Recombinant plant-based meat alternatives are a kind of product that simulates animal meat with complete structure by assembling plant-tissue protein and other plant-based ingredients. The market is growing rapidly and appears to have a promising future due to the broad culinary applicability of such products. Based on the analysis and summary of the relevant literature in the recent five years, this review summarizes the effects of raw materials and production methods on the structure and quality of specific components (tissue protein and simulated fat) in plant-based meat alternatives. Furthermore, the important roles of tissue and simulated fat as the main components of recombinant plant-based meat alternatives are further elucidated herein. In this paper, the factors affecting the structure and quality of plant-based meat alternatives are analyzed from part to whole, with the aim of contributing to the structural optimization and providing reference for the future development of the plant meat industry. As the global population continues to grow and natural resources are progressively reduced, the demand for protein supplies is increasingly strained ,2. TradiPlant-based meat alternatives, a type of artificial meat, are composed of plant protein processed either with or without the addition of auxiliary materials. This class of meat substitutes is considered similar in texture, flavor, morphology and other characteristics of animal meat products. In comparison to some of the other currently available meat substitutes, plant-based meat alternatives have gained a certain consumer base and are easily accepted by the public. Moreover, the production and processing of plant-based meat alternatives exert low impact on the environment, which is consistent with the concept of sustainable development and contributes to the realization of ‘carbon neutrality’ in the food industry . The marRecombinant plant-based meat alternatives are produced by adding blocks of simulated fat or simulated fat binder to the basis of tissue protein to simulate structurally intact animal meat . CompareThe factors affecting the structural quality of plant-based meat alternatives are complex and diverse. Previous studies and related reviews have focused on the general characteristics of plant-based meat alternatives in a broad sense, so there is currently a lack of analytical data regarding specific types of plant-based meat alternatives. ,14 AdditTissue proteins or textured vegetable proteins (with a lean fiber-like structure and chewiness) form the main skeletal structure of plant-based meat alternative products. These can be obtained through techniques such as extrusion, shearing, spinning, freezing structuring and three-dimensional (3D) printing . The maiProtein is the most important factor impacting the structural quality of tissue protein, as it is the main component of the fibrous structure that mimics meat products. Protein melting and denaturation are prerequisites for the formation of histochemistry. During processing, the combined actions of temperature, shear, pressure and water melt the plant proteins completely, with the molecular chains unfolding in the flow direction, exposing the sulfhydryl groups and creating new disulfide bonds through oxidation. In this way, the protein denatures, losing its original structure, and depolymerizes from the natural spherical aggregated state to form an anisotropic structure. This molten state includes the action of molecules with other substances, in which the protein molecules are rearranged. After sufficient homogenization and oriented arrangement, the mixture is cooled down and characterized, with the protein having been converted into a tissue protein with a structure similar to that of animal meat fibers . Thus, tLegumes are currently the most important source of protein in the production and processing of tissue protein. Soybeans and other legumes differ in their main components, nutritional values and processing characteristics. For example, soybean is extremely low in carbohydrates, high in fat and protein, and has a more balanced amino acid ratio. Protein content in pea is slightly lower than soybean, but it is one of the major plant protein resources due to the high yield. This section summarizes and analyzes the potential of legume proteins to be used in the production of tissue proteins, using two different categories of legumes as examples.Soybean protein is a common, high-quality plant protein that can readily replace animal protein . The oriAmong legume proteins, pea protein is another of the main raw materials occupying an important position in the plant-based meat alternatives market. This is due to its characteristics of being a non-genetically modified organism (GMO), non-allergenic and estrogen free, with high bioavailability, local cultivation, high yield and low cost . Peas alThe protein content of grains is lower than that of legumes, with poor digestibility and unsatisfactory amino acid composition, particularly in the lack of lysine and threonine . Indeed,Following hydration, wheat protein forms a 3D protein network structure maintained by disulfide bonds, which plays an important role in simulating the fibrous structure of meat, increasing the viscoelasticity and hardness of the product, and improving its color stability, juiciness and water retention . In one Although fungi are not essentially plants, meat analogs made from fungal proteins are often defined as plant-based meat alternatives. Fusarium, Agaricus bisporus and other fungal proteins are sources of high-quality plant-based meat alternatives . MyceliuAlgae proteins, such as chlorella primordialis and spirulina, have also been tested in the manufacturing of plant-based meat alternatives to improve the product quality . MicroalDuring processing, lipids can impact the structural quality and sensory properties of textured proteins. Lipids can act as plasticizers or lubricants during the tissue protein molding process, which can reduce particle interactions in the material and lessen the friction between processing machine and material, resulting in improved texture, viscosity and integrity in the tissue protein product. The combination of different lipid concentrations and processing conditions can produce tissue proteins with differentiated structural characteristics to meet the specific needs of a variety of products .The addition of lipids can also improve the organization of the legume tissue protein, resulting in a product with good fibrous structure and suitable hardness and flexibility. Jia et al.  found thIn general, the addition of lipids has varying effects on process kinetics. Further investigations are required to provide insight into the effects of lipids on processing conditions, protein polymerization, matrix rheological properties and their microstructure, as well as to clarify optimal quantities of lipid additions for various target products. The appropriate proportion of fats and oils can effectively improve fibrous strength and structural quality in the manufacture of organized products with different processing characteristics.Moisture affects both the fibrous structure and texture of tissue protein, contributing to the unfolding and arrangement of protein molecules during processing and promoting the formation of fibrous structures. It has previously been found that moisture content is a crucial factor affecting the formation of fibrous structure via the extrusion of lupin protein . When moMoisture levels are also important for the production, storage and transportation of final products. In the most common extrusion process, for example, low moisture extrusion (with a moisture content of less than 40%) produces a relatively poor-tasting tissue protein, while high moisture extrusion (with a moisture content of more than 40%) retains moisture and produces a highly textured, elastic, tough, fresh and high-quality tissue protein with a texture similar to animal meat and an appearance and taste similar to cooked meat . FurtherIn recent years, extrusion technology is still the main way to process tissue protein because it can form a fibrous structure similar to meat, and the research is relatively mature, in which high moisture extrusion has a better taste and gradually becomes the better choice. 