed. Drosophila is one of the most fascinating observations in biology that has intrigued scientists for many decades attP2, Bloomington stock number 38957. The 2nd-chromosome maternal GAL4-driver line V32 was obtained from the Perrimon lab and was used for all over-expression and RNAi experiments at 29°. Flies were fed with standard fly food (Bloomington recipe) and were exposed at 29° C to boost the efficacy of the maternal GAL4 driver.Canton-S stock from Bloomington No. 64349) was used as control. The 49 was us° C for 3–5 days, as described [Embryos were collected in 24 h. interval, incubated > 36 h., dechorionated in 50% bleaching solution, fixed in 25% formaldehyde for > 5 h., devitellinized, mounted in Hoyer’s medium and incubated at 65escribed .1,2 to detect freshly-made Tubulin [Embryos were heat-fixed for immunostaining. Rabbit antibodies against wispy and PAP2 were obtained from M. Wolfner and M. Simonelig, respectively, and were both used at 1:1000. The monoclonal antibody against Eve (DSHB) was used at 1:250. Rabbit-anti-Bcd antibodies  were use Tubulin  was used Tubulin .The protocol for fluorescent in situ hybridization was adopted from , except bcd gene-specific primer set giving a defined length and serving as control for integrity and concentration of the RNA (left side) and one primer set to amplify the gene of interest including the poly A tail (right side). The bcd gene-specific forward primer was 5’-CATTTTGCGCATTCTTTGACC-3’ and the bcd gene-specific reverse primer was 5’-TGTGTAGTTAGTCACAATTTACCC-3’. For poly A tail length determination, the bcd gene-specific forward primer again was 5’-CATTTTGCGCATTCTTTGACC-3’, identical to that described in [bcd and 30 bp from the anchor.Embryos were mounted in OCT perpendicular to the blade of a Cryostat. Consecutive sections of 60 μm starting from the tip of 10 nc 13 wild-type embryos similar to the method as described by  were perribed in , while tAll images were recorded using a Zeiss LSM 710 confocal microscope. Images were post-processed with Adobe Photoshop and Adobe Illustrator. Image J was used to measure the length of the embryos, the distance between the anterior tip, the anterior border of each Eve stripe and the posterior border of Ems. All data was analyzed with Analysis of Variance (ANOVA) and two-tailed Student’s t-tests. Data are reported as means ± SE.


Icing and formation of ice crystals is a major obstacle against applications ranging from energy systems to transportation and aviation. Icing not only introduces excess thermal resistance, but it also reduces the safety in operating systems. Many organisms living under harsh climate and subzero temperature conditions have developed extraordinary survival strategies to avoid or delay ice crystal formation. There are several types of antifreeze glycoproteins with ice-binding ability to hamper ice growth, ice nucleation, and recrystallization. Scientists adopted similar approaches to utilize a new generation of engineered antifreeze and ice-binding proteins as bio cryoprotective agents for preservation and industrial applications. There are numerous types of antifreeze proteins (AFPs) categorized according to their structures and functions. The main challenge in employing such biomolecules on industrial surfaces is the stabilization/coating with high efficiency. In this review, we discuss various classes of antifreeze proteins. Our particular focus is on the elaboration of potential industrial applications of anti-freeze polypeptides. Ice crystallization and ice formation are major drawbacks that considerably hinder the productivity of industrial machinery and equipment with a cooling system. The ice nucleation activity and freezing particles significantly reduce the system efficiency in a wide range of industrial applications including refrigerators, heat exchangers, wind turbines, helicopters, and aircraft [1]. It is therefore essential to integrate ice preventing mechanisms with the less energy requirement and more efficiency to prevent ice formation at interfaces or elsewhere. The formation of ice introduces excessive thermal resistance as a result of the outer ice layer resulting in deterioration in machine functioning . On the Anti-icing and de-icing processes can be categorized into passive and active modes. The passive method takes advantage of physical properties of the surface, while the active method relies on external systems, which require an energy supply. Since AFPs are passive agents, this review focuses on passive methods and solutions. In principle, because of their anti-adherent property, ice-phobic coatings prohibit ice from adhering to the surface, whereas super-hydrophobic coatings do not allow water to stay on the surface due to repulsive features . To miniEpoxy or polyester matrix composites reinforced with glass and/or carbon fibers are currently being used by many producers. However, inexpensive polyester and glass fibers are still the material of choice for manufacturers. Current research in chemical anti-icing research is focused on nanocomposite coatings, reinforced particles, and nanometer-scale polymers, which provide high water contact angles . PreventA notable diversity within various species of AFP families and their arbitrary placement in the respective phylogeny revealed their evolutionary history back to almost 10–30 million years ago when the sea level began to freeze in Antarctica; however; the degree of expression of ice-binding proteins differs in various species . The invChen and his colleagues concluded that antifreeze linked glycoproteins in antarctic fish are homologous to those in arctic fish, and they were results of evolution through a series of independent molecular pathways from non-coding DNA regions . AccordiThere are more than 60 identified heterogeneous plant species with AFP activity, where eleven of them have been isolated and characterized by high ice recrystallization inhibition activity (IRI) and low thermal hysteresis. According to these studies, all of the plant-based AFPs evolved from the OsLRR-PSR peptide sequence descendent from almost 36 million years ago . The disThere are three categories of Ice-binding proteins (IBPs). The formation of the ice crystals is controlled by ice-nucleating proteins (INPs) at high subzero temperatures, while the antifreeze proteins (AFPs) decrease the freezing point of body fluids in organisms to avoid freezing. The third category is Recrystallization Inhibition Proteins (RIPs) with insignificant TH activity. While INPs contribute to the generation of ice crystals at the freezing temperature, AFPs effectively mitigate freezing around the organism’s body fluids, and RIPs inhibit the recrystallization of ice in freeze-tolerant organisms that could freeze and yet survive the freezing temperature . AccordiLeucosporidium sp., an arctic yeast, Lee and his team demonstrated that various classes of AFPs from fish (I-IV), plants, and insects shared similar ice-binding domains, which were adequately consistent and hydrophobic; however, the distinct folds in each one were significant , and Ice Recrystallization Inhibition (IRI) phenomenon. Insects and fish exhibited high TH with no or mild IRI activity. Bacterial and similar microorganisms demonstrated adequate amounts of both IRI and TH; however, in contrast, plants demonstrated low TH and high levels of IRI. The excretion of AFPs in a substantial amount helps various microbes to endure the sub-zero condition, and the AFP secretion assists them to gather sufficient nutrients and oxygen . Since Aactivity . The intactivity . The IRI E. Desv , and Wooylvatica , which svia the hydrogen atoms present in threonine side chains with oxygen atoms towards the ice lattice; thereby suppressing the freezing point and eventually preventing further enlargement of planes in the pyramidal of the crystal ice  such that their hydroxyl groups were aligned in parallel and proximate distances with an axis in the direction of the ice. According to this hypothesis, such an arrangement