The current trend in dietary supplements and functional foods is the use of lipophilic bioactive compounds. The sea buckthorn (Hippóphae rhamnoídes) contains some such compounds: polyunsaturated fatty acids, tocopherols, and carotenoids. Lipophilic components are best distributed using oil-in-water emulsions, which ensures their high bioavailability. A significant property of emulsions is colloidal and oxidative stability, so the choice of emulsifiers that have both surface-active properties and antioxidant activity is an important area of research for making new types of food emulsions. The purpose of this study is the development and refinement of an emulsified biologically active food additive containing sea buckthorn products  and stabilized with soy phospholipids. We studied the fruits of Chuyskaya, Orange, and Prevoskhodnaya sea buckthorn varieties growing in the Altai Territory. As we analyzed their composition, we chose the Chuyskaya variety for making the emulsion. The fruits contain 5.30 ± 0.1% of lipids including 16.8 ± 0.5 mg/100 g of carotenoids and 10.5 ± 0.5 mg/100 g of tocopherols. To choose the emulsifier we studied the fractional and fatty acid composition of the soy and sunflower phospholipids with different hydrophilic-lipophilic balances (HLB). We made the emulsions containing sea buckthorn oil and pulp of its different layers, soybean oil, and phospholipids by dispersion using an HG-15D homogenizer. The study of the colloidal stability showed that the most stable (99.5%) are the emulsions containing a mixture of hydrolyzed soybean phospholipids (HLB = 7) and fractionated soybean phospholipids (HLB = 3). The best ratio is 40:60. We examined the oxidative stability of the emulsions by provoking accelerated oxidation. The emulsions containing 1.5% of a soy phospholipids mixture showed the best oxidative stability. The resulting direct oil-in-water fine emulsion contains polyunsaturated fatty acids (PUFAs), tocopherols, β-carotene, and essential phospholipids. For this reason, the emulsion can be used to make biologically active food supplements  and as part of special nutrients. It has been shown that nutrition directly affects the risk of lipid and carbohydrate metabolism disorders changing glucose and cholesterol levels, blood pressure, and body weight ,2.In terms of lipid metabolism disorder prevention, lipophilic biologically active substances including polyunsaturated fatty acids (PUFAs), phospholipids, fat-soluble vitamins, and antioxidant provitamins are of particular interest ,4,5,6,7.The sea buckthorn (Hippóphae rhamnoídes) is an organic product containing multiple lipophilic components ,9,10,11.Carotenoids and tocopherols can be produced from organic sources, including sea buckthorn fruits, to be used as nutraceuticals. There are several issues with their applications in foods and beverages since they are virtually insoluble in water. It usually means they should be dissolved in oils or dispersed in other suitable matrices before use in food ,16,17.Lipophilic components are best distributed as ultrafine oil-in-water emulsions with high bioavailability and biological efficacy compared to classical emulsions, where, due to the large size of the drops, it is difficult to protect the emulsion during the delivery ,21,22,23The choice of the emulsifier is a major challenge in making food and pharmaceutical emulsions. It depends on the type of emulsion ,26,27,28Currently, organic surfactants are preferred. They are lactylated esters, glycerol and fatty acid esters, incomplete glycerides and derivatives, sucrose esters, polyoxyethylene sorbitan and fatty acid esters, and phospholipids (lecithin and lecithin derivatives) ,33.Phospholipids are amphiphilic molecules with a polar phosphate-based hydrophilic head and two non-polar hydrophobic fatty acid tails bound by a glycerol molecule. Both animal and plant phospholipids can contain many fractions depending on the substituent bound to the phosphorus acid group. The key phospholipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and phosphatidylglycerol. The most valuable fraction is phosphatidylcholine. Its molecules have both non-polar (two fatty acid groups) and polar (phosphorylcholine) parts . It is tThe development of emulsified bioactive products and dietary supplements containing lipophilic ingredients requires more basic and applied research. Researchers and manufacturers should focus on the search for new, unconventional sources of target substances virtually not found in traditional oils. The production of lipophilic components from such sources is low-volume and requires a specific technology to process the raw material and its biologically active components .The purpose of this study is the development and refinement of an emulsified biologically active food additive containing sea buckthorn products  and stabilized with soy phospholipids. The study is structured as follows: First, an analysis of the composition and properties of the sea buckthorn fruits of different varieties cultivated in the Altai Territory is carried out to provide the rationale for selecting the sources of biologically active components; the composition and properties of the phospholipids obtained from different sources are studied; and finally, the effects of the added components on the final emulsion properties are also studied.There are many studies on the composition and properties of sea buckthorn varieties ,13,14,43Our study will produce knowledge about obtaining biologically active components from organic sources and ways of adding them to dietary supplements and food products. The experimental results can be used in pharmaceutics and food production to make dietary supplements and health-boosting foods.We studied the fruits of the Chuyskaya, Orange, and Prevoskhodnaya sea buckthorn varieties grown in the Altai Territory (2021 harvest) and purchased them at the local market. We also studied phospholipids purchased from Juvix-Pharm . The manufacturer’s specifications listed the hydrophilic-lipophilic balance (HLB) of the emulsifiers. The following samples were selected: hydrolyzed soybean phospholipids (HLB 7); hydrolyzed sunflower phospholipids (HLB 8); fractionated soybean phospholipids (HLB 3); and fractionated sunflower phospholipids (HLB 2). The HLB for the simulated complex emulsifiers was determined by summing up the HLB values of the individual emulsifiers multiplied by the mass fraction of the emulsifier in the mixture.All the laboratory kits and reagents used in the experimental research were purchased from Delta Origin .In the first stage, we studied the composition and properties of the Chuyskaya, Orange, and Prevoskhodnaya varieties of sea buckthorn fruits grown in the Altai Territory. We studied the chemical composition of the juice, seeds, seed oil, and sea buckthorn pulp. We measured the moisture content, proteins, lipids, total sugar, vitamins, ash, acidity, and the physical and chemical parameters of the oil.We used an OM-350M-02F laboratory chopper (homogenizer) for grinding the sea buckthorn fruits (and to separate the seeds from pulp and shells). The unit consists of a drive; a mashing assembly; a paddle + a 3 mm mashing disk. Its rated mashing performance is 400 kg/h, the cell diameter is 3 mm, and the rpm is 300 ± 10.−The bottom layer is a clarified juice (65%)−The middle layer is the middle layer pulp (30%)−The top layer contains the greatest amount of lipids (5%).The unit fine grinds the shell and pulp while the seeds do not pass through the gap and remain intact. The resulting crushed sea buckthorn pulp is separated into three layers when stored for 6 h:To analyze the chemical composition of the layers the separated pulp was frozen at −18 °C and then cut with a knife.The mass fractions of the dry substances, reducing sugars, and moisture in the pulp layers was determined by near-IR spectroscopy with a MATRIX IR spectrometer from Bruker Optics .We determined the content of macro- and microelements in the sea buckthorn fruits  by atomic emission analysis using a Plasma 1000 instrument from Perkin Elmer . The instrument is equipped with an automatic sampler and is computer controlled.2 extraction on a pilot extraction unit with a five-liter stainless steel vessel. The extraction conditions were 28 °C, 7 MPa, and 120 min, respectively; high-purity (99.999%) carbon dioxide was used as a solvent. We decided to apply this extraction method since it gently treats the thermolabile compounds in sea buckthorn.We made sea buckthorn oil from the dried and crushed pulp and seeds by subcritical COWe measured the density of the sea buckthorn oil samples obtained from the pulp and seeds with an areometer. The instrument was dipped into the oil and the reading was recorded.The iodine number of the sea buckthorn oil was measured with the Wijs method. It uses a solution of iodine monochloride in glacial acetic acid as a halogen reagent.The acid number of the sea buckthorn oil which indicates the content of free fatty acids was determined by dissolving lipids in an ether-alcohol mixture (2:1), followed by rapid titration of the sample with a sodium hydroxide solution in the presence of a phenolphthalein pH indicator until a weak pink coloration was achieved.The peroxide number of the sea buckthorn oil was measured by analyzing the reaction of the lipid oxidation products (peroxides and hydroperoxides) with potassium iodide in acetic acid and chloroform, followed by assaying of sodium thiosulfate.The composition of the fatty acids and phospholipids in the lipid phase of the samples was studied by gas-liquid chromatography. A chromatogram of the fatty acid methyl esters of the sample’s lipid phase is made followed by the identification and assaying of the components by peak area. Each component of the fatty acid methyl ester mixture is characterized by a specific retention time (from the sample introduction to the peak on the chromatogram). An Agilent 7890A gas chromatograph was used. The Polychrome software processed the results.The total content and fractional composition of tocopherols in the sea buckthorn oil, vitamin content, and the content of carotenoids and flavonoids were identified by IR spectrometry with a MATRIX IR spectrometer from Bruker Optics, Bruker .Direct emulsions (oil in water) (100 g each) were made in a lab environment. The aqueous phase consisted of sea buckthorn juice and middle layer pulp (53%). The fat phase consisted of sea buckthorn oils derived from the pulp and the seeds mixed with soybean oil and phospholipids (47%). The emulsions were prepared in stages. The aqueous phase was made from the clarified juice and middle layer pulp by magnetic stirring and pasteurization at 60 °C for 30 min. The pasteurization temperature was chosen to account for the high thermolability of carotenoids and ascorbic acid contained in the sea buckthorn pulp. Concurrently, the emulsifier was prepared for introduction into the fat phase. The soybean phospholipids were dissolved in refined deodorized soybean oil and heated to the phospholipid granule’s complete melting temperature (65 °C). Then, the aqueous phase was cooled to 20 °C to make a coarse emulsion by gradual addition of the sea buckthorn seed oil, pulp oil, and emulsifier dissolved in soybean oil while stirring with a lab stirrer. Then, the emulsion was homogenized by an HG-15D homogenizer  at 15,000 rpm. Three emulsions were prepared with various phospholipid content . The mass fraction of phospholipids was controlled by reducing the sea buckthorn oil amount. The final emulsions were stored in a refrigerator at 6 ± 0.5 °C for 24 h before other studies.V is the volume of intact emulsion, cm3; 10 is the sample volume, cm3.To determine the colloidal and thermal stability of the emulsions, we filled lab tubes with the emulsion samples to the top mark and centrifuged the tubes for 5 min at 1500 rpm. Then, the tubes were placed in a boiling water container. The water level was equal to the level of the samples in the tubes. The tubes were kept there for 3 min while preventing the water from intense boiling and then centrifuged again for 5 min at 1500 rpm. Then, the volume of the intact emulsion was measured by the scale on the tube with a separated aqueous or fat phase. The emulsion stability as a percentage of the intact emulsion by volume was estimated asThe oxidative and hydrolytic stability of the emulsions under accelerated oxidation was studied by estimating the peroxide and acid numbers of the lipid phase separated from the emulsion. The emulsion samples were stored for 14 days in a transparent glass container with free access to oxygen and light and at 25 °C. Samples were taken every day.