3D printing technology is gaining the attention of scholars studying plant-based meat alternatives because of its personalization and nutritional customization and its ability to create products with complex structures or geometric shapes. Many scholars are gradually researching it in depth, expecting it to become an industrial production technology for plant-based meat alternatives ,61,62. SExtrusion technology integrates the mixing, homogenizing, aging and forming of materials through thermomechanical treatment and shear flow to form a fibrous structure that is similar to meat. It is dependent on the moisture content of the material, and therefore, when the moisture content of the material is lower than 40%, it is a low moisture extrusion process, and if it is higher than 40%, then it is a high moisture extrusion process . Figure 3D printing technology, also called additive manufacturing technology for rapid prototyping, refers to the manufacturing of solid 3D structures created from virtual models through computer-aided design . In thisShear cell processing technology uses shear flow to mix raw plant protein materials and water. The processing time is controlled by adjusting the cylinder speed and temperature of the equipment. With anisotropic tissue proteins and a simple combination of shear force and heating, plant protein can be processed into homogenous, layered and multilayered fibrous structures . Shear cElectrostatic spinning technology refers to the process in which nano-level oriented fibers are formed from plant protein polymer solutions in a nonwoven state under the breakdown of a high-voltage electrostatic field. However, the technological difficulty of this process is relatively high because proteins have a complex advanced structure, making them more difficult to destroy during spinning, and the pre-treatment requirements for raw materials are stringent . These tRefrigeration structure technology produces porous fibrillar structures by, first, freezing protein emulsions and then removing the ice crystals to obtain tissue proteins that resemble the structure of animal muscle fibers and consist of many parallel and highly connected lamellar proteins . The comSimulated fat is an oil-containing protein-polysaccharide composite that mimics the structure and chewiness of animal fat, but it has a significantly lower lipid content. It can provide a filler structure and improve the texture and taste of recombinant plant-based meat alternatives. There are two main types of simulated fat, one that simulates entire fat with a distinct structure, and another that simulates the dispersed fat between tissue proteins and acts as an adhesive . The disPlant oils are suitable raw materials with which to simulate animal fats, as they are a relevant flavor source, providing proportional fatty acid composition for health benefits  as well The right proportion of lipids can effectively increase moisture retention in simulated fat, thereby improving the tenderness, texture and juiciness of recombinant plant-based meat alternatives . In one Different kinds of plant oils also possess different processing characteristics. The selection of a suitable oil based on the target product can improve the applicability and overall quality of simulated fat in recombinant plant-based meat alternatives. These different effects include those on the texture of simulated fat. In one study, as the concentration of palm oil increased, the hardness and viscosity of simulated fat gradually increased and the structure became more compact, whereas, with the increase in the concentration of soybean oil, simulated fat became softer . The ratPolysaccharides are an important raw material in the processing of simulated fat. A protein-polysaccharide gel network structure can be obtained via the compounding with protein, providing good water and oil-holding properties and resulting in simulated fat with juicy and lubricious textures. In this section of the review, the characteristics and optimization of different polysaccharides in previous studies are analyzed from the viewpoint of the influence of polysaccharides on the structural quality of block simulated fat with adhesive effect, providing a foundation for the selection of polysaccharide-based raw materials for simulated fat. Starch is a polysaccharide raw material that commonly acts as a filler, facilitating the combination of protein, water and lipid components in the product, maintaining structural stability, and generating a mimicked fat with good formability by combining water and lipids, mostly through physical means . It has Methylcellulose and konjac glucomannan are also important raw materials in the production and processing of simulated fat. Methylcellulose absorbs water, swells during processing, and has good water-holding and gelation properties. After heating, the viscosity increases significantly, which is conducive to improving the texture and juiciness of simulated fat, which makes it a popular binder for the formulation of simulated fat in plant-based hamburger . Huang eTransglutaminase (TG) is a commonly used synergist and cross-linking agent in simulated fat processing, in which it can play a bonding and cross-linking role, thereby promoting molding and improving the resulting simulated whole animal fat . TG actsTissue proteins can be assembled and combined with simulated fat and binders to obtain quality characteristics that match the product attributes of particular meat products. This is made possible by combining the analysis of animal meat composition with an understanding of the histochemical ability of plant proteins . Tissue Tissue protein in plant-based meat alternatives provides structure and chewiness similar to those of lean meat fiber. It is also the skeleton structure for building recombinant plant-based meat alternative products and has a decisive role in stimulating the structural characteristics of animal meat products . SuitablUnder the same processing conditions, the soybean tissue protein after silk tearing is thicker, with greater toughness and hardness, suitable for the production of chewable recombinant plant-based meat alternatives with a stronger fiber structure . Wheat-bThe simulated fat helps to refine the structure of the recombinant plant-based meat alternatives to adequately mimic the taste of animal meat products. It has been demonstrated that by optimizing the formulation, block simulated fat with sensory properties similar to pork fat, such as hardness, flexibility and chewiness, can be created for the assembly of reconstituted plant-based meat alternatives with improved sensory qualities . SimulatSimulated fat based on plant oil enables recombinant plant-based meat alternatives to maintain similar organoleptic properties to those found in animal fat, reducing the negative impact of the process on organoleptic properties and improving product structure and quality stability. Nieto et al.  preparedThe recombinant plant-based meat alternatives market is currently expanding worldwide, with the number of commercially available products growing steadily. Likewise, the manufacturing processes are becoming more industrialized and advanced; however, in comparison to animal meat, recombinant plant-based meat alternatives still have insufficient flavor, a large structural gap, and poor sensory attributes and chewing sensations. Its structural quality is one of the important factors affecting consumers’ preferential purchasing decisions. Furthermore, there are relatively few whole reconstituted plant-based meat alternative products available that are suitable for a variety of cooking methods, and the sector is largely undeveloped, with significant research gaps and market potential.(1) The future development of recombinant plant-based meat alternatives should be devoted to optimizing the structural quality and sensory characteristics of tissue protein. There is a need to expand the sources of plant proteins, explore in depth the new characteristic proteins and improve the quality of tissue proteins via the use of different processing of characteristic proteins for compounding, through enzyme modification and other methods. Moreover, the quality of tissue proteins could be improved by adding proteins with better gel ability to assist the main protein raw materials.(2) For the optimal molding of tissue protein, it is necessary to improve the inclusiveness of production equipment for raw materials and the level of processing technology, and to reduce production costs, as well to explore the simulation of meat fiber structure and chewing sensation in depth. Furthermore, the application of high moisture extrusion and 3D printing technologies with excellent production quality and good development prospects in the production of tissue protein should be promoted.(3) The nutritional composition and processing characteristics of different varieties of lipids and polysaccharides warrant in-depth investigation, and the optimal additions and related processes should be clarified to optimize their use in simulating fats and binders. It is also important to explore more abundant sources of lipids and polysaccharides, and to optimize the nutritional ratios, functional properties and specific applications of fat substitutes, as an important direction for the future development of plant-based meat alternatives. In addition, the effect of TG on simulated fat structures and related mechanisms should be researched to promote the application of TG to improve structural quality and enhance simulation effectiveness.(4) According to the plant-based meat product, different types of simulated fat or binders should be selected to optimize structural quality and to obtain high-quality plant-based meat alternatives with good juiciness and chewiness. The development of simulated fat binders is under consideration for our own future study to obtain a protein–fat interphase in recombinant plant-based meat alternatives with good texture, while developing the hierarchy of a complete meat structure.(5) Whole recombinant plant-based meat alternatives offer a wide range of culinary applicability and scenarios, and they should be developed in-depth. In the future, the simulation accuracy of tissue protein, fat and the skin-layering structure of whole meat should be improved to ensure that it can undergo corresponding changes in cooking while retaining its distinct taste and texture. Recombinant plant-based meat alternatives are an important future research direction in plant meat technology that deserves further investigation.