allows the polypeptide chain to bind in a zipper-like configuration with ice lattice stal ice . HoweverCheng and Merz proposed a mechanism, which explained the clathrate hydrate generation on the protein surface resulting in hydrogen bonding and hydrophobic effects, which subsequently reduced the dissolved solution’s free energy . Hence, Nicotiana tabacum and Arabidopsis thaliana are designed to produce AFPs from cold-loving species of Winter flounder  and perform remarkably to promote the process of cell ablation in cryotherapy . Moreoveervation .3 exist and have hexagonal, cubic, rhombohedral, tetragonal, monoclinic, and orthorhombic crystal structures . The subcooling temperature initiating the process is correlated with the chemical potentials as follows:via lowering the interfacial free energy . The interface geometry also affects the correction factor ). This confined affix results in a greater surface curvature, as the ice enlarges, subsequently leading to lowering the freezing point caused by the Gibbs-Thomson effect, and ultimately obstructs the ice formation  are needed to reach the thermodynamic stability at the curved interface. As stated before, AFPs activity is attributed to their binding to the specific surfaces on the ice seed crystals, which causes modification in ice embryo growth morphology. These proteins prevent the growth of the ice crystals into the water by creating a layer around the solid ice. The adsorption-inhibition theory is the most accepted mechanism for the freezing point reduction and ice formation inhibition of AFPs . This thormation . When thormation .Antarctomyces psychrotrophic, revealed that the protein structure with 6-ladder ß-helices was responsible for generating a remarkable water network, which formed a well-tuned binding. This network was supported by a particular side of hydrophobic residues within AFP polypeptide and plans of ice crystal prism , defined as the number of nucleation per time per volume, can be calculated as:kink parameter can be used to characterize the kinetics of retardation of ice nucleation process as follows  from the same species  and Ice Recrystallization Inhibition (IRI) activity. In thermal applications such as refrigeration, even small changes in the inception temperature of the phase change process affect the system efficiency and energy output. Therefore, AFPs with higher thermal hysteresis (TH) are desired. On the other hand, in turbomachinery applications such as air intake or turbine blades, it is important to maintain small ice crystal sizes. Thus, it is recommended to use AFPs with greater Ice Recrystallization Inhibition (IRI) activity.The current applications of APFs are majorly focused on agriculture, food, and cryomedicine industries. AFPs are promising supplements for frozen food  includinvia biomaterials such as polydopamine (PDA) (3-glycidoxypropyl) methyldimethoxysilane (GOPTS), and biopoly amino acids such as poly l lysine  is an important issue but the presence of ice during the operation also negatively affects the blades and results in an increase in power loss (up to 50% annularly) and mechanical failure (due to vibration and load increase). Additionally, measurement sensors and devices including temperature sensors, wind vanes, and anemometers are influenced by ice. For example, the wind speed can be miscalculated with a large error margin of 60% due to this effect .Ice formation (frost) on the surface of evaporators is a common and undesired phenomenon, which remarkably reduces the system efficiency. t = 3 min. Here, some big ice crystals grow up to frost branches. At time t = 9 min, the crystals start to form larger units. However, these branches are not large enough to pass over the fins. As crystals grow larger, the branches start to grow, which is shown at time t = 31 min. At this stage, the ice crystals touch the neighboring branches and form thick and high-density ice layers, which block the fin surface. The frost formation on the evaporator is shown in The degree of icing depends on the height above the ground level. Icing types include rime ice, glaze ice, and mixed ice, which is a mixture of rime ice and glaze ice. Rime ice is nonhomogenous, consisting of powdery ice particles, shaped into finger-like structures. It is soft and less dense and happens at very low temperatures when 100% impinging droplets freeze. On the other hand, Glaze ice is a homogenous transparent hard continuum solid, and it is harder and denser. Glaze ice happens close to the freezing temperature when the freezing fraction of impinging droplet is not 100% and some droplets run along the blade surface as a very thin water film. The concentration of water droplets and ice embryos changes with altitude. The risk of icing is high at altitudes up to 10,000 feet. Ice formation on the surface of aircraft including wings, airfoils, and rotors might cause catastrophic problems . Ice on During take-off and landing at sufficient speeds, a thin layer of ice might form on the windshield, which might cause frosting, thereby remarkably reducing the visibility of the runway during a critical time in flight. The structural surface with thin leading edges such as antennas, horizontal stabilizers, rudders, and landing gear struts are the first locations, which experience ice accumulation and result in faults in radio antennas, pilot tubes, and static vents. The thin air temperature gauge, altimeter, airspeed, climb rate, gyroscopic instruments, pilot tube, and static pressure ports are flight instruments, which might not operate correctly or cease operation due to ice accumulation. Not only all communications between VHF Omni-Directional Range reception and ground might be affected by ice, but the ship antennas might also break under the accumulated ice weight.The freezing of water on the pavement surfaces causes slippery conditions, which will impair the safety of travel and transportation. This condition is of great importance in several applications such as airport runways, railways roads, and highways. Although de-icing chemicals and agents are mostly being used as a solution to reduce the risk of casualties, a significant problem is wastewater originating from de-icing pavements. Airport operators worldwide are under scrutiny to cope with the regulations focusing on the environmental impacts of de-icing practices. Chemical substances used for winter maintenance of airports are the main source of water and land pollution. After use, most of the de-icing and anti-icing agents get typically mixed with stormwater runoff and enter ground and water near the airfield pavements .Spraying superhydrophobic/icephobic materials is the widely used method to decrease surface energy. A two-step coating method  can be applied to increase the adhesion of such coatings . The icethe road . The utithe road . The larthe road .The stability and durability of the proteins are determined by their folded structure formed from the interactions between different amino acid residues within their primary structures. Generally, the overall stability of proteins depends on their core subunit interactions within tertiary and quaternary structures which are defined by various bindings such as hydrophobic interactions, van der Waals, electrostatic, and disulfide bridges . The dynperennial ryegrass, Lolium perenne, remained intact at the boiling temperature and demonstrated higher ice recrystallization inhibition and lower antifreeze as compared to its counterpart in antarctic fish. This protein has a unique sequence of amino acids with minimum homology with other antifreeze proteins and exhibited extremely hydrophilic properties abundant in asparagine, serine, and valine and less number of residues with hydrophilic or aromatic side chains  at 28.5°C (pH 7.4) with one disulfide, was shown to have kinetic stability beyond its melting temperature and ability to revert to its native state at 70°C (D. Canadensis with Tm 84°C remained with no activity changes once exposed to a much higher