−Sample 1: Lipid phase of emulsion before emulsification (reference);−Sample 2: Lipid phase of the emulsion containing 1.0% of phospholipids;−Sample 3: Lipid phase of the emulsion containing 1.5% of phospholipids;−Sample 4: Lipid phase of the emulsion containing 2.0% of phospholipids.The lipid phase of the emulsion not mixed with the aqueous phase and not containing phospholipids was the reference sample. The following samples were prepared:3 conical flask with a ground joint, and 15 cm3 of ethyl alcohol was gradually added to the sample and stirred with a glass rod until a suspension was obtained. Then, 30 cm3 of chloroform was added to the flask while stirring. An HCN-300-29/32 glass spiral refrigerator (made in Russia) was used to cool it down. The mixture was then boiled in a water bath for 1 h while stirring in a gentle circular motion. After, the sample was cooled to room temperature. Then, the sample was filtered through a glass funnel with a filter pre-moistened with the extraction mixture. Solvents were removed from the extract first by evaporation in a water bath, then in a desiccator for 30 min at (105 ± 5) °C. The crucible with the fat phase was cooled at room temperature for (20 ± 5) min and weighed to measure the mass fraction of fat.After the storage and accelerated oxidation, the fat phase was extracted as follows: a 5 g emulsion sample was placed in a 250 cmPeroxidation of the emulsion lipid phase was estimated using the reaction of lipid oxidation products (peroxides and hydroperoxides) with potassium iodide in acetic acid and chloroform, followed by assaying the sodium thiosulfate.The acid number of the emulsion lipid phase was estimated by dissolving the lipids in an ether-alcohol mixture (2:1), followed by rapid titration of the sample with sodium hydroxide solution in the presence of a phenolphthalein indicator until a weak pink color was reached.p ≤ 0.05).All the studies were repeated three times. The results were processed with the standard mathematical statistics methods and the Statistica 10.0 software . Variance in the phytochemicals content was analyzed by the use of ANOVA with Tukey’s test for homogenous groups. The differences between the mean values were considered significant when the confidence interval was less than 5% . The analysis identified the Chuyskaya variety as the one with the highest content of dry matter (14.5 ± 0.2%) and lipids (5.30 ± 0.1%), including carotenoids (16.8 ± 0.5 mg/100 g) and tocopherols (10.5 ± 0.5 mg/100 g). Furthermore, out of the studied varieties, Chuyskaya has a higher content of ascorbic acid and polyphenolic substances. The differences between the varieties in the content of the target substances cannot be called significant. However, the Chuiskaya variety still demonstrated a slight superiority in all respects. For further research, this particular variety was chosen. In addition, we were guided by the greatest availability and prevalence of this variety of sea buckthorn in the region.The study detected significant differences in the chemical composition of the Chuyskaya, Orange, and Prevoskhodnaya sea buckthorn (Hippóphae rhamnoídes) varieties grown in the Altai Territory. In general, the result agrees with the data obtained by Olas, B. (2016) ,11 TelesAt the next stage, we investigated the products obtained by crushing the sea buckthorn fruits of the Chuiskaya variety.After removing the seeds, the sea buckthorn pulp was poured into PET bottles for storage. After 6 h, the pulp formed three layers: clarified juice, bottom pulp, and top pulp. 3.The chemical composition of sea buckthorn seeds was also investigated. The mass fraction of moisture was 11.0 ± 1.0%, nitrogenous substances: 8.0 ± 0.5%, oil: 10.0 ± 1.0%, ash: 2.0 ± 0.2%, fiber: 20.0 ± 2.0%.The lipid fraction isolated from the pulp contained 320 mg of carotenoids per 100 g and 90 mg of tocopherols per 100 g. The carotenoids composition study identified α, β, γ-carotenoids, lycopene, poly-cis-lycopene-3, and zeaxatin. The content of the most physiologically active β-carotene in the fruits was 45% of the total amount of carotenoids. Buckthorn tocopherols are overwhelmingly represented by the most physiologically active α-tocopherols . γ- and δ-tocopherols were also found.The chemical composition of the seed oil differs significantly from the oil obtained from the pulp in terms of fatty acid composition and the content of the key lipophilic components. For instance, 11.1 ± 0.05 mg/100 g of carotenoids and 160.0 ± 0.1 mg/100 g of tocopherols were found in the oil obtained from the seeds by subcritical extraction. These results agree with the data provided by Li Zheng et al. (2017), who investigated the composition of sea buckthorn oil obtained by subcritical extraction. They found that the seed oil had a carotenoid content of 15.1 ± 0.1 mg/100 g, the oil obtained from the pulp had 104.6 ± 2.0 mg/100 g, while the tocopherol content was 245 ± 1 mg/100 g for the seed oil and 198 ± 1 mg/100 g for the pulp oil [To make a food additive as an emulsion, we used sea buckthorn pulp, sea buckthorn juice, sea buckthorn oil obtained from the pulp as a source of carotenoids, and seed oil as a source of tocopherols. We also used surfactants with relatively low hydrophilic-lipophilic balance suitable for stabilizing oil-water emulsions.A significant property of emulsions is colloid and oxidative stability and the ability to preserve and deliver biologically active components. The development of emulsifiers that have both surfactant properties and biological efficacy is an important area of research for making new fine, biologically active emulsions.To select the most suitable surfactant with the required processing properties and biological activity, we investigated some samples of soybean phospholipids (SBP) and sunflower phospholipids (SFP) with different hydrophilic-lipophilic balances, widely used in food and pharmaceutical emulsion production.The fractional composition of phospholipids was investigated .As reported in the available sources, the surface activity of lecithin is due to the presence of a phosphatidylcholine fraction. Its molecule has non-polar (two fatty acid groups) and polar (phosphorylcholine) parts ,34. We fNext, the fatty acid composition of soybean and sunflower phospholipids was examined .The study of the fractional and fatty acid composition of soy and sunflower phospholipids confirms the data from such researchers as Caysen A. et al., (2022) .It is feasible to add soy phospholipids to the emulsion thanks to the presence of 1,2-dilinoleoyl-phosphatidylcholine, which is not synthesized by the human body. The advantage of this special phospholipid form compared to endogenous ones is the presence of two essential polyunsaturated fatty acids as glyceride compositions. It facilitates easy incorporation into hepatocytes when consumed, and the restoration of the protective cell wall phospholipid layer protective properties ,39,40.Two samples of soy phospholipids were selected for further studies: hydrolyzed soy phospholipids (HLB 7) and fractionated soy phospholipids (HLB 3).The reference oil-in-water emulsions containing sea buckthorn pulp, oil pulp, and sea buckthorn seed oil mixed with soybean oil and phospholipids were made in a laboratory environment. First, a coarse emulsion was obtained with a lab stirrer; then the emulsion was homogenized with an HG-15D lab homogenizer. Two types of soybean phospholipids in different amounts as well as a mixture of phospholipids with different HLB values served as surfactants. The colloidal stability of the resulting emulsions was estimated .The study showed that the emulsions made with a mixture of hydrolyzed soybean phospholipids (HLB = 7.40%) and fractionated soybean phospholipids (HLB = 3.60%) have the best colloidal stability. The hydrophilic-lipophilic balance of the resulting complex emulsifier is 4.6, an optimal value for stabilizing oil-in-water emulsions.The presence of the aqueous phase plus polyunsaturated fatty acids of the fat phase can provoke oxidative and hydrolytic processes during the emulsion storage, resulting in the breakdown of the fatty acids, oxidation of the triglycerides and isomerization of the original acids. Accumulation of such oxidation products as peroxides, hydroperoxides, aldehydes, ketones, and oxy compounds can contribute to the deterioration of organoleptic and rheological properties and reduce its physiological and biological value. For this reason, we studied the oxidative and hydrolytic stability of the emulsions and the effects of the phospholipid content on these properties. 2/kg. The peroxide number of the emulsion’s fat phase with 1% phospholipids added increased from 1.5 to 13.1 mEqO2/kg in two weeks of storage under accelerated oxidation conditions. The samples containing 1.5% and 2% of phospholipids showed the highest oxidative stability: their peroxide number increased from 1.5 to 9.2 mEqO2/kg. A study of the acid number (AV) variation showed that the lowest acid number at the end of the accelerated oxidation period was also found in the samples containing 1.5% and 2% of phospholipids. Since increasing the mass fraction of phospholipids in the emulsions had no significant effect on the oxidative and hydrolytic deterioration change rate, we concluded that increasing the mass fraction of phospholipids in emulsions to 2% is inexpedient.The graphs show that the peroxide number (PV) of the emulsion lipid phase without phospholipids increased from 1.5 to 15.9 mEqOThe results also confirmed that soy phospholipids have antioxidant properties and can slow down the oxidation process in polyunsaturated fatty acids, which predominate in the fat phase of the emulsion. The available research literature presents studies on the combined effect of tocopherols and phospholipids on the oxidative stability in lipids. The data we obtained do not contradict the results presented by such researchers as McClements DJ and Decker E. (2018) , Xu N etWe investigated the quality factors of the emulsions obtained in a laboratory environment. The results are presented in The oil-in-water emulsion we made contains polyunsaturated fatty acids (PUFAs), tocopherols, carotenoids, and essential phospholipids. With such content, the emulsion is suitable for the production of biologically active food additives, also encapsulated, and health-boosting foods.As a result of this study, a food emulsion containing pulp, juice, pulp oil, and oil of sea buckthorn seeds stabilized with soy phospholipids was obtained. After analyzing the chemical composition of the fruits of Chuyskaya, Orange and Prevoskhodnaya sea buckthorn varieties grown in the Altai Territory, the Chuyskaya variety was selected for making the emulsion. The variety of fruits contains 5.30 ± 0.1% of lipids, including 16.8 ± 0.5 mg/100 g of carotenoids and 10.5 ± 0.5 mg/100 g of tocopherols. To choose the emulsifier, we studied the fractional and fatty acid composition of the soy and sunflower phospholipids with different hydrophilic-lipophilic balances (HLB). This study has some restrictions since the design and methods were chosen to match the specific chemical composition of certain species and varieties. It is assumed that the content of bioactive compounds in sea buckthorn samples of other varieties, or Chuyskaya, Orange, and Prevoskhodnaya varieties harvested at a different time and location, and phospholipids obtained from other sources, may have a significantly different chemical composition which requires making changes to the application of such components in foods and food supplements. The study of emulsion colloidal stability showed that the most stable (99.5%) emulsions contain a mixture of hydrolyzed soybean phospholipids (HLB = 7) and fractionated soybean phospholipids (HLB = 3) in the 40:60 ratio. By analyzing the fractional and fatty-acid composition of phospholipids, we chose the most suitable sample of soy phospholipids containing 1,2-dilinoleoyl-phosphatidylcholine not synthesized by the human body to be added to the emulsion. The studies of the emulsion oxidative stability with various phospholipid contents also confirmed that the highest antioxidant activity of the substance is achieved at 1.5% wt. concentration. The study showed that a further increase in the phospholipid content to 2% does not affect the oxidative and hydrolytic stability of the emulsions.The resulting direct oil-in-water fine emulsion contains polyunsaturated fatty acids (PUFAs), tocopherols, β-carotene, and essential phospholipids. For this reason, the emulsion can be used to make biologically active food supplements  and as part of special nutrients.The study results will be used in further research to develop efficient methods of adding plant lipophilic components to food systems for making food, beverages, and pharmaceutical products.The use of processed sea buckthorn and plant phospholipids for making therapeutic, health-boosting foods and dietary supplements will increase the nutritional and therapeutic properties. Regular taking such products will reduce the risk of metabolic diseases.