Diabetic kidney disease (DKD), an emerging global health issue, is one of the most severe microvascular complications derived from diabetes and a primary pathology contributing to end-stage renal disease. The currently available treatment provides only symptomatic relief and has failed to delay the progression of DKD into chronic kidney disease. Recently, multiple studies have proposed a strong link between intestinal dysbiosis and the occurrence of DKD. The gut microbiota-derived short-chain fatty acids (SCFAs) capable of regulating inflammation, oxidative stress, fibrosis, and energy metabolism have been considered versatile players in the prevention and treatment of DKD. However, the underlying molecular mechanism of the intervention of the gut microbiota–kidney axis in the development of DKD still remains to be explored. This review provides insight into the contributory role of gut microbiota-derived SCFAs in DKD. AccordiDKD is a complex disease often related to four main characterized pathological changes, including mesangial proliferation, glomerular basement membrane thickening, podocyte loss, and glomerular sclerosis . The facOver recent years, the research community has attached great importance to the idea of the “gut–kidney” axis, first introduced by Meijiers in 2011 . The incShort-chain fatty acids (SCFAs), a type of saturated fatty acid containing less than six carbon atoms produced by the fermentation of the gut microbiome, are the most studied metabolite . The low22.1via the activation of the generated advanced glycation end product (AGE) pathway, protein kinase C pathway, and polyol pathway, which promotes the expression of cytokines  related to inflammatory pathways and macrophage infiltration leading to insulin resistance, proteinuria, and renal interstitial fibrosis . However, the higher expression of mTORC1 is often detected in rodents with DKD, which leads to the inhibition of autophagy , activation of the protein kinase C (PKC) pathway, and enhanced polyol pathways, which play a significant role in promoting the development of DKD . Long-te33.1Intestinal microbiota dysbiosis was observed in patients with DKD, which was attributed to the limited uptake of foods containing high fiber and fruits, resulting in the insufficient production of SCFAs . SCFAs, 3.2Although a tight control of hyperglycemia is effective against DM and is beneficial to the delay in the progression of DKD, in one study, some patients with diabetes did not reach the stage of DKD even after poor control over hyperglycemia . Multipl3.3via the recruitment of immune cells   . Meanwhi (C5aRA) . The C5- (C5aRA) . In conc3.4Under normal circumstances, the tight junctions, intestinal epithelial cell membranes, mucus secretion, and gut innate immune defensive mechanisms are the four main parts constituting a complete gut barricade defense system, which is of great importance to preclude the entry of hazardous substances or microorganisms from the intracavity to the circulation system ; It is b3.5The human body in a healthy state can tolerate a small number of toxic products derived from gut microbiota. However, the increased production of toxic metabolites damaged the gut barricade due to the disorders of the gut environment and alteration in the composition of the gut microbiota, which led to insulin resistance, energy metabolism disorder and immune-inflammatory response in those DKD diagnosed. Trimethylamine N-oxide (TMAO) is a waste product derived from the digestion of red meat by gut microbiota, which has recently emerged as a gut microbiota-dependent metabolite linked to DKD. TMAO participates in the regulation of lipid and glucose metabolism and influences the gut, liver, kidney, and heart through a physiological connection. Multiple studies indicated that the increased concentration of TMAO in the bloodstream is positively correlated with the occurrence of atherosclerosis, thrombosis, and diabetes . The eleThe above-discussed mechanism of intestinal microbiota dysregulation in the pathogenesis of DKD is summarized in 4Sufficient production of SCFAs ensures the host’s health by exerting multiple protective effects against various diseases, including diabetes, obesity, and cardiovascular disease. This beneficial outcome is contributed by SCFAs in regulating energy metabolism, inhibiting inflammation and oxidative stress, and regulating the immune response. Studies on the role and related mechanisms of SCFAs in the prevention and treatment of DKD are summarized in 4.1via SCFA-mediated GPCR activation  provides a large amount of fermentable substrate for intestinal microbiota and regulates the health of the host in an indirect manner, which is recognized as a promising method to delay the progression of DKD . Becausecrobiota .5.2Lack of exercise, obesity, and stress are the major causes leading to the rising prevalence of DM. It is estimated that about 27% of DM, 30% of ischemic heart disease, and 21%–25% of breast cancer can be attributed to a lack of physical activity . Relevanvia enhancing the upregulated tight junction proteins (Compared with the less active group, the level of bacterial endotoxin was lower in well-trained athletes, accompanied by a higher concentration of heat shock protein, which contributed to reducing intestinal permeability proteins .5.3via significantly downregulated levels of CRP and MDA plus enhancing GSH expression (Clostridium butyricum and corn bran played a positive regulatory role in reducing the abundance of pathogens while enhancing the growth of SCFA-producing bacteria, which further led to an increase in acetate and isovalerate (via interacting with NOD2 (via limiting the production of LPS and TMAO. The intake of probiotics, synbiotics, and postbiotics also helped to suppress the activation of signaling pathways that were responsible for oxidative stress, inflammation, and insulin resistance.Probiotics are a series of indispensable living bacteria or beneficial microorganisms for human health that participate in the synthesis of various vitamins, the regulation of food digestion, the inhibition of the proliferation of pathogenic bacteria, and the degradation of toxic substances. A survey of 340 DKD patients receiving probiotic intervention had shown that the administration of probiotics exerted beneficial effects on reducing the expression of genes that are responsible for the generation of inflammation and oxidative stress biomarkers pression . It was pression . Synbiotvalerate . Postbiovalerate . Furtherith NOD2 . In shor6DKD is a global health problem with a complex mechanism of pathogenesis. A full understanding of the mechanisms underlying DKD is of great significance for laying out an effective treatment plan for the disease. Inflammation, oxidative stress, and autophagy play negative roles in accelerating the progress of DKD. Treatments involving inhibition of inflammation, reducing oxidative stress, and restoring autophagy