temperature at 100°C (P. americanus with dimeric structure lost its activity due to a complete denaturation of the tertiary and quaternary structure once exposed to room temperature (22°C)  .Nevertheless, since AFPs evolved with different structural integrity and there is a lack of sequence homology among their various types, they are likely to exhibit different characteristics in their physical properties . SimilarSaccharomyces cerevisiae, with an anti-freezing activity enhanced the dough cold tolerance and gassing rate quality which eventually led to the production of high-quality dough texture  are viable for industrial-scale production, and these bio-based materials are sought in the industry from sustainability and environmentally friendly aspects. Although the development of the coating of the AFPs on industrial surfaces is adaptable i.e., dip coating , the temvia microbes.This review discusses various sources of antifreeze proteins, their potential advantages, and their need for industrial applications. We reviewed different approaches to implement these highly active polypeptides to form a futuristic applicable coated material. What makes these macromolecules highly suitable is their biocompatibility, low cost, and scalability, in addition to their environmentally friendly nature, which is aligned with the current global demand. Although coating and immobilization of AFPs on engineering surfaces may be a challenge for adaptation of such technology for energy applications, promising laboratory results are reported in the relevant industry. The effective coating of AFPs using metal-associated polypeptides on various industrial equipment such as refrigerators and wind turbines guarantees future advancement in energy conservation and saving. Among the suggested methods of antifreeze coating, what is most noticeable is based on simple molecular biology techniques to alter and clone the gene encoding for these proteins using microbial sources attached to the metal-binding peptides. The infinite microbial source ensures a convenient, biocompatible, and controllable approach for large-scale production. Biobased materials such as trehalose and chitin that are recommended for the protection of the coated surface could be also generated


The number of deviated screws above G3 was one in the third cervical vertebra, three in the fourth cervical vertebra, one in the fifth cervical vertebra, and one in the sixth cervical vertebra. The number of deviated screws was 6 out of 183, and the deviation rate was 3.2%. Conclusions: In this study, the LLMS deviation rate was 3.2%, and strong fixation was possible without any complications. We measured the screw length and screw deviation rate in cases in which LLMS were actually inserted.Introduction: Lateral mass screws (LMS) have been widely used for the posterior fusion of the cervical spine. Even though LMS are safe, the screws are short and postoperative fixation is uncertain. Therefore, we measured and reported a technique using long lateral mass screws (LLMS), a new method of screw insertion, using a Zed spine from LEXI . Materials and Methods: In this study, we evaluated the outcomes of 35 patients who underwent surgery using LLMS at our hospital from 2019 to 2021. Operative time, blood loss, complications, inserted screw length, screw length based on gender differences, and screw deviation rate were evaluated. The Mann–Whitney U test was used to determine the gender differences in screw length. Screw deviation was evaluated by postoperative CT and a Zed spine to determine the screw insertion angle. Results: The mean operative time was 185 ± 51 min (120–327 min), and the mean blood loss was 236 ± 316 g (10–1720 g). The total number of screws was 183. The screw length was 22.2 (16–28) mm for males and 20.8 (16–28) mm for females, with an average length of 21 ± 2.7 mm. No gender differences were observed in terms of screw length ( Lateral mass screws (LMS) have been widely used for the posterior fusion of the cervical spine. LMS are safe, but the screws are short, and there are concerns about postoperative fixation. Therefore, we measured and reported a technique using long lateral mass screws (LLMS), a new method for inserting screws longer than LMS, using a Zed spine from LEXI . A Zed spine is a 3D preoperative planning software for posterior spinal fusion that uses CT images. The LLMS can be inserted without damaging the vertebral artery by inserting the screw at an angle of 50 degrees cephalad and 30 degrees outward  Figure . This swp > 0.01 NS). Furthermore, the LLMS were able to be inserted without damaging the vertebral artery by inserting the screws at an angle of 50 degrees cephalad and 30 degrees outward. Operative time, blood loss, complications, screw length, and screw deviation rate were evaluated. The Mann–Whitney U test was used to determine any gender-related differences in the screw length. To evaluate screw deviation, we examined the screw insertion angle by means of postoperative CT and the Zed spine. For screw deviation, we classified the screws that could be inserted without any problems as G0, and those that deviated as G1 to G4, with G1 being a slight perforation of the vertebral artery wall, G2 being a perforation that was less than 1/2 of the screw diameter, G3 being a perforation of more than 1/2 of the screw diameter, and G4 as a perforation that was the same size as entire screw diameter . The mean age of the patients was 70 ± 11.3 years (32–88 years), and the sample included 22 males and 13 females. Reported diseases included 23 cases of cervical spinal cord injury, 5 cases of the ossification of the posterior longitudinal ligament of the cervical spine, 4 cases of cervical myelopathy, 1 case of destructive spondyloarthropathy, 1 case of a metastatic spinal tumor, and 1 case of rheumatoid arthritis. Angio-CT scan were not taken before the procedure because the screws are not interested into C1 and C2 level. The insertion point and path of the LLMS were measured from the third cervical vertebrae to the sixth cervical vertebrae using the Zed Spine in 50 patients who underwent surgery or CT for diagnostic purposes. The insertion point of the LLMS was 2 mm cranial and 2 mm medial to the medial margin of the intervertebral joint (Inter laminar V), and screws with a minimum length of 20 mm and a maximum length of 28 mm could be inserted. The difference in screw length from left to right was evaluated using the Wilcoxon test, but there was no significant difference (diameter . Categorp > 0.01 NS). The number of deviated screws above G3 was one in the third cervical vertebra, three in the fourth cervical vertebra, one in the fifth cervical vertebra, and one in the sixth cervical vertebra. A total of 6 out of 183 screws deviated. The screw deviation rate was 3.2%. No complications, such as neurovascular injuries, (The results showed that the mean operative time was 185 ± 51 min (120–327 min), and the mean blood loss was 236 ± 316 g (10–1720 g). There were 183 screws. The screw length was 22.2 mm (16–28) for males and 20.8 mm (16–28) for females, with an average length of 21 ± 2.7 mm. No gender difference in screw length was observed A 69-year-old female patient had obtained a central cervical spinal cord injury due to a fall. A simple cervical CT scan showed the segmental ossification of the cervical posterior longitudinal ligament from the second to the sixth cervical vertebrae, and an MRI scan showed narrowing of the spinal canal and spinal cord compression in the same region . LLMS weThe purpose of internal fixation is to stabilize the spine and correct deformities. In the literature, the first cervical internal fixation was reported in 1891 by Hadra ,3. HadraWe reviewed cases of LLMS. The mean screw length of the LLMS was 21 mm. The screw deviation rate of the LLMS was 3.2%. We were not able to achieve a swing angle of 30 degrees in cases where there was severe deviation. The ideal LLMS insertion angle was fine for the Zed spine measurements.