Abiotrophia defectiva is a rare causative agent of infective endocarditis associated with pre-existing heart disease, immunocompromised and prosthetic valves. Dental procedures are also a common predisposing factor.Henoch-Schonlein purpura is the most common vasculitis in childhood, usually triggered by an upper respiratory tract infection and rarely observed in infective endocarditis patients. Abiotrophia defectiva combined with recurrent Henoch-Schonlein purpura. A 10-year-old girl with uncorrected congenital heart defects and Henoch-Schonlein purpura developed a purple petechial rash again. Transthoracic echocardiography evaluation revealed multiple irregular vegetations on the right ventricular side of the ventricular septal defect and on the tricuspid valve leaflets. Blood cultures grew Abiotrophia defectiva. The girl received cardiac surgery for vegetation resection as well as congenital heart defect correction and tricuspid valve replacement. Five months after the surgery, the patient was in satisfactory condition without any signs of endocarditis or valve insufficiency and her purpuric rash disappeared.We present the first pediatric congenital heart disease case of infective endocarditis caused by Abiotrophia defectiva belongs to the streptococcus with a high virulence. In addition, cardiovascular surgery is often required for pediatric infective endocarditis associated with Abiotrophia defectiva, and bioprosthetic valve replacement is considered feasible for irreparable tricuspid valve in children.The coexistence of recurrent Henoch-Schonlein purpura and infective endocarditis is possible.  Abiotrophia defectiva rarely causes IE. In this paper, we report an extremely rare case of IE caused by Abiotrophia defectiva with ventricular septal defect (VSD) and recurrent HSP in a child and no cases involved the association of IE due to Abiotrophia defectiva with recurrent HSP in medical literature in English. Because of the large vegetations, VSD and irreparable tricuspid valve, vegetation eradication, congenital heart defect correction and tricuspid valve replacement were performed.Henoch-Schonlein purpura (HSP) is a common systemic vasculitis in childhood, characterized by palpable purpura on the lower limbs, arthralgia, abdominal pain and renal disease. Upper respiratory infections caused by streptococci and staphylococci are considered to be potential predisposing factors, while a few cases lack a clear trigger . InfectiAbiotrophia defectiva. Laboratory results are in Table A 10-year-old girl, with uncorrected congenital heart defects, was initially presented to our pediatric outpatient clinic because of the diffuse purple petechial rash on her lower extremities. The first clinical evaluation showed a positive urinalysis for red blood cell (RBC) with 1  but a negative for protein, she was diagnosed with Henoch-Schonlein purpura and received an oral cetirizine and prednisolone. Two months later, the petechial rash disappeared with negative urinalysis for RBC and protein. Prior to this admission, the girl had two fevers with pharyngeal hyperemia in 7 months. She received an oral cefprozil but did not follow the doctor's prescription for a transthoracic echocardiography (TTE). It was not until 3 days prior to admission that the girl was again advised to do the TTE due to a recurrence of the petechial rash Fig.  showed sOn the 18th day, transthoracic cardiac surgery was performed with cardiopulmonary bypass. Intraoperatively, PFO and perimembranous ventricular septum defect (3 mm × 5 mm) were observed with the floppy and yellowish vegetations on the all 3 leaflets and chordae tendineae of the tricuspid valve  [Abiotrophia defective [Abiotrophia defectiva is also a rare invasive organism predisposed to valve destruction, heart failure, systemic embolization and death as compared to other streptococci [Abiotrophia defectiva require surgical intervention despite treatment with sensitive antibiotics [IE is infrequent in children with an incidence of 0.3 to 3.3 per 100, 000 children and adolescents . In the cedures) , 8. Atypefective , 10. In ptococci . ApproxiAbiotrophia defectiva is quite rare in pediatric cases, and it seems to be more devastating when compared to in adults. Since the definition of Abiotrophia defectiva was revised in 1995, including this case, only a total of 10 patients have been reported in the English medical literature [IE caused by terature . The anaValve replacement in children is always a challenge for cardiac surgeons because of the initial valve implant, small patient size, anatomy and growth potential. Without question, valvular repair is the goal of intervention because restoration of valvular anatomy and physiology using native tissue allows for growth and a potentially better long-term outcome .Due to the high morbidity of complications associated with tricuspid valve replacement, it is rarely required to replace the tricuspid valve, particularly in children. In this case, the tricuspid valve structure suffered irreparable destruction, so valve replacement became inevitable. A bioprosthetic tricuspid valve replacement was used as an alternative for this girl.Abiotrophia defectiva is a rare but devastating organism in children with IE and it is necessary to perform a comprehensive cardiac and pathogenic evaluation as early as possible in children with atypical symptoms, especially those with high-risk factors such as CHD. Notably, bioprosthetic valve replacement is considered feasible for irreparable tricuspid valve in children.The coexistence of HSP and IE is possible, although the underlying pathogenesis remains unclear.