activation are shown to protect podocytes and decrease proteinuria. Recent progress in the knowledge of the “gut–kidney” axis is a novel perspective for elucidating the relationship between gut microbiota and DKD. SCFAs are beneficial end products derived from gut bacteria, which regulate a variety of host physiological functions. Nonetheless, the mechanism of attenuation of DKD-related complications by the administration of SCFAs is still in its infancy. The beneficial effects of SCFAs may be associated with their ability to regulate energy metabolism, reduce the inflammatory response, and suppress immune effects. It is apparent that the beneficial properties of SCFAs act as a promising biomarker to diagnose and treat DKD. Still, a few questions need to be addressed before the clinical application of SCFAs.First, the binding of SCFAs to GPCRs is necessary for their diverse functions. But there are still many unknown receptors with GPCR-like effects yet to be discovered. Discovering these not-yet-known receptors and developing specific targeted drugs will contribute to solving the puzzle of DKD. Second, most of these SCFA-induced beneficial results were observed in animal experiments and have rarely been performed in humans. Further research should be conducted to confirm whether these benefits could be repeated in humans. Lastly, SCFAs undoubtedly have an irrefutable role in attenuating DKD; any possibility that other metabolites would produce the same beneficial results needs to be investigated.We are confident that these difficulties will and must be overcome to elucidate the detailed mechanism of the function of SCFAs and their promising medical value to mankind.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Some malignancies have very few technologies available for screening, and advancements in cancer therapy have not been as effective in lowering death as those for other chronic diseases. The major method for decreasing cancer incidence is primary avoidance through dietary and environmental changes. The potentially reversible risk factors were projected to be responsible for cancer-related mortality worldwide. Of these fatalities, many of the cases occurred in high-income nations, whereas very few cases did so in low- and middle-income countries. Risk factors in Europe and Central Asia were responsible for the majority of cancer mortality in low- and middle-income regions. Smoking, drinking alcohol, and eating few fruits and vegetables were some of the primary factors that contributed to cancer mortality both globally and in low- and middle-income countries. In high-income countries, alcohol consumption, smoking, and obesity were the main cancer-causing factors. The sexual transmission of the human papillomavirus is one of the leading risk factors for cervical cancer in women in low- and middle-income countries. Mortality rates from all malignancies decreased by 32% globally between 1991 and 2019 in both men and women. This decrease was smaller than the drop in cardiovascular disease death rates, which fell by 9% and 14% in males aged between 30 and 69 and more than 70 and by 15% and 11% in women of similar age ranges, respectively [Changes in risk factor exposure and the accessibility of screening instruments and medical care aﬀect mortality rates from chronic diseases in particular age groups. The death rate from cardiovascular illnesses has decreased throughout the recent decades, at least in industrialized countries, because of advancements in primary and secondary prevention and treatment . HoweverEven in industrialized nations, the impact of treatment on community patterns in cancer mortality is constrained by the availability of therapeutic medicines for other malignancies and access and usage constraints. While combining screening and therapy is improving at lowering cancer-related mortality, this is still the case. Therefore, primary prevention by changes in lifestyle and environment may be the most excellent alternative for reducing the heavy and rising burden of malignancies globally. It is necessary to conduct accurate and comparative evaluations of the impact of cancer risk components at the community level to develop programs and policies to carry out such interventions.The World Cancer Report summarizes several publications that have measured the impacts of the risk factors on cancer incidence and death . HoweverMethodologyAssessed Risk FactorsTo control the cumulative impact of risk variables on cancer deaths, in addition to meta-analyses and comprehensive reviews of the literature on risk components, exposure, and comparative risk conducted as part of the relative risk assessment project ,22, we aProceduresAn expert working committee evaluated published studies and additional sources in depth and methodically to obtain data about risk component disclosure and relative risk for each factor (R.R.). Additionally, the groups collected primary data, reanalyzed primary data sources, and conducted epidemiological investigations of meta-analyses. References listed in Table Analytical StatisticsWe used the equation given in the panel or its discrete counterparts for hazard elements and express publicity information to determine the attributable populace fractions (PAF) for certain hazard factors. We calculated the proportional lower in particular site cancer mortality that might increase if exposure to every risk had been reduced to the counterfactual distribution using PAF for every danger element and most cancers webpage. The theoretical-minimum-chance publicity distribution is the publicity distribution that can bring about the lowest population threat which was used in this observation because of the counterfactual (opportunity) situation Table . This meBecause most cancers are due to many threat factors, PAFs for several threat components for the equal cancer region may overlap and account for more than 100%. For instance, solid fuels  are used in more than 70% of Chinese households for heating and cooking  while >6We calculated all individual and combined PAFs after dividing the population into eight age groups. We extended the PAF by the overall local website-particular cancer deaths for the year 2001 (from WHO databases) for each global financial institution, place, age, sex, and cancer website to decide the wide variety of website-particular most cancers deaths that may be without delay attributed to a hazard element or group of hazard factors. For presentation, fatalities that might be attributed have been combined into three age categories.Role of the Funding SourceThe funding of the study had no bearing on the design, assembly, analysis, explanation, or writing of the research report. Whether to submit the survey for publication rested solely with the corresponding author who had complete access to the study’s data.Outcome measuresTable Cervix-uterine, lung, and esophageal cancer had the highest percentages of cancers (>60%) which