This work studies ultrasonic propagation in liquid and ice water drops. The effect of porosity on attenuation of ultrasonic waves in the drops is also explored. The motivation of this research was the possible application of ultrasonic techniques to the study of interstellar and cometary ice analogs. These ice analogs, made by vapor deposition onto a cold substrate at 10 K, can display high porosity values up to 40%. We found that the ultrasonic pulse was fully attenuated in such ice, and decided to grow ice samples by freezing a liquid drop. Several experiments were performed using liquid or frozen water drops with and without pores. An ultrasonic pulse was transmitted through each drop and measured. This method served to estimate the ultrasonic velocity of each drop by measuring drop size and time-of-flight of ultrasonic transmission. Propagation of ultrasonic waves in these drops was also simulated numerically using the SimNDT program developed by the authors. After that, the ultrasonic velocity was related with the porosity using a micromechanical model. It was found that a low value of porosity in the ice is sufficient to attenuate the ultrasonic propagation. This explains the observed lack of transmission in porous astrophysical ice analogs. This material originated in the local interstellar cloud and during the solar nebula epoch that evolved toward our current solar system. The ice mantle accretion process in space occurs by condensation of molecules onto the cold dust surfaces. At the lowest temperatures near 10 K, this accretion is expected to form highly porous ice, as occurs in laboratory simulations. This leads to a fluffy cometary ice structure since comets are made by agglomeration of icy dust particles . The por CO2 ice ,4 or up  CO2 ice .This work explores the feasibility to study porosity by means of ultrasonic measurements in astrophysical ice analogs using a pulse–echo technique . After sUltrasonic techniques allow non-destructive and non-intrusive analysis of samples . The ultThe drop experiments and numerical simulations are described below.2 bubbles was used to grow ice drops with porosity. Ultrasonic propagation was monitored without and with porosity in the liquid and ice water drops. A liquid ethanol drop experiment was performed to study the effect of a different liquid composition. The CO2 gas content of the bottle containing sparkling water was estimated as follows. The sparkling water bottle was weighed before opening. After the experiments it was filled with regular water and weighed again. The difference in weight was converted to the number of CO2 moles, which was 2.92 mol, and a density of 1.024 g cm−3 was obtained (2.3% higher than regular water). This value for the density falls in the range of sparkling water with CO2, i.e., a 2–3% increase. We refer to the highly cited work of J.E. García for details on this topic [2 in the sparkling water samples is near 10.2% of the total volume, the average porosity of the drops should be similar to this value.The top surface of a 10 MHz frequency transducer was covered with water. Two series of experiments were carried out. First, the transducer was covered by water  and allowed to freeze, forming compact ice drops. Second, sparkling water containing COis topic . BecauseThe equipment used for the emission, amplification and reception of ultrasonic signals was the AIMS pulser/receiver. A Panametrics DHC713-R  dual element transducer with nominal frequency of 10 MHz, 66% bandwidth with 6 mm diameter was used. The two piezoelectric ceramic elements of this transducer act as emitter and receiver. It was excited with a voltage spike of 200 V. The received signal was amplified by 30 dB and a pulse envelope detector was applied. This transducer was selected taking into consideration the sample dimensions (drop size) and to avoid the interface pulse between the transducer and the drop overlapping with the pulse echo background. A four-channel Handyscope HS6 oscilloscope  was used to digitize the ultrasonic signals with a sample rate of 200 MHz and a 12 bit resolution, see x-axis and the intensity of the signal is provided in the y-axis of the A-scans.The ice is grown inside a freezer for around 20 min. After the drop is frozen, the transducer was connected to the ultrasonic pulser and an oscilloscope to obtain the ultrasonic signals. Amplitude-scans (A-scans) were measured in this way by emitting a single ultrasonic pulse that was typically attenuated as it propagated along the drop, reaching the drop surface and bouncing back to the transducer, thus causing attenuation of the ultrasonic signal that can be measured. This is commonly known as the pulse–echo technique. The propagation time of the signal, known as time of flight (ToF) or pulse–echo time is represented in the In order to better understand the wave propagations in compact and porous water and ice, several simulations were made with in-house SimNDT software ,19. The The inspection setup consists of one transducer (6 mm in diameter) operating in pulse–echo mode and drives a raised cosine signal at 10 MHz, A set of simulations was made consisting of a liquid water or ice drop without and with pores, see The relationship between the porosity and the ultrasonic velocity in ices was also studied using a micromechanical model . The micC) presented in [C11 and C44, in reduced notation, obtained by symbolic notation introduced by Hill [m and i refer to matrix and inclusions, respectively, and ν represents the volume fraction of the phases. In this case, the inclusions are the pores, which can be full of air or carbon dioxide. ijklT are the components of Wu’s tensor [This micromechanical biphasic model considers that the sample material consists of two phases: solid matrix and pores. Assuming that the ice sample is an isotropic material, an equivalent isotropic medium is defined. From the expression of elastic constants tensor  and shear (Vt_eff) velocities as:effρ is the density of the composite and mρ and iρ are the densities of matrix and pores, respectively.The components of the elastic constant’s tensor of the composite (liquid water/ice with bubbles in our case) are related to the effective longitudinal  to freezing temperatures. The height of the drop is assumed to stay constant for its liquid phase (between R.T. and 0 °C). From the received ultrasonic signal, the peak that corresponds to the top of drop surface was identified, see h, as follows:The ToF related to the height of the drop was subtracted from the ultrasonic data by the time difference between the surface of the transducer signal and the first peak that was observed. Generally, the peaks generated by compact ice are steep and easy to spot, making the determination of the ToF for this type of drop straight forward. The ToF was determined from the maximum of the first peak. The deviations of these velocity values with those published in other works for water films are less than 2% for the three temperatures, see In total, 22 out of the 52 drop experiments were performed with compact ice. Not all of them had readable ToF’s and in some of them, visible cracks in the ice made the ultrasonic signal disappear. The useful experiments were used for the statistics reported in The results in 2 in the sparkling water sample corresponds to a gas volume of 10.2% of the total volume (water + CO2), see To make water ice porous under atmospheric conditions, sparkling water was used. The molar mass of COFor this type of experiments, the determination of the drop height is done in the same manner as for the drops with compact ice. The left and right