Several studies in animal models of intestinal inflammation have been performed with the aim of understanding the mechanisms of action of anti-inflammatory proteins and peptides that reduce TNF-α. In order to present the best targets, effects and strategies for the treatment of intestinal inflammation in experimental models, this systematic review (SR) aimed to answer the following question: what are the mechanisms of action of molecules with anti-TNF-α activity on the intestinal barrier? The SR protocol was registered in the International Prospective Register of Systematic Reviews  and guided by the methodological procedures used for the elaboration of the SR. Articles that were part of the SR were selected considering the eligibility criteria according to the PICO  and were searched in the PubMed, Scopus, Web of Science, Excerpta Medica Database (EMBASE) and ScienceDirect databases. Twenty-five articles reporting studies in rats and mice were selected and the risk of bias was assessed using the tool from the SYstematic Review Center for Laboratory Animal Experimentation (SYRCLE). A descriptive synthesis of the results obtained was carried out. Based on the results, the anti-inflammatory molecules that reduced TNF-α acted mainly on the TNF-TNFR1/TNFR2 and TLR4/MD2 complex signaling pathways, and consequently on the NF-κB pathway. This improved the aspects of the inflammatory diseases studied. In addition, these mechanisms also improved the macroscopic, histological and permeability aspects in the intestine of the animals. These findings point to the potential of protein and peptide molecules that act on inflammatory pathways for medical applications with specific and promising strategic targets, aiming to improve inflammatory diseases that affect the intestine. This systematic review also highlights the need for more details during the methodological description of preclinical studies, since this was a limitation found. In addition to being involved in food processing and digestion, the gastrointestinal tract is a space for dynamic interactions between host cells and the external environment. Together with the chemical barrier and the cellular immune system, the epithelial cell layer plays an essential role as the first barrier against external factors and maintains a symbiotic relationship with commensal bacteria. Therefore, an imbalance in the structure of this barrier can trigger an immune reaction and uncontrolled microbial growth and is related to inflammatory bowel disorders, extra-intestinal autoimmune diseases, obesity and metabolic disorders .Intestinal homeostasis is maintained through the strict balance between cell death and proliferation to prevent intestinal inflammation and tumor formation. In the regulation of this balance, tumor necrosis factor-alpha (TNF-α) is a central inflammatory cytokine that plays a crucial role .TNF-α modulates multiple signaling pathways, being vital in the immune response, regulating the immediate inflammatory reaction with innate immune involvement, and cell activation with subsequent proliferation and programmed cell death or necrosis. However, because of this broad spectrum of cellular effects, TNF-α is also implicated in various disease states, such as inflammatory bowel diseases, infectious diseases, intestinal wound healing and tumor formation .Due to the participation of TNF-α in the pathogenesis of several inflammatory disorders, anti-TNF-α molecules have been used over the past few years to treat inflammatory bowel diseases and other conditions that include high concentrations of this cytokine, such as rheumatoid arthritis and psoriasis , althougAnti-TNF-α therapy consists of the use of antibodies that act to neutralize soluble TNF-α, but each agent has specific actions due to its distinct pharmacological properties .The side effects and risk of loss of effect of classic anti-TNF-α therapy, however, have stimulated the search for alternatives for anti-inflammatory treatment, with the development of new classes of antibodies , includiSeveral preclinical studies with models of intestinal inflammation aimed to understand the mechanisms of action of these anti-inflammatory proteins and peptides that reduce TNF-α. Such studies describe the mechanisms involved in the action of these molecules to develop the best strategies for treating these diseases –12. TherThe protocol of this systematic review (SR) was prepared following the guidelines described in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P)  and regiThe initial proposal of the study considered the inclusion of publications without delimitation of the type of molecule evaluated, and if it reduced TNF-α, as described in the systematic review protocol . HoweverInitially, preliminary equations were tested using keywords indexed in the Medical Subject Headings (MeSH), with the objective of developing search strategies with high sensitivity. The search equations  were conAlthough a more open research question was used and, consequently, broader search strategies, only original articles resulting from experimental studies that met the eligibility criteria based on the PICO strategy  were included in the review .This work did not include review articles, case reports, comments, editorials, letters to the editor, theses, publications in annals of congresses; studies in humans or other animal/cell models; studies in other organs or in the context of other inflammatory diseases that do not affect the intestinal barrier; studies with other types of molecules; studies with only healthy animals as control group; studies that did not evaluate the reduction in TNF-α concentrations or its activity after treatment; studies that did not address the mechanisms of action of the molecules studied to obtain the effects found in the intestinal barrier.The articles were imported into the Mendeley Reference Manager (1.17.11), duplicates were excluded and the reading of titles and abstracts was performed following the eligibility criteria for this step . Then, tData extraction was performed by M.S.R.L. and V.C.O.L., independently, in a previously standardized way, creating a database containing bibliographic information , data on the study population , experimental model studied , intervention studied  and outcomes on TNF-α and on the intestinal barrier, in addition to the mechanisms discussed for the effects found, according to Lima et al. .Following the protocol by Lima et al. , the risUsing the search equations  in each Some data obtained during the extraction step were described, including the characteristics of the animals and the models of inflammation used, the description of the molecules tested and their dosages, the effects on serum/plasma and intestinal TNF-α, in addition to the main findings described in studies on the results of treatment in the intestinal barrier of the animals .Escherichia coli. Some used CD4+CD45RBhi T cell adoptive transfer (n = 3), ischemia and reperfusion (n = 2) or other less used ways, such as body burn (n = 1), recombinant TNF-α (n = 1) and indomethacin to induce jejunoileitis (n = 1) (Most of the studies (n = 20) used mice, mainly of the BALB/c and C57BL/6 strains, and only five studies were with rats, mostly Wistar (n = 4). Regarding the inflammation induction model, most studies (n = 9) used dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS) to induce colitis. Many studies (n = 8) also induced intestinal inflammation through the use of lipopolysaccharide (LPS) or microorganisms, mainly  (n = 1) .All included studies were with peptides (n = 10) or proteins (n = 15), mainly immunoglobulins (n = 12). Due to the different nature of the molecules, this variable was used to classify the studies, facilitating the presentation and discussion of results, in: 1) anti-TNF-α antibodies, 2) anti-IL-17 antibodies, 3) other types of antibodies, 4) other types of proteins, 5) hybrid peptides and 6) other peptides.TNF-α was evaluated in most studies by Enzyme-Linked Immunosorbent Assay (ELISA) or Reverse Transcription Polymerase Chain Reaction (RT-PCR) techniques, in serum or intestinal tissue, and the results showed a reduction in this cytokine after treatment with anti-inflammatory molecules. Only two studies did not measure TNF-α, as the molecules were antibodies, therefore they evaluated the reduction in TNF-α activity.Regarding treatment results on intestinal parameters, studies have shown an improvement in colitis, consequently, in clinical disease scores, as well as a reduction in histological damage scores. The results also included reduction of inflammation markers, such as cytokines , chemokines , leukocyte infiltration and apoptosis markers . Some studies have shown an increase in anti-inflammatory components  and increased gene/protein expression of structural and functional components  of the intestinal barrier.Regarding the assessment of the risk of bias in the studies, using the SYRCLE tool, no study clearly detailed the methods used to generate a random allocation of animals (Q1). Most articles presented baseline characteristics that showed pairing between animals (Q2). However, none was clear about the blinding of evaluators during allocation (Q3), random housing of animals during the experiment (Q4) and blinding of researchers in relation to the treatment received by each group (Q5).Regarding the questions related to the analysis of results, no article described whether there was random selection of animals (Q6) and nearly half reported that the result evaluator was blinded (Q7). As for the presentation of results, most studies did not adequately address incomplete outcome data (Q8), but most were also free from biased reporting of results (Q9). Regarding other potential sources of bias, most articles with a “no” answer had a conflict of interest (Q10) .By observing the studies included in this SR , it is cRegarding the assessment of the risk of bias of the studies included, in many questions of the SYRCLE tool, the score attributed to the studies was equivalent to the “not clear” option, as the articles did not contain sufficient details to allow the risk of bias to be properly reported. In this regard, the authors of the tool themselves recognize that the quality of current reports of studies with animals is low, lacking many details about the conditions in which the animals are kept or about the moment of evaluating the results. For this reason, the role of this tool in increasing researchers’ awareness of the importance of improving the internal validity of animal studies .Another important aspect is that the research question of this review was focused on the mechanisms of action of molecules with anti-TNF-α activity. In this regard, it is important to consider that some articles included in the review to address this issue presented different characteristics and strategies. There are articles that have as direct objective specifically to target TNF-α (mainly using antibodies). In this case, it was easier to establish mechanisms of action linked to the inhibition of production/activity of the cytokine.In other articles, the primary objective/strategy was not specifically to target TNF-α. In these last studies, although a reduction in the production/activity of the cytokine was achieved, this is described as one of the effects, together with changes in other cytokines and molecules. Thus, the mechanisms of action of treatments associated with a decrease of production/activity of TNF-α were provided, but not all the articles included in this review have the explanation of how specifically a given treatment produces the effect on TNF-α.Anti-TNF-α therapy, performed by anti-TNF-α antibodies, is the most classically used therapy in the treatment of inflammatory diseases that affect the intestinal barrier . Bhol etThe results of established colitis treatment showed that the neutralization of TNF-α by the antibody generated a reduction in CD68 and myeloperoxidase (MPO) markers, indicating a reduction in macrophages and neutrophils in the colon tissue. There was also a reduction in TNF-α and TNF-α-directed pathway markers, both in relation to their protein and mRNA expression, which shows that AVX-470m inhibits not only the gene and protein expression of TNF-α, but also of several cytokines from TNF-α-directed pathways of inflammation.highCD4+ T cells and treated them with an anti-TNF-α monoclonal antibody. The authors found that early and late treatments reduced inflammation, infiltration of leukocytes (CD4+ T cells and macrophages) and epithelial lesions in the colon, significantly reducing histological scores. Immunohistochemical analyzes for CD4, F4/80, CD80, CD54 and CD40 showed significantly reduced expression compared to control, suggesting that anti-TNF treatment can block leukocyte recruitment and the expression of costimulatory molecules in inflamed sites.In another study with experimental colitis, Liu et al.  induced CD4+ T cells from lamina propria from