may be attributed to these factors. Smoking, alcohol use, a poor diet of fruits and vegetables, and sexual transmission of HPV that results in chronic infection of oncogenic viruses were the greatest risk factors for these malignancies. The malignancies with the lowest cumulative PAFs, leukemia (9%) and colorectal cancers (13%), had a high number of risk factors and diverse and unmeasured exposure patterns among groups, likely reflecting considerable genetic susceptibilities.Lung cancer accounted for 37% of all risk factor-related mortality, liver cancer accounted for 11%, and esophageal cancer accounted for 27% , showing that significant numbers of cancer-related deaths were aﬀected by the union of relatively large joint PAFs  of all cancer mortality in high-income countries; the remaining 72% of cancer fatalities were due to other malignancies. This shows the effectiveness of some preventive measures as well as the disproportionate growth in lung cancer deaths in many high-income countries where smoking incidence is still high.Europe and Central Asia (ECA), which had the greatest joint PAF for all cancer sites combined in low- and middle-income nations, had a 39% rate, whereas sub-Saharan Africa (SSA), as well as North Africa and the Middle East (MENA), had the lowest joint PAF at 24% . ECA had the highest joint PAF among men (50%) and the lowest joint PAF among SSA (19%), with smoking, drinking, being overweight, and obesity having a big influence on both. SSA had the greatest combined PAF for women (29%), while MENA had the lowest (19%). The high mortality rate from cervix uteri cancer, which is significantly impacted by the lack of access to cervical screening and the sexual transmission of HPV, was a major contributor to the high PAF for women in SSA.The majority of fatalities in low- and middle-income countries that could be attributed to the hazards listed in Table MENA and SSA had the lowest cancer fatalities that might be directly associated with the risk variables we studied due to the lower overall number of cancer mortality and the lower PAFs resulting from these hazards.According to estimates, smoking alone is responsible for 21% of cancer mortality globally. Another 5% was caused by drinking and having inadequate amounts of fruits and vegetables. Due to shorter smoking history and a lower incidence among women, smoking caused a huge percentage of cancer mortality in high-income nations (29%) compared to middle- and low-income regions (18%). However, due to the higher overall number of cancer mortality in middle- and low-income countries , the number of smoking-related cancer fatalities was higher. Because there were so many fatalities in ECA and EAP that might be attributed to alcohol consumption, the all-cancer-site PAF for alcohol use was comparable in the two regions. The most fatalities from alcohol-related causes in EAP were related to cancers of the liver and esophagus.Smoking and alcohol use are hazards with proven treatments that have considerably increased cancer deaths in middle- and low-income regions. In hospital settings, contaminated injections caused 108,000 fatalities throughout the six regions, with EAP accounting for 91,000 of those fatalities. In total, 16,000 lung cancer fatalities were caused by indoor smoke from solid fuels, which was exclusively an issue in the EAP and SAR regions where coal is utilized .Obesity and inactivity were the leading causes of all-site cancer deaths in ECA, showing that this group was significantly exposed to these lifestyle-related risk factors, which coexist with the risk from smoking and alcohol use. The risk variables included in this study contributed to 41% of cancer deaths in men compared to 27% in women, or almost two times as much cancer mortality in men as in women. For males, lung cancer accounted for 45% of all fatalities attributed to risk factors, and for women, cervix uteri cancer . Diﬀerences in PAFs and attributable fatalities between men and women were significantly influenced by smoking and alcohol use. Except for cervix, corpus uteri, and breast cancers and their specific risk factors, which virtually exclusively impact women, the total number of fatalities from major cancers and the number attributed to the primary hazards were higher in men than in women Figure . This agDiscussionNine potentially modifiable risk factors account for more than one in three of the 7 million cancer-related deaths globally, with drinking and smoking contributing significantly in both low-and high-income countries. A significant risk component for women is the sexual transmission of HPV, which can result in chronic infection and the spread of cancer-causing virus strains. This is especially true in the poorest regions where access to cervical cancer screening is scarce. This fraction may significantly rise for different cancer regions depending on other potentially modifiable risk variables not mentioned here. Doll and Peto’s  estimateHelicobacter pylori in food, UV radiation exposure, and ambient cigarette smoke. Due to the diﬃculties in obtaining accurate exposure estimates from available data, we mostly disregarded these factors from our analysis.Additionally, just the United States was included in their calculations. Consequently, there is no direct comparison between Doll and Peto’s estimates and ours. Some significant malignancies, such as kidney, prostate, lymphomas, and melanoma, were not linked to any of these risks we examined. These malignancies have several known or improbable behavioral and environmental hazards, as well as varied exposure patterns that make it challenging to quantify exposure and risk. Additionally, we did not evaluate several well-known risk variables, including professional exposure, which caused 102,000 cancer mortality worldwide, exposure to Our estimates are subject to several sources of uncertainty, particularly those that entail extrapolating exposure and risk from one group to another. The explanations about the data sources of each risk factor go into further depth -31. TherH. pylori are currently being researched [In affluent countries, decades of biomedical research have produced several successful therapies that reduce the incidence and mortality of cancer. Examples include the hepatitis B vaccine for liver cancer, screening methods for cervical cancer ,38, fecasearched ,46. The The fixation on healing advanced diseases has hampered the fight against cancer. Additionally, the accessibility and uptake of preventative, screening, and treatment interventions, factors that heavily depend on the cost and the characteristics of the health system, will only impact population statistics if implemented. These obstacles prevent these therapies from being used widely in places with limited resources. These limitations highlight the significance of our findings for programs and policies that alter social and environmental conditions to reduce cancer incidence.