images of From the ultrasonic data, the A-scans were analysed. The reflected signals exhibit peaks that look different compared to the compact ice drops with no bubbles. The intensity of the peaks dropped and the amplitude of the noise and intermediate reflections that originate from the bubbles increased. In the case of the experiments with compact water, the top of the drop surface can be identified as the first peak that appears in these plots, but when comparing the ToF of the first peak in the experiments with bubbles to the one expected from the literature, one could see that the first peak that appears is usually located before the literature ToF value, see (no−bubbles)) is shown as the dark green dashed line in the graph. The effective ultrasonic velocity in porous water or ice is slower causing the peak corresponding to reflection on the top of the drop surface to appear behind the green dashed line. This is why any peak on the left of the green dotted boundary can be considered a consequence of the bubbles that are present in the drop. The signature of the bubbles is pointed at by the red circle in these A-scans. The two lighter green dashed lines are the positions in the graph for which the theoretical literature ToF of a compact drop is 25% and 50% more. Following the analogy applied on the compact ice drops in Equations (5)–(7), the ultrasonic velocity for the drop can be found. This results in VR.T. = 1352.11 m/s, V0 °C = 1347.35 m/s and V−18 °C = 3468.96 m/s for the two liquid water drops with different temperatures and the ice drop. This corresponds to 91.30%, 96.03% and 89.21% of the ultrasonic velocity with respect to water without bubbles at R.T., at 0 °C and to ice at −18 °C, respectively.For Drop Experiment 1, the procedure of defining the top of the water and ice drops is clarified by the graphs in As mentioned above, a second porous ice drop experiment is discussed to highlight differences between A-scans. For the compact ice drop experiments, all experiments showed a similar and predictable behaviour in their measured ultrasonic response, the porous ice drop experiments showed less consistency and higher unpredictability. Similar to the previously discussed experiments, the determination of the drop height can be seen in R.T. = 1358.84 m/s, V0 °C = 1247.47 m/s and V−18 °C = 3667.41 m/s for the two liquid water drops with different temperatures and the ice drop, respectively. This corresponds to 91.75%, 88.91% and 94.31% of the ultrasonic velocity with respect to water without bubbles at R.T., 0 °C and ice at −18 °C, respectively.This indicates that the drop would have grown in the freezer and the height measurement of the liquid drop in The useful experiments performed with porous liquid/ice drops and the statistical outcome are summarized in To benchmark the technique on substances other than water, ethanol was used with a purity of 96%. From From the ultrasonic ToF of plot in a.At 0.10 µs, a longitudinal ultrasonic pulse is transmitted from the transducer and the wave front penetrates into the ice drop.b.At 0.57 µs, the wavefront encounters the curvature of the drop surface after which the longitudinal wave is reflected. These reflections contribute to a horizontal propagation within the drop. The undisturbed part of the longitudinal wave continues to propagate towards the top of the drop surface.c.At 0.75 µs, the first horizontal reflected wavefronts meet in the centre of the drop and start interfering with each other.d.At 0.94 µs, the undisturbed wavefront of the original pulse reaches the surface at the top of the drop. Meanwhile, the horizontal wavefronts from the reflections dominate the reading of the transducer. This is the reason why reflective signals are observed in the experiments before the reflection peak coming from the top of the drop.e.At 1.32 µs, the reflection from the top of the drop is travelling towards the transducer.f.At 1.70 µs, impact of the reflection of the ultrasonic signal from the top of the drop onto the transducer. This impact creates the first large peak in in the measured ultrasonic response.g.At 1.79 µs, after the impact, this strong signal will be reflected back to the top of the drop.h.At 2.45 µs, the reflected signal approaches the surface of the top of the drop again. Once more, disturbing interactions of other pressure waves interact with the transducer.iAt 2.55 µs, after this second impact it can be seen that the reflected signal is significantly attenuated by this impact.j.At 2.83 µs, interference of a curved wave front moving towards a common centre point makes the amplitude locally higher, resulting in a dark-red dot in the simulation frame moving towards the transducer now.k.At 3.21 µs, close to the transducer the wave front is more dispersed, which decreases its intensity.l.At 3.30 µs, the second impact on the transducer of the reflected wave from the top of the drop. This creates a second, smaller peak.The average height for the ice drops from the experiments was 2.96 mm. x-axis of this graph. For comparison, the simulation corresponding to a liquid water drop of the same size is shown in the left panel of this figure. Right after 0 µs, the longitudinal pressure wave sent by the transducer can be seen. As explained above, the first large peak at 1.70 µs can be attributed to the top of the drop surface. The second large peak at 3.30 µs is a reflection of the first large peak that came from the top of the drop. In between and in front of the first peak, acoustic disturbances are present as a result of the drop geometry. It is important to clarify that this simulates a wave front that covers the entire bottom of the drop. In the experiments the receiver is half of the surface of the transducer. This would make the propagation within the drop look more chaotic because the propagation of the longitudinal waves will have more horizontal movements, but the ToF of the large peak would not change significantly.This simulation entails the ultrasonic reading as seen from the transducer as well, which is presented in The ToF values in these simulations agree very well with those measured in the liquid and ice compact water drop experiments presented in a.At 1.1 µs, the wave front is dispersed by pores near the transducer forming numerous peaks in the A-scan.b.At 1.5 µs, the wave front is thus considerably attenuated by the pore interactions on its way to the drop surface.c.At 2.0 µs, arrival at the top of drop surface, the weakened front wave travels back in the direction of the transducer.d.At 2.4 µs, the reflected wave front A is about to merge with two intense wave fronts; on the left, B, and on the right side, C. Wave front C was also reflected from the drop surface, while wave front B was a residual that did not reach the drop surface.e.At 3.1 µs, the coalescence of these waves produces a reinforced new front wave.f.At 3.3 µs, this new front wave interacts with the pores located around the center of the drop.g.At 3.6 µs, the new front wave is heavily dispersed by the pores.h.At 4.0 µs, the first fragment of the dispersed front wave arrives to the transducer.A similar set of numerical simulations were performed for porous liquid and ice drops to understand the influence of porosity in the sparkling water experiments. The simulation creates a cross section of the drop with randomly filled porosity bubbles that have random radii between 0.05–0.15 mm. The drops were filled with bubbles until a certain percentage of surface area was covered by these bubbles. a.At 0.3 µs the wave front is dispersed by pores near the transducer forming numerous peaks in the