mice treated with anti-TNF-α were also shown to secrete less TNF-α, IFN-γ and IL-2, suggesting that anti-TNF-α regulates Th1-mediated mucosal inflammation through inhibition of inflammatory cytokine secretion, without evidence of Th2 change. IL-23p19 and IL-17 mRNA expressions were significantly lower in colon tissue of mice treated with anti-TNF-α, indicating that anti-TNF-α blocks mucosal inflammation by down-regulating their expression.hi cells. The main objective of the study was to examine the interaction between the Fc region of the anti-TNF antibody and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) would increase the effectiveness of anti-TNF against colitis. The antibodies were able to reduce the severity of colitis, decreasing histological damage in the colon and mRNA expression of inflammatory cytokines, in addition to increasing regulatory CD206 macrophages. For this, the interaction between anti-TNF and FcγR was necessary. In addition, anti-TNF antibody hypofucosylation showed that the enhancement of binding to FcγRIIIa generated by this modification can make it more effective against colitis, as it significantly improved the therapeutic outcome.Still considering anti-TNF molecules, Bloemendaal et al.  studied Evaluating an anti-TNF-α monoclonal antibody in the treatment of indomethacin-induced jejunoiliitis, Nandi et al.  found thTNFR1 and TNFR2 genes were double knocked out, which showed that TNF-α is critical for epithelial barrier dysfunction associated with total parenteral nutrition, with involvement of both TNFR1 and TNFR2 receptors.Feng and Teitelbaum , in turnBased on the analysis of the results of studies that used anti-TNF-α antibodies, the reduction in intestinal inflammation from TNF-α blockade is the result of lower expression of inflammatory cytokines, including TNF-α, and the lower recruitment and infiltration of leukocytes in the colon, achieved through downregulation of the NF-κB pathway.Interleukin 17 (IL-17) is an inflammatory cytokine produced by Th17 lymphocytes . The rolSong et al.  also evaAlthough evaluated in different models of intestinal inflammation induction, the neutralization of IL-17 through antibodies generated a reduction in inflammation, acting in the reduction of inflammatory cytokines, such as IL-1β and IL-17 itself.Considering the anti-inflammatory effect of other types of antibodies, Ungaro et al.  used theHowever, in the late treatment (days 7 and 10), there was an impairment to the mucosal healing of the animals, due to the reduction of repair mediators and intestinal homeostasis  because TLR4 is also important for mucosal repair, suggesting that its blockage is most effective during the early stages of intestinal inflammation.Tool like receptors (TLRs) are transmembrane receptors that play an important role in the regulation of innate immunity by recognizing pathogen-associated molecular patterns (PAMPs), such as LPS present in the cell wall of gram-negative bacteria. The interaction of TLR4 and its MD-2 co-receptor with LPS triggers signaling cascades mediated by pathways that are or not MyD88-dependent, which lead to the translocation of the nuclear factor kappa B (NF-κB) transcription factor and the production of inflammatory cytokines , 45. Thuhi T cells to lymphopenic mice colonized by Helicobacter typhlonius. Colitis was reversed by depleting colitogenic T cells with anti-CD4 antibodies, leading to decreased leukocyte infiltration to sites of inflammation and reduced serum and colonic TNF-α concentrations. Anti-CD4 treatment was associated with significant changes in intestinal gene expression profiles, with a rapid reduction in expression levels of several inflammatory mediators and chemokines. Colitogenic T cell depletion was also associated with restoration of colonic epithelial integrity and regeneration of the mucus layer, probably due to the recovery of globet cell function in response to mucosal healing, which was correlated with the restoration of physical separation between commensal bacteria and intestinal epithelial layer.Brasseit et al.  studied -/- H+ receptor mice were fully protected from colitis.However, the effect of disease remission by anti-CD4 treatment cannot be attributed only to the drop in systemic and intestinal TNF-α, because when a therapeutic treatment with anti-TNF-α antibody was performed, the concentrations of other inflammatory mediators in the serum and colonic tissue from colitic mice were not affected, indicating that these mediators can compensate for the absence of TNF-α bioactivity and, therefore, mediate disease exacerbation. These discrepant findings in the literature can be attributed to the extent of intestinal inflammation present when anti-TNF-α treatment was started, as when anti-TNF-α administration was done together with the adoptive transfer of colitogenic T cells (preventive treatment), RagIn the study by Souza et al. , intestiOther results from the same study showed that the effects of IL-1 seemed to work via modulation of IL-10 production, with a consequent reduction in tissue damage and in TNF-α dependent lethality. The reduction in TNF-α production in tissue was attributed to the inhibition of neutrophil influx caused by treatments with IL-1, anti-IL-1ra and IL-10. The authors concluded that, in the studied model, TNF-α is central to the pathogenesis of injury and lethality associated with reperfusion, but both IL-1 and TNF-α seem to trigger anti-inflammatory cascades; and that IL-10 production is mainly under the control of IL-1, while TNF-α seems to activate IL-10 and another distinct molecule, not yet recognized .Fiorucci et al.  evaluateClostridium difficile. Treatment with anti-GM-CSF reduced the inflammatory cytokines TNF-α and IL-1β, as well as iNOS, and the expression of the neutrophil chemokines CXCL1 and CXCL2. Consistent with a decrease in the expression of neutrophil-attracting chemokines, fewer neutrophils were observed in histological sections and a reduction in gene expression of secretory leukocyte protease inhibitor (SLPI), a tissue anti-protease that protects against damage by neutrophil elastase.McDermott et al.  also obsIn addition to the molecules already presented, other proteins such as thrombin and its specific receptor, the protease activated receptor 1 (PAR1), act as important triggers of inflammation. Tsuboi et al.  evaluateBacillus amyloliquefaciens (BA SOD) improved disease severity, increased the activity of antioxidant enzymes, reduced the infiltration of inflammatory immune cells and, consequently, the production of inflammatory cytokines.Hwang et al.  evaluatein vitro study with human colon epithelial cells transfected with SOD1 siRNA, the authors also found a reduction in p-p38 and p-p65 when the cells were treated with BA SOD, in addition to a reduction in apoptotic cells and caspase 3 and an increase in Bcl-2. Together, the results suggest that BA SOD administration suppresses oxidative stress, inflammatory response and apoptosis through inhibition of p38-MAPK-NF-κB signaling.The authors also observed that administration of BA SOD reduced the expression of phosphorylated p38 and p65 (p-p38 and p-p65). The p38-MAPK pathway is a key regulator of cell apoptosis and this signaling promotes cell death, and the NF-κB signaling pathway activates caspase 3 and leads to apoptosis in intestinal epithelial cells. In an Xu et al.  evaluateThe results of Yin et al.  showed tThe peptide hybridization method has also been applied to antimicrobial peptides (AMP), because despite the anti-inflammatory effects of these peptides, its development faces several obstacles, mainly due to its toxicity for eukaryotic cells, impairing its clinical impact. Hybridization, however, has increased anti-inflammatory potential and stability and minimized AMP toxicity .Zhang et al.  construcThe results of the study showed that the hybrid peptide LTA improved anti-inflammatory activity with minimal toxicity, prevented intestinal inflammation in the jejunum epithelium and leukocyte infiltration in a murine model of LPS-induced intestinal inflammation. There was a reduction in inflammatory cytokines, increased expression of tight junction proteins ZO-1 and occludin, and reduced cell permeability and apoptosis in the jejunum. The results of the quantification of NF-κB pathway proteins suggest that this is one of the ways in which LTA modulates the immune system of animals. Additional flow cytometry and molecular dynamics assays demonstrated that the interface of MD2 that binds to LPS was compared to that of LTA, suggesting that this hybrid peptide blocks the binding of LPS to the TLR4/MD2 complex.Zhang et al.  evaluateAnother hybrid peptide containing the functional core region of LL-37 was evaluated by Wei et al. , this tiEscherichia coli (EHEC) in mice [This same hybrid peptide was studied in a model of intestinal inflammation induced by infection with enterohemorrhagic  in mice . In addiAccording to Li et al. , AMP havIn addition to the hybrid peptides presented, other AMP–the defensins–were among the molecules evaluated in the studies included in the review. Defensins are an important family of host defense peptides, as are also called AMP, expressed predominantly in neutrophils and epithelial cells. β-defensins have shown a range of biological functions, such as antimicrobial and immunomodulatory, acting as important components of innate defense , 49.Escherichia coli was evaluated. Treatment with DEFB188 alleviated intestinal inflammation and epithelial injury, which was associated with reduced expression of genes involved in the inflammatory response–including NF-κB and TLR4 –and with apoptosis in the small intestine, in addition to improved intestinal morphology and barrier functions.In the study by Lin et al. , the actXie et al.  evaluateStreptomyces albulus during the fermentation process, which exerts antimicrobial activity and activates calcium-sensitive receptors (CaSR) by allosteric binding. Treatment with PL in a murine model of DSS-induced colitis reduced the severity of this inflammation, prevented colon shortening and improved its morphology. In addition to the reduction in the relative expression of TNF-α mRNA in the colon, there was also a reduction in the relative expression of IL-6, IL-17, IL-1β and IFN-γ mRNA. Many of these effects were abrogated in animals receiving NPS-2143, an antagonist used to block CaSR activation, suggesting that PL exerts anti-inflammatory effects in the gut mediated primarily by allosteric CaSR activation. The CaSR, in the intestine, are mainly involved in the modulation of absorption and secretion, in the control of ionic transport and in the repair of the intestinal barrier.Mine and Zhang  studied Sigalet et al.  induced Bombyx mori, called gloverin A2 (BMGlovA2). It is a peptide belonging to the cecropin family that has been shown to inhibit the growth of bacteria by binding to LPS on the surface of the bacterial membrane, which increases the permeability of this membrane. In the study, mice were treated for 6 days with BMGlovA2 and on the seventh day an intraperitoneal injection containing enterotoxigenic Escherichia coli (ETEC) was given. The treatment attenuated the inflammatory response and the damage to the intestinal mucosa, improving the morphology and integrity of the intestinal epithelium. The expression of genes related to apoptosis in the jejunum was reduced, while the expression of genes related to mucosal barrier functions, such as MUC2 and MUC1, was increased. Thus, the authors concluded that BMGlovA2 had antibacterial and anti-inflammatory functions that make it a potential candidate to replace conventional antibiotics.Lin et al.  evaluateIn general, the molecules belong to different groups, there is a great diversity in the evaluated outcomes and, consequently, in the pathways involved in the reduction of TNF-α and improvement of aspects related to the intestinal barrier, even within of the same class of molecules evaluated. However, most studies reaffirmed the important role of TNF-α in intestinal inflammation induced by different ways, as a cytokine that mediates an inflammatory cascade that is related to effects on the intestine, including the production of other cytokines and chemokines and recruitment of defense cells that culminate in changes in the integrity of the intestinal barrier.The anti-inflammatory molecules studied acted by reducing TNF-α mainly by acting on the TNF-TNFR1/TNFR2 and TLR4-MD2 complex signaling pathways and, consequently, on the NF-κB signaling pathway . The regThe results obtained in this review point to the potential of protein and peptide molecules that act on inflammatory pathways for medical applications, aiming to improve the inflammatory diseases that affect the intestine. The importance of this study is highlighted, since these diseases have become a global health problem with an increasing incidence in some populations.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.