Next-generation sequencing (NGS) applications have flourished in the last decade, permitting the identification of cancer driver genes and profoundly expanding the possibilities of genomic studies of cancer, including melanoma. Here we aimed to present a technical review across many of the methodological approaches brought by the use of NGS applications with a focus on assessing germline and somatic sequence variation. We provide cautionary notes and discuss key technical details involved in library preparation, the most common problems with the samples, and guidance to circumvent them. We also provide an overview of the sequence-based methods for cancer genomics, exposing the pros and cons of targeted sequencing vs. exome or whole-genome sequencing (WGS), the fundamentals of the most common commercial platforms, and a comparison of throughputs and key applications. Details of the steps and the main software involved in the bioinformatics processing of the sequencing results, from preprocessing to variant prioritization and filtering, are also provided in the context of the full spectrum of genetic variation . Finally, we put the emphasis on selected bioinformatic pipelines behind (a) short-read WGS identification of small germline and somatic variants, (b) detection of gene fusions from transcriptomes, and (c) de novo assembly of genomes from long-read WGS data. Overall, we provide comprehensive guidance across the main methodological procedures involved in obtaining sequencing results for the most common short- and long-read NGS platforms, highlighting key applications in melanoma research. BRAF, NRAS, KIT, and NF1 genes . I. I106]. From here on, each step of a typical pipeline for cancer genomics will be briefly described, indicating the most common tools and peculiarities for different types of experiments.Paired-end read sequence data is generally provided as two files in FASTQ format, each file representing one end of the read. The sequence data is stored in the FASTQ files as plain text and contains the sequence of the read and the per-base quality scores. In a typical pipeline, the sequence files are aligned to the reference sequence using an aligner. Different builds of the human reference genome are available, with GRCh37 (hg19) and GRCh38 (hg38) being the most popular. In practice, hg19 is still the most widely adopted, as most vendors provide their probesets in hg19 coordinates . RecentlTo perform short-read alignment of gene panel, WES, or WGS data, BWA-MEM  is one oDepth of coverage is a key metric to evaluate, often defined as the average number of non-duplicated reads that align across the target region. The target region can be the exons of a gene panel, the targeted exons across all genes, or even the entire genome. The level of coverage often determines whether variant discovery can be performed with a certain degree of confidence at a given genomic position. Typically, the coverage needed for an NGS experiment is determined by the method being used and the characteristics of the experiment. This metric should be calculated on both normal and tumor BAM files and can be easily obtained using tools such as Mosdepth . For genThe alignment results in BAM format are subsequently examined for the presence of any type of somatic variation. The accurate identification of mutations is of critical importance. Numerous variant callers are available for this purpose. A list of the most widely used SNV and indel callers can be found in Sequencing the tumor and normal sample genomes allows not only the identification of variants with greater fidelity but also allow the finding the of potential therapies , several tools allow the use of a “panel of normals” (PoN), made out of sequencing data from normal unrelated individuals (N ~50). The PoN can be used to filter out variant calls associated with recurrent technical artifacts, systematic noisy positions, and germline variants, although its effect is limited since this approach does not eliminate the germline variants of the individual.The performance of somatic variation detectors varies widely, as demonstrated in various benchmarking studies, each showing strengths and weaknesses ,151. Thehttps://precision.fda.gov/challenges/22, accessed on 3 September 2022) aim to improve indel detection by validating and benchmarking indel calling pipelines across laboratories.Indels have an important role in tumorigenesis, especially if they affect the coding region, where they can significantly disrupt the reading frame and lead to changes in protein function. Because indels have not been studied as thoroughly as SNVs, tools, and methods for indel detection typically need to be fine-tuned and optimized. In this context, initiatives such as NCTR Indel Calling from Oncopanel Sequencing Challenge , encoding metadata and variant records for each sample. VCF files are often compressed and indexed so that they take up less disk space and can be handled more efficiently by applications. Widely used tools for managing VCF files are BCFtools  and VCFtStructural alterations, including large insertions and deletions, duplications, inversions, translocations of at least 50 bp in size, and gene fusions, have been associated with cancer pathogenesis. Large deletions and amplifications, occasionally spanning genes, or even entire chromosomes, sometimes lead to alterations in gene copy number. This type of SV is usually referred to as CNVs and copy number aberrations (CNA). In most cancer types, including melanoma, a remarkable number of somatic CNAs accumulate during the progression of the disease and have been associated with cancer prognosis and development. CNAs have been directly associated with the expression of driver genes, where copy number changes may increase the expression of oncogenes and decrease the expression of tumor suppressor genes ,154,155.For SV calling based on NGS paired-reads data, SV detection tools typically rely on one or a combination of the following approaches: (a) coverage depth (RD), in which changes in coverage may imply an SV, (b) discordant read pairs (RP) in the alignment, where read pairs map at unexpected distances or orientations, (c) split-read mapping (SR), in which part of the read aligns to either side of an SV, (d) and the assembly approach (AS), which detects SVs by assembly-based sequence reconstruction. The best-performing detection tools usually leverage a combination of some of the above methods .Popular tools for SV detection include Manta , DELLY , LUMPY , GRIDSS In targeted sequencing of a gene panel and WES, only the approach based on the variation in depth of coverage (RD) can be applied; hence, only CNVs can be reliably detected in such experiments. The sparse distribution and small size of exon targets make the relationship between copy number and depth of coverage more complex, making CNV detection less successful. This discontinuous data is skewed by technical limitations arising from GC content bias, non-uniform sequencing depth, and PCR amplification artifacts. In order to mitigate some of these limitations, CNV callers for target sequencing/WES usually perform multi-sample normalization and implement several model-based approaches by using samples sequenced on the same equipment and with the same sequencing kit for better results. Some of the most used CNV detection tools in these experiments are ClinCNV , CNVKit Similar to somatic SNV and indel calling, combining the results of at least two of the tools based on different approaches results in an optimal strategy for somatic SV/CNV calling . In the Although the detection of SNVs and indels based on NGS data can be considered routine, the detection of SVs even with WGS data still poses many challenges. This is mainly because a large fraction of SVs are found in difficult-to-map regions of the genome, such as repetitive regions or tandem duplications, which impose uncertainty during the aligning process. Additionally, short reads are often insufficient to resolve complex SVs and long insertions, as these can be smaller than the SV sizes. All these may result in miscalling events or provide false positive and false negative calls. For this reason, linked reads and long-read-based sequencing are increasingly being applied to the detection of SVs to achieve higher levels of sensitivity and specificity in the studies.This is an active area of interest but is still unresolved. Initiatives such as the precisionFDA challenges are aimed to benchmark the state-of-the-art variant callers in challenging genomic regions, especially those important for medical sequencing ,161.After variant calling, the identified variant callset, including SNVs, indels, CNVs, and SVs, needs to be annotated with functional information to assess the biological implications. The accurate identification of somatic variants is essential to provide potential candidates to be used in targeted cancer therapy . This prAfter the annotation step, the variant calls should be filtered to remove common alignment artifacts and reduce the number of false-positive somatic calls. The annotation information is very useful because it enables the filtering the variant callset. Population filtering is also a common strategy for identifying and filtering likely germline variants from somatic mutation callsets. However, this step must be performed carefully, as common databases such as dbSNP and gnomAD contain several mutations from human tumors, whereas somatic variant catalogs, such as COSMIC, contain germline variants. Similar to SNV and indel calling, CNVs and SVs can be filtered against a PoN to remove variants in highly variable regions and artifacts. Germline SV databases such as gnomAD-SV can also be used to filter SVs that are variable in otherwise healthy human populations.A manual review of tumor and normal sequencing alignments using visualization tools such as IGV  