A-scan.b.At 0.6 µs the wave front is considerably attenuated by the pore interactions on its way to the drop surface. Numerous high-intensity waves arrive continually to the transducer producing peaks in the A-scan. The intensity of the received signal is, however, highly variable.c.At 0.8 µs the wave front arrives at the top of drop surface. The weakened wave front then travels back in the direction of the transducer.d.At 1.0 µs, unlike the liquid drop case, there is no obvious merging of wave fronts and the reflected wave front does not find its way back to the transducer.The simulation corresponding to the equivalent ice drop with 3 mm height and 5% porosity is presented in The plots in When comparing the results of experiments of compact drops with porous drops, it was observed that the average ultrasonic velocity in porous drops is lower than in compact drops. A summary of the results is reported in 2 gas volume in the sparkling water sample used in our experiments. This is compatible with the results presented in This work served to study in detail the propagation of ultrasonic waves in liquid and frozen waters drops, either compact or showing different values of porosity. An estimate of the ultrasonic velocity in various drop experiments could be made, showing the expected decrease in velocity in porous drops. The drop porosity in the experiments was expected to be around 10.2% in most cases, since this was the COThe numerical simulations allowed the study of wave propagation in the liquid/frozen water drops as a function of the time of flight, and for three porosity values of 5%, 10% and 15%. These simulations show that only for the case of 5% porosity in the liquid drop, an attenuated peak was measured corresponding to reflection from the top of the drop surface. Higher porosity values of 10% and 15% only displayed small peaks due to reflections of the bubbles since the wave front in these drops did not reach the top of the drop surface. This explains the lack of peak detection in our experiments with ice films grown from vapor deposition under vacuum, in order to reproduce fluffy cometary ice, with expected porosities up to 40%. We conclude that ultrasonic measurements can probably not be used for the study of such high porosity ices, but they might serve to detect a transition from porous to compact ice, which occurs during ice warmup. This issue deserves more research work. In particular, the hemispherical drop shape in our experiments causes reflection of the wave front to occur mainly along the symmetrical axis of the drop, and therefore pores along this axis lead to strong attenuation. This effect will not occur in flat ice films grown in vacuum chambers by vapor/gas deposition to mimic interstellar ice. Finally, ultrasonic measurements allowed the detection of bubbles in the drop experiments. This finding, in combination with the decrease in ultrasonic velocity measured in porous drops, are a direct consequence of porosity in liquid and frozen drops.


We assess the geographical accessibility of COVID-19 vaccination sites—including mass vaccination centers and community-level provision—in England utilizing open data from NHS England and detailed routing data from HERE Technologies. We aim to uncover inequity in vaccination site accessibility, highlighting small-area inequality hidden by coverage figures released by the NHS. Vaccination site accessibility measures are constructed at a neighborhood level using indicators of journey time by private and public transport. We identify inequity in vaccination-site accessibility at the neighborhood level, driven by region of residence, mode of transport , rural-urban geography and the availability of GP-led services. We find little evidence that accessibility to COVID-19 vaccination sites is related to underlying area-based deprivation. We highlight the importance of GP-led provision in maintaining access to vaccination services at a local level and reflect on this in the context of phase 3 of the COVID-19 vaccination programme (booster jabs) and other mass vaccination programmes.  The UK became the first country to approve a COVID-19 vaccine, kick starting a nationwide programme of immunisation beginning on 8th December 2020 . This neHealth service providers such as the NHS continually make strategic decisions regarding the balance between service efficiency, the allocation of scarce resources and accessibility of facilities. With finite resources available, it is impossible to provide ubiquitous coverage of vaccination sites or any other health service which is delivered from fixed locations or which benefits from economies of scale. In a primary health care setting, facility accessibility has been defined as ‘‘the ease with which the residents in a given area can reach a particular service or facility”  (p. 70).In seeking to vaccinate large population sub-groups—some of which are elderly, extremely vulnerable or suffer limited mobility or financial resources-in a very short period, the geographical accessibility of vaccination sites is important in the efficient administration of COVID-19 vaccinations. Depending on the vaccine used, most recipients initially required two doses administered 8 to 12 weeks apart, with recipients invited to book their vaccination appointments via an online and telephone ‘National Booking Service’ (NBS), enabling them to choose a preferred vaccination site and appointment dates/times based on local availability. Some patients were also contacted directly by their registered General Practitioner (GP) with the option to receive their vaccination at a local GP surgery (which may not be their regular surgery) if their surgery was part of a primary care network-led vaccination service. Under this scheme, vaccines were administered at a single GP surgery on behalf of a network of local GPs, hereafter termed ‘GP-led’. Vaccinations were also administered at hospitals (predominantly targeting in-patients and staff), residential care settings, pharmacies, community medical facilities and also via mass vaccination centers specifically set up for this programme, some capable of vaccinating thousands of people each day. In this paper, a ‘vaccination site’ refers to any location administering COVID-19 vaccines, whilst a ‘vaccination center’ is a specific category of vaccination site offering vaccinations at scale—typically thousands per day. Whilst NHS England claimed that 99% of the population lived within 10 miles of a vaccination site at the peak of the vaccination programme in July 2021 , the medWhilst there are many studies of vaccination uptake for COVID-19  and for other routine vaccination programmes, there are a deficit of studies on vaccination site accessibility. In an isolated international example, Guhlincozzi and Lotfata  considerEven routine forms of vaccination—such as the annual winter flu programme, or vaccinations predominantly offered to travelers—are notably absent from studies of health service accessibility, with the only known example related to accessibility of Yellow Fever vaccination sites . StudiesThe following sections draw on the unique context of the COVID-19 vaccination rollout in England to assess—at the neighborhood level—the accessibility of COVID-19 vaccination sites. Applied research across a variety of domains and international contexts have given rise to a number of approaches to capture geographical accessibility, which can be calculated at the level of individual facilities (‘how accessible is this vaccination site?’) or small geographic areas (‘how accessible are vaccination sites to the households in this