We aimed to define downstream drivers of the KDM5D effect. Using chromatin immunoprecipitation sequencing (ChIP‐seq) of the LNCaP cell line (androgen‐sensitive human prostate adenocarcinoma) with and without silenced KDM5D, MYBL2‐binding sites were analyzed. Associations between MYBL2 mRNA expression and clinical outcomes were assessed in cohorts of men with localized and metastatic hormone‐sensitive prostate cancer. In vitro assays with silencing and overexpression of MYBL2 and KDM5D in androgen receptor (AR)‐positive hormone‐sensitive prostate cancer cell lines, LNCaP and LAPC4, were used to assess their influence on cellular proliferation, apoptosis, and cell cycle distribution, as well as sensitivity to androgen deprivation, docetaxel, and cabazitaxel. We found that silencing KDM5D increased histone H3 lysine K4 (H3K4) trimethylation and increased MYBL2 expression. KDM5D and MYBL2 were negatively correlated with some but not all clinical samples. Higher MYBL2 expression was associated with a higher rate of relapse in localized disease and poorer overall survival in men with metastatic disease in the CHAARTED trial. Lower MYBL2 levels enhanced LNCaP and LAPC4 sensitivity to androgen deprivation and taxanes. In vitro, modifications of KDM5D and MYBL2 altered cell cycle distribution and apoptosis in a cell line‐specific manner. These results show that the transcription factor MYBL2 impacts in vitro hormone‐sensitive prostate cancer sensitivity to androgen deprivation and taxanes, and lower levels are associated with better clinical outcomes in men with hormone‐sensitive prostate cancer.Loss of the histone demethylase KDM5D (lysine‐specific demethylase 5D) leads to  Higher levels of the transcription factor MYBL2 increase the aggressiveness and decrease the sensitivity of hormone‐sensitive prostate cancer to androgen deprivation and docetaxel. ADTandrogen deprivation therapyARandrogen receptorATCCAmerican‐type culture collectionChIPchromatin immunoprecipitationCRPCcastration‐resistant prostate cancerCSScharcoal‐stripped serumctlcontrolDHTdihydrotestosteroneDOCdocetaxelFFPEformalin‐fixed, paraffin‐embeddedHBSSHank's balanced salt solutionHRhazard ratioINTiodonitrotetrazolium chlorideLVlentivirusmHSPCmetastatic hormone‐sensitive prostate cancero.e.overexpressionOSoverall survivalPrCaprostate cancerRIPAradioimmunoprecipitation assaySCANsingle channel array normalizationscrscrambleTCGAThe Cancer Genome Atlasde novo presentation with high‐volume disease) have a marked and consistent improvement in OS with the addition of docetaxel to ADT. By contrast, the addition of an extragonadal androgen synthesis inhibitor (abiraterone) or potent direct androgen receptor (AR) inhibitor  to ADT imparts clear OS benefits for patients with both low‐ and high‐burden disease [While some men with metastatic hormone‐sensitive prostate cancer (mHSPC) treated with testosterone suppression  alone have a rapid tumor progression and die within months from starting therapy, others will have durable responses lasting more than a decade. Clinical features associated with poorer overall survival (OS) with ADT alone include a higher disease burden and presenting with metastatic disease at the time of initial diagnosis , 2, 3. N disease , 5, 6.RB1 are strongly associated with shorter time to castration resistance and OS, and preliminary data suggest that cell cycle alterations might be enriched in poor‐prognosis high‐volume disease [To date, little is known regarding the biological basis for an aggressive course and who benefits from the addition of docetaxel, abiraterone, enzalutamide, or apalutamide to testosterone suppression. Alterations in cell cycle control mechanisms including  disease , 8, 9.KDM5D expression was associated with a higher risk of metastasis after prostatectomy and progression to castration resistance as well as shorter OS. One potential cause of low KDM5D expression in advanced prostate cancer is complete copy number loss given its localization on the Y chromosome.In prior preclinical work  we foundTo identify potential downstream mediators of the effects of KDM5D, we deployed chromatin immunoprecipitation (ChIP)‐sequencing and discovered that KDM5D physically interacts with AR in the nucleus and regulates its transcriptional activity by demethylating H3K4me3‐active transcriptional marks . Thus, lMYBL2 levels impact resistance to androgen deprivation and docetaxel. It has been previously reported that higher levels of MYBL2 are associated with poor prognosis in both hematological and solid tumors[In this study, we demonstrate that MYBL2, an MYB transcription factor family member, is one of the key mediators of KMD5D's biological effects and that id tumors, 14, 15 id tumors. In addiid tumors. We hypom of L‐glutamine at 37 °C in 5% CO2. The viability of cells treated in each experiment was determined using the Cell Titer‐Glo Luminescent Assay  and incubating the cells in a 96‐well format for 10 min with a luminescent reagent in a 1 : 1 medium according to the manufacturer's protocol.The prostate cancer cell lines LNCaP, 22RV1, and DU145 were obtained from the American Type Culture Collection (ATCC). The LNCaP‐C42 cell line was obtained from ViroMed Laboratories. The E006AA cell line was provided by John T. Isaacs , and the LAPC4 cell line was provided by Charles Sawyers . Cells were maintained in a medium (RPMI 1640) containing 10% FBS  or 10% charcoal‐stripped serum (LNCaP and LAPC4) supplemented with 2 m6) at room temperature were cross‐linked for 10 min with 1% paraformaldehyde and the reaction was terminated by the addition of 1 mL of 1.25 m glycine for 5 min, followed by extracting the nuclear fraction using the hypotonic lysis buffer. Cross‐linked chromatin was transferred to AFA fiber tubes (Covaris) and sonicated in 0.2% SDS buffer. Sonicated chromatin was centrifuged for 5 min at 16 000 g and diluted to 0.1% SDS concentration. After preclearing with Dynabeads Protein G  for 1 h, chromatin–protein complexes were immunoprecipitated with 5 μg of antibodies overnight at 4 °C. The next day, Dynabeads Protein G was added for 2 h, and beads were washed in the buffer. Precipitated chromatin was then eluted from the beads in 300 μL elution buffer  for 1 h at room temperature followed by de–cross‐linking at 65 °C overnight. After RNase A and proteinase K treatment, ChIP and input DNA were extracted by phenol‐chloroform extraction. Fragment sizes (200–300 bp) were evaluated by a High Sensitivity DNA Kit  on an Agilent 2100 Bioanalyzer. Specific enrichment was analyzed by qPCR and as percent input. For ChIP‐seq library preparation, ThruPLEX DNA‐seq Kit  was used with 2 ng of DNA input in 10 cycles of PCR amplification. The products were size‐fractionated and purified by a polyacrylamide gel. Fragment sizes and library concentrations were validated as above before sequencing on Illumina HiSeq 2500 (PE50)  at the MSKCC Integrated Genomics Operation core. ChIP‐seq raw data were mapped to the Human Genome 19 (NCBI build) by Bowtie using default parameters (This method is similar to that described in our previous paper [Cells 7.5 × 106 at roomSequences of short hairpin RNAs (shRNAs) used are listed in Table Total RNA was isolated from cells using TRIzol  according to the manufacturer's protocol and quantified using a NanoDrop spectrophotometer. 1 μg of RNA was reverse‐transcribed using a High Capacity cDNA Reverse Transcription Kit . TaqMan gene expression assays  were used for relative gene expression. Transcript levels were normalized to GAPDH. Proteins were extracted by radioimmunoprecipitation assay (RIPA) buffer with proteinase inhibitor mixture  and sonicated using a Bioruptor Standard for 5 min. RIPA buffer and protein concentration was determined by the Bradford method. Equal amounts of protein were loaded and resolved by SDS/PAGE, then transferred to polyvinylidene difluoride membranes for immunoblotting. All antibodies are listed in Table A soft‐agar assay was performed in 6‐well cell culture plates in triplicates. 20 000 live cells  were plated in 2 mL 0.3% Noble Agar mixture media onto 2 mL 0.6% Noble Agar media. Cells were incubated for 2 weeks, changing media every 3 days, and stained with 0.1% iodonitrotetrazolium chloride (INT) for 1 h. Colony numbers were counted and photographed by using Gel Count .m TRIS‐HCI pH 7.4, 150 mm NaCI, 1 mm EDTA, 1% Triton X‐100, 1% sodium deoxycholate, and 0.1% SDS) supplemented with protease and phosphatase inhibitors (ThermoFisher Scientific). Protein concentrations were measured using the Bradford protein assay. Western blot was performed using specific antibodies (Table Cells were washed with HBSS and lysed in radioimmunoprecipitation assay (RIPA) buffer unless otherwise noted (50 mes Table . For BRCThe matrigel invasion assays were performed in Matrigel invasion chambers (Fisher Scientific) in triplicates. 20 000 live cells (LNCaP sh‐control and overexpression of MYBL2) were plated on the top of chambers in serum‐free media. 10% FBS in the lower chamber was used as a chemo‐attractant. After 48 h incubation, cells in the bottom chamber were fixed in methanol and stained with crystal violet. Invaded cells were counted and photographed under phase‐contrast microscopy. In one chamber, colonies stained with crystal violet stain were visually counted.Cells were cultured in 6‐well plates to 50% confluency, then transfected with siRNA. After 72 h, cells were collected in media with 1% FBS, mixed with Muse Annexin V and Dead Cell Reagent, and analyzed using a Muse Cell Analyzer .LNCaP‐wild‐type, lenti‐control plus si‐control, and overexpression of MYBL2 plus si‐KDM5D cells were cultured in a medium containing 10% CSS . LAPC4‐wild‐type, lenti‐control plus si‐control, and overexpression of KDM5D plus si‐MYBL2 cells were cultured in 10% CSS medium for 72 h, and 1 million cells were harvested followed by washing with Hank's balanced salt solution (HBSS) and fixation with ice‐cold 70% ethanol overnight. Cells were then washed with HBSS, stained with 200 μL of PI/RNAse reagent  for 30 min, and the cell cycle distribution was analyzed using the Muse Cell Analyzer (Millipore).Among men with mHSPC who participated in the CHAARTED trial, consent and IRB approval were obtained for genomic and gene expression studies. Formalin‐fixed paraffin‐embedded prostate biopsy tissues from before initiation of androgen deprivation therapy were profiled for whole‐transcriptome gene expression using the Affymetrix human exon 1.0 ST microarray , as previously described , 19, 20.KDM5D and MYBL2 levels were compared as continuous variables using Spearman correlation unless detailed otherwise in the results. Follow‐up for disease‐free survival including biochemical recurrence started at cancer diagnosis in cohorts of localized disease and randomization in CHAARTED. Overall survival and time to castration resistance were assessed using the Kaplan–Meier method and Cox proportional hazards regression. Patients who had tumors in the highest quartile of MYBL2 expression were compared with outcomes of patients with lower MYBL2. In CHAARTED, multivariable models included ECOG performance status, disease volume , and de2 of the tumor with at least 60% tumor cellularity was required for the assay. RNA was extracted using the RNeasy FFPE kit , converted into cDNA and amplified using the Ovation FFPE kit , and hybridized to the Human Exon 