can helpBesides manual review, a subset of the detected variants can be independently validated by orthogonal approaches, such as Sanger sequencing. Despite the filtering protocols that can be implemented, most NGS methods detect many more candidate variants with likely functional effects than it is possible to validate experimentally as part of a project. Variant prioritization is a common practice to obtain a manageable set of variants. Although variants can be ranked based on various parameters, the prioritization of candidate variants that may be related to the disease is a multifactorial problem, and generally represents a bottleneck in cancer genomics. Open access databases such as the Clinical Interpretation of Variants in Cancer (CIViC)  have accHigh-throughput sequencing enhances the study of tumor evolutionary patterns, enabling the deciphering of all mutations in tumor clones . Tumor cAssuming that almost all somatic variants in tumor cells are heterozygous, the proportion of tumor cells with the same mutation is twice the VAF value. This means that a specific variant present in 80% of tumor cells will have a VAF value of 40%. Using a VAF density plot, tumor clones and the present variants in each clone can be represented by each peak in the plot, helping us to infer and identify clonal progression . InitialGene fusions  can be caused by somatic chromosomal rearrangements involving large SVs or chromosomal translocations. Gene fusions have been involved in the progression of a variety of cancers , includiThe most common way to shed some light on cancer-related gene fusions is to run de novo assembly and annotation of transcripts using short reads from RNA-Seq . To carrThe massive amount of data generated by NGS technologies need to establish standardized procedures, guidelines, and rigorous QC steps to ensure accurate results. More importantly, these steps are key for the implementation of NGS technologies in clinical areas, where high-quality data and reliable results are fundamental . Many QCTraditionally, pedigrees were the gold standard to infer relatedness, but the advent of new technologies and the availability of different genetic markers make it possible to evaluate relatedness using many approaches. Tools to infer relatedness exist for the SNP array technologies, such as KING , REAP 1, or KINDThe genetic inference of the sex of a sample from its sequence obtained in an NGS experiment is a mandatory QC step to detect errors in the metadata, which helps to improve sample traceability. Several bioinformatic tools are available to infer the biological sex of the sample based on the proportion of reads aligning to X and Y chromosomes, such as XYalign  Figure . Some otA broad range of long-read sequencing technologies, also known as TGS, is flourishing and being used to improve our knowledge of complex regions and SVs that were difficult to resolve or were missed by short-read sequencing analysis . Long-reSeveral long-read sequencing technologies have been developed recently . HoweverONT has marketed a number of platforms such as the MinION, GridION, or the PromethION, among others, which are capable of sequencing ultra-long fragments of DNA or directly of RNA, offering also real-time analysis . The MinPacBio uses proprietary SMRT  technology , a methoAnother approach, known as linked-read technology , was devIn 2020, the T2T announced the first gapless de novo assembly of a human X chromosome using ultra-long DNA reads from ONT sequencing of the T2T-CHM13 genome . In 2022Long-read sequencing offers several advantages over short-read sequencing approaches. They are particularly useful in de novo assembly strategies because, by solving the low complexity and repetitive regions, they can reconstruct accurate and high-resolution genome assemblies . This alAs previously mentioned, TGS has several technical advantages and improvements compared to the traditional NGS. However, there still exist some limitations and challenges that are associated with these technologies, which may explain why their use in cancer research is not that widespread yet, particularly in the case of melanoma . The maiAs discussed before, long-read sequencing platforms offer advantages for the de novo assembly of genomes compared to the classical NGS methods . ONT andONT provides a comprehensive range of DNA and RNA library preparation kits, offering high throughput with low DNA input, fast modes for library preparation, and long reads. This technology has a wide variety of solutions, including WGS, targeted sequencing, and RNA sequencing, among many others. Whereas with other sequencing technologies the read length is limited by the technology itself, in the case of long reads this limitation is given by the quality and size of the starting DNA material. Thus, the challenge is in how to improve DNA extraction to preserve DNA purity and integrity. For analyzing human cancer WGS, one can proceed with the ONT ligation-based library preparation kit (SQK-LSK110) and a PromethION platform, which can generate up to 150 Gb of sequence per flow cell and offers 30× coverage WGS for less than 1000 dollars .Signals from the sequencing process are stored within FAST5 format files and could be processed by basecalling algorithms, such as Guppy or Bonito (ONT), to decode the sequence into FASTQ files. Basecalled reads can be inspected using tools such as pycoQC  Figure  or NanoPIn recent years, the research activity involving long-read technologies has grown rapidly ,241. ThiDe novo assembly of genomes is one of the main uses of the long reads generated by ONT and PacBio sequencing. The main goal of de novo assembly is to attempt to reconstruct the whole genome sequence without information from a reference genome ,262 or iThe quality of the assemblies could be improved by combining data from different sequencing technologies, such as long reads from ONT and short reads from Illumina, Inc. With this approach, tools such as MaSuRCA  run hybrIdentification of small variants (SNVs and indels <50 bp) is essentially well resolved with short reads. However, the difficulty increases when it comes to detecting larger sequence mutations with this technology , and it SV methods with long reads are mainly focused on the following two strategies: one based on aligning reads to a reference and another based on de novo assembly. Approaches that use de novo assembly are based on assembling reads to longer sequences, namely, contigs or scaffolds, and discovering SVs by comparing aligned reads to the assembled sequences and to a reference . On the A large number of tools rely on these methods, and their comparison helps in the selection of the optimal tool and the strategy for SV calling . The appNGS platforms have made a huge impact in the characterization of human tumors, helping to understand and identify several types of cancer and establish new targeted treatments. Moreover, despite the technical challenges that are due to the quality and quantity of the tumor samples, advances in sample preparation methods have enabled the full characterization of cancer genomes, transcriptomes, and epigenomes . These aBioinformatics pipelines for the analysis and clinical interpretation of cancer genomic results have been implemented in many platforms and laboratories. Likewise, multiple computational tools have been developed for the analysis of oncological NGS data. Most of these tools are used through the command line, sometimes being complex to parameterize and optimize. The choice and application of these tools depend on the characteristics of each specific project. Thus, the optimal configuration must be determined empirically for the sequencing strategy, the sample type, and the computational resources.Advances in NGS technologies, improvements in the variant detection algorithms, and further development of specific human cancer databases and functional annotations of the genetic variants, as well as the reduction of the cost of sequencing, will impact the field of cancer genomics, bolstering the development of better treatments . All of Optimal annotation and prioritization of variants is also a bottleneck in current precision oncology. This process requires databases containing curated variants as well as links with mechanistic effects and potential drug interaction data. However, simple and completely automated methods to carry out these processes are not yet available. In addition, large datasets of cancer patients, including their response to therapy, are needed so that effective machine-learning-based algorithms can be designed.Long-read sequencing enables more comprehensive analysis of cancer genomes, solves complex genomic aberrations, improves the study of long transcript isoforms, and epigenomic modifications. The use of long reads in a multitude of cancer types is widespread, despite it not yet being leveraged in cutaneous melanoma. However, this technology still has several limitations to solve, including the higher error rates and the difficulty of obtaining high-molecular weight DNA material in sufficient quantities from the most common types of biobanked tumor samples .Because of the multiple advantages, the tremendous growth of applications, and the steep reductions in the cost per base, NGS has revolutionized research in melanoma and cancer genomics in the past decade. Despite the recent advances in the technology, including moving towards leveraging longer reads to optimally assess SVs, which are central in cancer, these improvements still lack a translation to the field. In order to continue pushing this field further, it will also be important to standardize the bioinformatics procedures and update the annotation databases with the most recent and complete references, as they will allow us to better identify somatic and germline sequence variation that is key in the pathogenesis of melanoma.