neighborhood?’). Approaches fit into two broad categories- ‘distance-based’ and ‘container-based’ . DistancBy contrast, container-based methods calculate supply to demand ratios within a geographic ‘container’ . In spite of the ease with which these metrics can be interpreted and compared (between areas or over time), container approaches have faced criticism due to their lack of consideration of distance and the inability of these methods to simulate flows  outside of the administrative or statistical zone within which they reside . Whilst The analysis undertaken in this paper considers only England owing to differences in both data availability and vaccination strategy between devolved nations of the UK. Data capturing the supply side (vaccination site locations) were obtained from NHS England. Demand side data were drawn from the 2011 Census of Population and Housing in England and Wales alongside corresponding geographic data for neighborhoods, considered in turn in the following sub-sections. Vaccination site data were supplied by NHS England. They were first published on 11 January 2021, with NHS England claiming that they would be updated weekly. Whilst weekly updates were not actually published, the data were regularly amended through to 26 July 2021 and accessed via a Freedom of Information request. With each update, NHS England revised their ‘coverage’ figure-the proportion of adults living within ten miles (16.1 km) of a COVID-19 vaccination site. Although the format of the vaccination site data did change slightly over time, each dataset included the name, address and postcode of each vaccination site. The data released on 22 January 2021, and each subsequent iteration also split the vaccination sites into categories capturing the site type, including hospital hubs, GP-led vaccination services, pharmacies and vaccination centers. In Most of the analysis presented in this paper uses the list of vaccination sites published on 6 July 2021. At that time—during the peak of phase 2 of the vaccination programme-2091 vaccination sites were listed . Five reIn England, as each age or risk-based cohort became eligible for the vaccine they were invited to book an appointment via the online ‘National Booking Service’ (NBS) or via a direct invitation from their registered GP. Although not reported fully here due to space constraints, the list of vaccination sites within the NHS Data was interrogated against the list of available vaccination sites presented to an individual when booking their vaccination. This revealed that hospital hubs should not be included as they gave priority for vaccinating hospital staff and in-patients and were generally not available for public appointments. All 274 hospital hubs were removed from the analysis. As a result, analysis was undertaken using 191 vaccination centers, 691 pharmacies and 911 GP-led vaccination sites within the supply side dataset. It also revealed that many  GP-led services were only available to patients registered at that practice. The analysis was also repeated without inclusion of the 911 GP-led services (as many of these are only available to registered patients), highlighting the impact of the eligibility of GP-led services on overall vaccination site accessibility. Households (which contain populations eligible for COVID-19 vaccination) are nested within geographical zones (which we term neighborhoods) which can be attached to a series of neighborhood indicators of population size and composition, car ownership, degree of rurality and measures of area-deprivation. We use Lower Layer Super Output Areas (LSOAs) to represent neighborhoods. LSOAs are an area-based statistical and administrative geography designed to be as consistent as possible in terms of population size and composition, containing between 400 and 1200 households [Whilst LSOAs represent geographic zones , drawn from the 2011 Census of Population and Housing in England and Wales. At the time of analysis, these were the age groups able to book a vaccination appointment via the NBS. Attribute information capturing an area-based classification of urbanity or rurality—the Office for National Statistics Rural Urban Classification for Small Area Geographies  were appThe journey time data used for these analysis see  enable rOnce supply and demand side data had been prepared, the bulk of the analysis was based on modelled journey times between neighborhoods (using PWCs) and vaccination sites. The analysis presented here considers the most accessible five vaccination sites to each LSOA. This recognizes that many people will not have received their vaccination at their closest site due to appointment availability, opening hours, personal convenience and a range of other individual and locally specific factors. In particular, many individuals may have been willing to travel further than their most accessible site in order to attend a mass vaccination center . This also reflects that many GP-led services were running on different timescales to the NBS , meaning that many individuals would not have accessed their vaccination at their most accessible site if that was a GP-led service. Travel times and travel distances were calculated for the interaction between each LSOA PWC and its most accessible five vaccination sites using HERE Technologies’ Routing and Public Transit APIs (both version 8). HERE is an established spatial intelligence provider who provide location data, including those data used within this analysis, under license to a number of major end-users which include Garmin, Facebook and Amazon . By combRecords (origin-destination pairs) for which no journey time was returned were checked manually. For car transport these affected only 10 routes  and were attributed to a single island for which travel time to the mainland could not be calculated. A greater number of routes (3877) could not be calculated within the Public Transit API . In many cases this was because the vaccination site was too close to the origin for public transport to be a viable option, and in these cases walking time (taken from the pedestrian transport mode in the HERE Routing API) was used instead. Where routes were multi-modal  these were summed to report a single total journey time value. The following section presents outputs from the journey time analysis, linking neighborhood-level vaccination site accessibility to the underlying demand side data highlighted in The number of sites providing COVID-19 vaccinations increased considerably during the phase 1 and phase 2 vaccination programme to July 2021 , with thNHS analysis summarized in Travel times for each neighborhood were calculated as the mean journey time from the LSOA population-weighted centroid to its closest five sites, to reflect that residents may not have booked into their closest site due to personal choice, lack of appointments or ineligibility (the limitations of GP-led services were discussed above). For those households with access to private transport, the median value of these travel times is just over 10 min. This suggests that the average drive time between any neighborhood (LSOA) in England and its five most accessible vaccination sites is approximately 10 min. The 75th percentile is just 13 min and 45 s and the maximum average journey time for all LSOAs is just under 58 min. All households with access to private transport therefore have at least one vaccination site that can be reached within less than an hours’ drive, whilst most have considerable choice of sites within this threshold, representing excellent access to vaccination sites.As summarised in