1.0 ST oligonucleotide microarray (Thermo Fisher), in a Clinical Laboratory Improvement Amendments‐certified laboratory facility (Decipher Biosciences). Quality control was performed using Affymetrix Power Tools, and normalization was performed using the single channel array normalization (SCAN) algorithm. The study methodologies conformed to the standards set by the Declaration of Helsinki. The experiments were undertaken with the understanding and written consent of each subject. The study methodologies were approved by the Dana‐Farber Harvard Cancer Centers.Complete descriptions of clinical specimens from the CHAARTED trial of mHSPC have been published in . In brieKDM5D in the HSPC cell line LNCaP caused a marked increase in H3K4me3 peaks on ChIP‐seq but no major changes in H3K4me2 or H3K4me1 at the promotor region of MYBL2 .We then quantified to what extent the negative expression correlation between 0.03, in ; r = −0.0.11, in ) and met0.06, in ), and inMYBL2 expression was associated with a greater risk of relapse after local therapy  in The Cancer Genome Atlas  and high‐volume (78% vs. 65%) disease. As expected, the treatment effects of docetaxel were similar in the analytical population.We then studied the association of MYBL2‐high tumors (31 deaths among men with tumors in the upper quartile of MYBL2) than among men with MYBL2‐low tumors , compared with the 120 men with MYBL2‐low tumors  were not substantially higher  than among men treated with ADT and docetaxel . By contrast, patients with MYBL2‐high tumors had poorer OS whether treated with ADT alone or ADT plus docetaxel. Specifically, there was no clear evidence of differences in associations of MYBL2 with OS by treatment arm  with adjusted HR for high MYBL2 of 1.64  in the ADT‐only arm and of 2.71  in the ADT plus docetaxel arm. High expression levels of MYBL2 were associated with a shorter time to recurrence and less effective treatment with ADT and docetaxel.Interestingly, among men treated with ADT only Fig. , high MYMYBL2 status and treatment arm. This facilitated an analysis of outcomes and potential treatment effects in the CRPC state with and without prior docetaxel given that OS is a composite of time in both states. Consistent with the modeling results above, patients with early docetaxel appear to have had a similar amount of time in the pre‐CRPC state regardless of MYBL2 level. By contrast, patients with no early docetaxel and high MYBL2 were more likely to have a short time in the pre‐CRPC state. Survival from the time of CRPC appeared similar regardless of MYBL2 levels and prior docetaxel.We also plotted the time to CRPC , without a detectable association of KDM5D with OS  cells increased approximately fourfold in the presence of DHT and about 2‐fold with androgen deprivation. The proliferation rate was also similar to wild‐type LNCaP when silencing KDM5D without silencing MYBL2 with a resultant modest increase but still lower MYBL2 levels than overexpressing MYBL2 in the LNCaP cells (KDM5DLo/MYBL2Lo).The influence of 2Lo Fig.  increaseLo/MYBL2Hi in wild‐type LAPC4). Namely, the LAPC4 KDM5DLo/MYBL2Hi cells lines had the most rapid proliferation in both DHT (+) and DHT (−) conditions whereas LAPC4 cells with KDM5DHi/MYBL2Lo and KDM5DLo/MYBL2Lo had the least proliferation in both DHT (+) and DHT (−) conditions. However, discordant with the LNCaP findings is that overexpressing KDM5D in LAPC4 cells with si‐control/no manipulation MYBL2 did not slow proliferation in DHT (+) and partially slowed down the cellular proliferation in DHT (−) conditions, indicating more autonomy of MYBL2 from KDM5D in LAPC4.The findings from altering MYBL2 and KDM5D in the LAPC4 cell line were mostly consistent with those observed with LNCaP, despite their reversed constitutive expression levels  decreased colony formation both in number and size of colonies, whereas overexpressing MYBL2 increased colony number and size in LNCAP cells . Whereas both low and high doses of siRNA had a minimal effect in LNCaP with minimal MYBL2, the higher siRNA doses caused an 80% and 90% inhibition of proliferation in LAPC4 and E006AA, respectively, and the lower siRNA dose had minimal effect in LAPC4 but an 80% inhibition in E006AA  had a greater sensitivity to androgen deprivation, DHT (−) plus docetaxel than their counterparts with KDM5DLo/MYBL2Hi (green curves). The same observation was observed  with and without DHT had about 1/3rd the proliferation compared with LNCaP with KDM5DLo/MYBL2Hi. The addition of docetaxel in DHT (−) and DHT (+) conditions resulted in the fewest number of cells on Day 3 in the LNCaP with KDM5DHi/MYBL2Lo whereas docetaxel had less of an effect in the more rapidly proliferating LNCaP‐KDM5DLo/MYBL2Hi cells (green bars) with and without DHT. Similarly, the addition of docetaxel to LAPC4 with KDM5DHi/MYBL2Lo showed caused 59% inhibition of cell proliferation whereas LAPC4 with KDM5DLo/MYBL2Hi showed only 13% inhibition of cell proliferation.When the absolute cell counts on Day 3 were assessed  were more sensitive to docetaxel without and with DHT  with ADT plus docetaxel. When the expression of MYBL2 in LAPC4 was decreased (and KDM5D not overexpressed), LAPC4 with KDM5DLo/MYBL2Lo were approximately as sensitive to docetaxel without and with DHT  and DHT (+) compared with their LNCaP control counterparts  and DHT (+) versus their control counterparts  and DHT (+) to another class of agents interacting with cell cycle, a CDK4/6 inhibitor, ribociclib  and DHT (+) conditions. In comparison, the LAPC4 . We noted  had a slight increase in the percentage of live cells as well as fewer dead and apoptotic cells compared with wild‐type LNCaP and vector control . Consistent with this, we then noted that the more slowly proliferating LAPC4 cells with overexpressed KDM5D/silenced MYBL2  had a more notable increase in early and late apoptotic cells and much fewer live cells than the more rapidly proliferating KDM5DLo/MYBL2Hi LAPC4 cells (wild‐type and vector control). It therefore followed that silencing MYBL2  increased PARP cleavage in both the LNCaP and LAPC4 cells . We did not detect an association with overall survival. The finding that elevation of MYBL2 had a more consistent association with poorer mHSPC outcomes than lower KDM5D is consistent with MYBL2 being a transcription factor controlled by other factors that promote many of the hallmarks of cancer [MYBL2 and KDM5D status jointly.Consistent with our prior ocetaxel , men tref cancer . The samin vitro cell line work was largely consistent with the observational data among patients. Specifically, AR+ HSPC cell lines, LNCaP and LACP4 and their derivatives with KMD5DHi/MYBL2Lo genotypes, and compared with their KMD5DLo/MYBL2Hi counterparts, that had the most rapid proliferation, were the least sensitive to androgen deprivation and had the least sensitivity to docetaxel with and without DHT. The intermediate genotypes of KMD5DLo/MYBL2Lo and KMD5DHi/MYBL2Hi showed that the presence of KDM5D modulated the impact of MYBL2 in LNCaP but not LAPC4. One explanation is that there is a little constitutive expression of KDM5D in LAPC4, and it could suggest that the MYBL2 effects may be independent from KDM5D to some degree [KDM5D and MYBL2 in clinical specimens.The e degree   than was seen with LNCaP (IC50 ~ 50 nm). By contrast, LNCaP KDM5DHi/MYBL2Lo had greater sensitivity to the ATR inhibitor, which was partially abrogated by increasing MYBL2 levels but had no effect on LAPC4.Finally, we characterized the type of cell death and cell cycle distribution upon silencing of  to G2/M . Notablyity Fig.  to docetin vitro and in vivo castration‐resistant growth and metastases by activating YAP1. The association between high MYBL2 levels and worse clinical outcomes across several cohorts that we examined and our corroborating in vitro findings suggest that further mechanistic studies exploring the cellular machinery driven by MYBL2 activation might lead to actionable insights on the progression of mHSPC.The limitation of our work is the paucity of mechanistic data and the limited experimental model systems. Findings in this study are supported by recent work by Li et al.  who showTaken together, high MYBL2 is consistently associated with poorer patient prognosis in localized prostate cancer and mHSPC and leads to more aggressive cell lines and resistance to androgen deprivation and anti‐cancer therapies impacting cellular replication. Increased MYBL2 is a poor prognostic marker for patients with mHSPC with ADT treatment alone and ADT plus alone or with docetaxel, with the potential as a predictive biomarker for treatment selection if confirmed by future studies. The development of an MYBL2 inhibitor for clinical use may improve the efficacy of ADT and ADT with docetaxel for the treatment of mHSPC.MYBL2 impacts both in vitro hormone‐sensitive prostate cancer sensitivity to androgen deprivation and taxanes and lower levels are associated with better clinical outcomes in men with hormone‐sensitive prostate cancer.The transcription factor Philip Kantoff: As of August 15th, 2021, PWK reports the following disclosures for the last 24‐month period: he has an investment interest in Convergent Therapeutics Inc, Cogent Biosciences, Context Therapeutics LLC, DRGT, Mirati, Placon, PrognomIQ, SnyDevRx, and XLink, he is a company board member for Context Therapeutics LLC and Convergent Therapeutics, he is a company founder for XLink and Convergent Therapeutics and is/was a consultant/scientific advisory board member for Anji, Candel, DRGT, Immunis, AI (previously OncoCellMDX), Janssen, Progenity, PrognomIQ, Seer Biosciences, SynDevRX, Tarveda Therapeutics, and Veru, and serves on data safety monitoring boards for Genentech/Roche and Merck. He reports spousal association with Bayer. Christopher Sweeney, MBBS: Consulting or Advisory Role: Sanofi, Janssen, Astellas Pharma, Bayer, Genentech, Pfizer, Lilly, Research Funding: Janssen Biotech (Inst), Astellas Pharma (Inst), Sanofi (Inst), Bayer (Inst), Sotio (Inst), Dendreon (Inst); Patents, Royalties, Other Intellectual Property: Parthenolide (Indiana University): dimethylaminoparthenolide (Leuchemix); Exelixis: Abiraterone plus cabozantinib combination. Stock or other ownership: Leuchemix;YY carried out the experiment with support from YM, GC, SHR, and RH. KHS, XVW, and YHC developed the theoretical formalism, performed the analytic calculations, and performed the numerical simulations. FB, SN, DF, GL, and MAC analyzed the data. ED and GSML were involved in planning this project. YY wrote the manuscript with support from CJS. Both HA and PWK conceived the study and were in charge of overall direction and planning. CJS supervised the project. All authors provided critical feedback and helped shape the research, analysis, and manuscript. All authors discussed the results and contributed to the final manuscript.https://publons.com/publon/10.1002/1878‐0261.13314.The peer review history for this article is available at Fig. S1. Western blot sheet.Fig. S2. Cell lines phenotype silencing MYBL2.Fig. S3. Clinical outcomes in CHAARTED.Fig. S4. Impact of KDM5D and MYBL2 on time to CRPC and OS in CHAARTED.Fig. S5. Doxycycline‐induced sh‐MYBL2.Fig. S6. Impact of KDM5D and MYBL2 on sensitivity to drugs in HSPC.Table S1. Materials.Table S2. Quantitative analysis of cell cycle profiles.Click here for additional data file.