Whilst private transport affords ‘better’ access to vaccination sites (quicker journey to nearest site and greater choice of sites available within threshold distances), there remain inequalities between neighborhoods which hold true irrespective of the mode of transport used. These can be illustrated at a variety of spatial scales and are considered here by region. Inter and intra regional inequalities revealed by To account for some of these vagaries around GP-led provision we undertook analysis, which simulated the removal of these services, in order to illustrate the important role that GP sites play in the provision of an accessible vaccination service. Our analysis reveals that once GP-led services are removed, the average adult faces a journey time that increases by 7 min, with increases of up to 15 min in some of the most remote rural areas. Removal of GP-led services reveals 2463 LSOAs from which the closest site was over an hours’ journey by public transport. Census data reveal that—within those neighborhoods—a total of almost 380,000 adults live in a household without access to a car. This echoes concerns raised at the beginning of the vaccination programme [Availability of private transport, degree of rurality and access to GP practices are also indicators used (among many others) to capture area-based measures of deprivation. Whilst literature and wider policy (including established measures of area deprivation ) recogniThe COVID-19 vaccination programme in England aimed to vaccinate as many of the eligible population as possible in a short period of time, bringing notions of accessibility and coverage to the fore on the premise that increasing the number of vaccination sites should help improve geographic accessibility and reduce access-driven inequalities. As highlighted by our analysis, the average drive time between any neighborhood (LSOA) in England and its five most accessible vaccination sites is approximately 10 min and the maximum average journey time for all LSOAs is just under 58 min. This suggests that overall, geographical accessibility of the vaccination site network is excellent and compares very favourably with other forms of primary, secondary and emergency care see for example ,27,28. In spite of NHS-published coverage figures which implied that almost all households lived within 10 miles of a vaccination site, our findings support media coverage, e.g., see  reportinCapturing travel time to the five most accessible sites allows vaccine recipients to exercise some personal choice over which vaccination site they visit  and highlights that lack of appointment availability at the most accessible site did not typically preclude accessibility. However, removal of GP-led services—which were typically available only to patients registered at that specific practice—does increase journey times, especially for those recipients who are reliant on public transport. This highlights that GP-led services are a very important component of the vaccination site network, especially in rural localities which may already be more remote from mass vaccination centers or other forms of community provision . During Phase 2 of the vaccination programme, there was considerable ambiguity around the availability of vaccinations from some GP-led sites, in part due to a lack of coordination between GP-led services and the NBS. This resulted in duplicated invites for many patients, with the faster paced roll out of invites via the NBS resulting in many patients travelling further than required to obtain vaccines via mass vaccination centers when they could have waited for an invitation for a vaccination appointment at their usual GP surgery . Whilst mass vaccination centres may address concerns regarding capacity (ability to vaccinate thousands per day), their centralized locations do not necessarily promote equity in accessibility. In spite of the points raised above, Haynes  reportedAlthough not directly related to accessibility, one of the key recommendations from this study is for a comprehensive and all-encompassing national booking system for future mass-vaccination programmes so that recipients have the opportunity to book an appointment at their most accessible sites, irrespective of the mode of delivery. Given the important role of GP surgeries in providing accessible vaccination sites in many localities, it is inevitable that the challenges and delays in being able to book vaccinations at these sites  resulted in vaccine recipients travelling further than required to receive their vaccination(s). In recommendations to the charity sector, the International Longevity Centre (ILC) suggest that vaccination uptake among some under-represented groups could be addressed via improvements to vaccination-site accessibility, yet their only practical recommendations for doing so involve better use of taxis or lift-sharing services and better signposting of vaccination opportunities . This paIn September 2021, phase 3 of the vaccination programme began in England, enabling individuals who had received their second dose COVID-19 vaccination at least 6 months beforehand to obtain a booster dose, again administered via GP-led services alongside vaccination centers and other community provision, including pharmacies. As with phase 1 and 2 of the vaccination programme, invites are sent by GPs alongside the NBS, and availability is also provided via a series of walk-in sites (for which no appointment is required). Whilst this range of provision may provide a number of opportunities for an eligible individual to obtain their booster vaccination, it may again result in individuals travelling further than required if there is a time lag in sending out invitations for local GP-led services. In common with phases 1 and 2 of the vaccination programme, GPs were not invited to administer COVID-19 booster jabs at the GP practice level, with clusters of GP practices (within primary care networks) favored given the economies of scale that can be achieved at these larger sites. The NHS cited considerable operational and logistical challenges associated with vaccination administration at a GP practice level (summarized in ). O’DowdCOVID-19 has highlighted the importance of effective vaccination programmes in promoting public health. This study, undertaken at the peak of phase 2 of the COVID-19 vaccination programme in England (July 2021), evaluates geographical accessibility of COVID-19 vaccination sites and highlights the importance of private transport and area type  in driving inequalities in vaccine site accessibility at a neighborhood level. We highlight the important role GP surgeries play in maintaining ‘good’ access to proximate vaccination sites, especially in remote rural localities and for those most-reliant on public transport. Whilst overall accessibility of vaccination sites is considered excellent, challenges in accessing these services among some population subgroups should not be overlooked in ongoing and future vaccination campaigns. We note that the decision not to offer phase 3 of the vaccination programme at an individual GP practice level misses a tremendous opportunity to improve access to these services. We also highlight the benefits of utilizing appropriate measures to capture key accessibility metrics and note deficiencies in NHS England’s  use of cTimely analysis of these nature is only possible due to the availability of publicly available data on vaccination site provision, alongside user-friendly and robust tools through which accessibility (in this case journey time) can be measured. Arribas-Bel et al.  highligh