Apart from being the subject of this Special Issue, what is nanometrology? The most straightforward definition would be that it is a measurement science related to anything at the nanoscale. This might include various subjects that can be very distinct: measurements of physical and chemical properties with very high spatial resolution, such as in electron microscopy; measurements of properties of various ensembles of nanoscale systems, such as aerosols or nanocomposites; or the development of metrology instrumentation that would be based on some nanoscale phenomena, such as in Surface Enhanced Raman Spectroscopy. Calling any of these disciplines “nanometrology” would make sense, and since this word started to be used in the 1990s, it has been used in very distinct contexts. From the perspective of national metrology institutes (NMI) that have been always very active in the dimensional aspects of measurements at the nanoscale, stimulated by the increasing needs of the semiconductor industry, nanometrology was initially related to providing metrological traceability to dimensional measurements and performing interlaboratory comparisons. In academia, the variety of topics became much wider, focusing on novel measurement principles, sometimes far from any metrological traceability. When looking at some older review papers related to nanometrology, e.g., , we can Starting from the opposite direction, we can determine which subjects have been related to nanometrology in scientific papers. By searching the Web of Science, we can find about 1300 papers related to the word “nanometrology”  with an average number of citations per publication around 10. Taking the most cited 10% of papers (having more than 25 citations), we can calculate statistics for their topics, materials and methods. Even if we classify the papers manually in order to prevent the largest errors, due to the fact that the relation to word “nanometrology” can be highly variable, the results of this survey are only very rough. However, we can still use the results to obtain a rough idea about how the whole subject has evolved over the years.Looking from the perspective of metrology institutes, we can see that 30% of papers have at least one author from NMI; 32% are related to metrological traceability and 58% to uncertainties and error sources, but only 12% deal with some kind of methodological comparisons between different instruments, methods or laboratories. The wide range of measurement topics related to optics  is covered by 37% of papers, while the much narrower field of Scanning Probe Microscopy is covered in about the same number, 36% of the papers. Classical dimensional metrology topics, such as building interferometers for providing metrological traceability up to the smallest movement scale, are discussed in 31% of papers. More complex analytical techniques such as X-ray-based surface and thin film analysis are found in only 21% of selected papers. Many of the expected topics have quite low numbers of papers, e.g., methods for thin film analysis are developed in only 6% of the papers . There is a lot of variety in the studied materials and sample types, out of which there is one larger group (21%) of papers related to the analysis of nanoparticles. Only 18% of the selected papers are related to the very broad area of life sciences, mostly focusing on nanoparticle toxicology. In So, were the expectations about the growth of nanometrology found in the old review papers met? From the above survey, we could conclude that they were met only partially. The yearly number of papers related to nanometrology is stagnating at a value of around 70 papers/year since 2005. Does this mean that nanoscale measurement research has not evolved? Most likely not. If we search for publications related to the term “nanoscale” together with the term “measurement science”, we see that yearly publication counts for both topics are constantly growing. In addition, when going through the selected papers discussed above, we can see that many existing and growing areas are under-represented—there are only a few papers about various electron microscopy techniques, not many papers about analytical techniques and no papers about super-resolution methods in biology. Such methods are certainly “measurement science at nanoscale”, and there is certainly a continuous development in them; however, the authors for some reason do not call such work “nanometrology”. This might sound like only a minor terminological difference, but it might also partly reflect gaps that exist in reality. The connection of national metrology institutes to the life sciences or to academia is, unfortunately, sometimes weaker than to industry, and we can often observe that people are talking different languages. So, even if the whole field of nanoscale measurement science is growing, it is important to constantly search for new interconnections between academia, industry and metrology institutes, raising the aspects of metrological traceability in the nanoscale world. The increasing variability in paper topics that we can see in In the past few years, nanometrology began facing new challenges, and many more are appearing even now. For example, projects related to the re-definition of the Si units were also targeting nanoscale measurements, either directly, e.g., by improving methods for silicon sphere surface and thin film measurements, or indirectly, e.g., by studying parameters such as silicon lattice spacing. This had led to a novel traceability route for nanometrology based on silicon lattice parameters , namely,As can be seen, the area of nanoscale measurements is very diverse, and as it is growing, it is becoming much broader than what the people who coined the term nanometrology could have anticipated. We are therefore happy that, in this Special Issue, we can present papers from various fields, coming from both academia and metrology institutes. This includes the aspects of traceability for analytical techniques, in particular X-ray-based techniques , electri


The coronavirus pandemic has revolutionized our world, with vaccination proving to be a key tool in fighting the disease. However, a major threat to this line of attack are variants that can evade the vaccine. Thus, a fundamental problem of growing importance is the identification of mutations of concern with high escape probability. In this paper we develop a computational framework that harnesses systematic mutation screens in the receptor binding domain of the viral Spike protein for escape prediction. The framework analyzes data on escape from multiple antibodies simultaneously, creating a latent representation of mutations that is shown to be effective in predicting escape and binding properties of the virus. We use this representation to validate the escape potential of current SARS-CoV-2 variants. Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accounted for more than 500 million infections and more than six million deaths worldwide according to World Health Organization . Though Although several antibodies and vaccines showed good clinical results, recognizing mutations that impact the escape from antibodies and vaccines is still a major question in the battle against SARS-CoV2. The receptor binding domain (RBD) region is a sub-region of the SARS-CoV-2 spike glycoprotein that mediates viral attachment to ACE2 receptors. The RBD is a major determinant of host range and a dominant target of neutralizing antibodies, promoting systematic studies of mutations to the RBD region and their impact on a variety of attributes including binding , antibodThe on-going emergence of variants with dozens of mutations motivates computational approaches to study the effect of multiple mutations. These include structural-based approaches  or approIn this paper, we try to combine the best of both worlds–aggregating escape data based on experimental data a la , while uWe developed a framework to assess the effect of mutations in the RBD on viral escape, both with a single-task approach and a multi-task approach. We tested our framework using experimental antibody escape data and compared the multi-task and single-task approaches. We demonstrate that multi-task learning helps reduce variance and improve performance. Moreover, we show that using multi-task learning yields an informative representation of the RBD sequence that can be subsequently used to predict multiple properties.Our main training data set is taken from After establishing the utility of our predictive model, We aim to further use it to find an informative representation of mutations that is more compact than the sequence of amino acids, while also preserving antibody escape information. Such a representation will allow us to test the predictive power of our model with respect to yet unseen properties. As a first test, we calculate viral escape of single amino-acid substitution from new, yet unseen antibodies: LY-CoV016, REGN10987 and REGN10933 As a second test, we checked the utility of the representation in predicting the effect of a single amino-acid substitution on the binding of the spike protein to ACE2. Specifically, binding affinity is given as the difference between the log of the dissociation constant of the mutation with respect to wildtype. As the binding is vital for viral entry, we assumed the learned representation could encompass useful information about it. Greaney et al. compiled a data set containing the escape information of about 2,000 single amino-acid substitutions in the RBD with respect to nine monoclonal antibodies The original escape information is given as probabilities. In order to create the viral-escape classification task from the continuous data, we followed Starr et al. n = 201 over an alphabet of size m = 20 (number of amino acids). The model applies one-hot encoding on every character, resulting in a binary vector of size m. It then applies a linear transformation to each vector to create a “character-level” embedding. These embeddings are concatenated and fed to a fully-connected layer, creating a “sequence-level” embedding. The final output layer is a linear layer with size equal to the number of prediction tasks k, followed by k task-dependent activation functions. In our case, k = 9, each task corresponds to an antibody in our train data, while our output activation functions are all sigmoids. This means our output will be 9 probabilities each corresponding to an escape probability of a different antibody.We use a neural network that receives as an input a string of fixed length k = 1, when k > 1 we refer to the model as a “multi-task model”. This means, that when comparing between multi-task and single task, we will have k single-task model, each corresponding to one antibody, while having a single multi-task model with k outputs. For training and evaluation we randomly split the data into 30% test and 70% train, run the model 100 times and report the performance distribution obtained using box-plots. Performance is measured in the binary case using the area under the ROC curve and in the continuous case using Pearson’s correlation between predictions and true value in test set. We use the Adam optimizer When we refer to a model as a “single-task model” it means that via logistic regression , meaning the task is identical to binding regression.Utilizing our multi-task model’s last hidden layer as a latent representation of mutations, we can predict other RBD properties such as binding. To this end, we add a linear layer after the embedding layer whose weights are trained using linear regression. When calculating escape probabilities, we use the sigmoid activation function in our output layer, so the training with fixed embedding is done https://github.com/bgmoshe/multi_tasking_antibodies.Code and data are available at In this paper we develop a computational framework that harnesses systematic mutation screens in the receptor binding domain of the viral Spike protein for escape prediction. Unlike  and Gre, who demIn order to showcase this attribute, We used our trained model to predict the escape probabilities of variants of concern  as shown in