Real-world evidence (RWE) is increasingly involved in the early benefit assessment of medicinal drugs. It is expected that RWE will help to speed up approval processes comparable to RWE developments in vaccine research during the COVID-19 pandemic. Definitions of RWE are diverse, marking the highly fluid status in this field. So far, RWE comprises information produced from data routinely collected on patient’s health status and/or delivery of health care from various sources other than traditional clinical trials. These sources can include electronic health records, claims, patient-generated data including in home-use settings, data from mobile devices, as well as patient, product, and disease registries. The aim of the present update was to review current RWE developments and guidelines, mainly in the U.S. and Europe over the last decade. RWE has already been included in various approval procedures of regulatory authorities, reflecting its actual acceptance and growing importance in evaluating and accelerating new therapies. However, since RWE research is still in a transition process, and since a number of gaps in this field have been explored, more guidance and a consented definition are necessary to increase the implementation of real-world data. Real-world evidence (RWE) research has received attention in the recent decade and is playing an increasing role in health care decisions . As showThe present review indicates, in its first chapter, current RWE gaps and highlights the chronological development of RWE, including the actions taken and guidelines released by the regulatory authorities. The second chapter describes the current situation of RWD in accelerating the orphan drug market and early drug assessment. In the third chapter, the contribution of real-world results during the COVID-19 pandemic and how it impacted the understanding of vaccine research is described; the fourth chapter reviews the importance of high-quality cancer registries as a growing RWE source in oncological research. The following two chapters, chapters five and six, explore the ways in which RWE facilitates RCTs and digital health technology, respectively. In chapter seven, four FDA-funded projects of RWE are introduced. The current review generates a chronological overview of RWE development over the past decade and contextualizes lessons learned from research in oncology and the COVID-19 pandemic with actual perspectives from clinicians.As RWE has received increased attention over the past decade ,7,8, sevThe Cures Act had the intention to accelerate medicinal product development and to effectively delivering medicinal advances to patients. Subsequently, the FDA released a framework in 2018 to evaluate RWE sources ,9.In the following years, a series of several other guidelines for the use and submission of RWE documents were released, such as the guidance on the use of RWE to support regulatory decision-making for medical devices (2017) and the industry guidance on the use of electronic health record data in clinical investigations (2018) ; see TabMeeting the gap of limited access to RWE due to multiple sources and interoperability, the European Medicines Agency (EMA) released a draft guidance on studies based on registry data as one source of RWD for marketing authorization applicants in 2020 ; see TabIt is believed that implementation of RWE in early assessments of newer therapies may speed up approval processes of effective therapies and reduce costs of drug development . To presDuring the COVID-19 pandemic, the number of scientific questions to be answered surpassed the capacity of the available means to conduct RCTs. RWE studies were the primary source of evidence during the COVID-19 pandemic that reported on patient symptoms and the influence of patient characteristics and risk of morbidity and mortality ,26,27. Ivice versa, feed data from healthcare into guideline updates [A first systematic review evaluated oncological new drug applications and biologics license application between 2015 and 2020  and foun updates . Already updates . In the  updates . Using h updates .In 2020, the MHRA released guidance on RCTs generating RWE to support regulator decisions . The perIt is widely acknowledged that RWE more likely reflects real treatment effects and is more generalizable than evidence from standardized clinical trial data ; see FigNewer RWE methods including digital health technologies (DHTs), data linkages, and registries have an expanding role in health care, offering considerable opportunities for drug development . DHTs coAlongside the FDA framework to explore RWE sources, in November 2020, the FDA announced four grant awards on the utility of RWD to accelerate regulatory decision-making ; see TabThe present review provides an overview of the different definitions, types, and utilities of RWE, how it has been developed over the last last decade, gaps and challenges, and the necessary regulatory guidances in this field to accelerate regulatory procedures and decision-making. In addition, it describes hurdles and potentials of RWE research, and contextualizes lessons learned from oncological research and the COVID-19 pandemic with the actual perception of clinicians. Hereby, the FDA in the US as well as the EMA in Europe play a pro-active role in exploring gaps and potentials of RWE and in guiding stakeholders in this field with the release of actual and appropriate guidelines. In addition, international RWE initiatives and registries have increasingly been developed supporting this process. Provided the data quality is robust and transparent, RWE research is complementing RCTs for identifying research gaps and for the evaluation of treatment effectiveness, especially in diverse patient sub-groups. Catalyzed by the COVID-19 pandemic, the general perception of RWE among clinicians is that it produces important data for informing treatment decisions. In addition, the ongoing implementation of RWE in early benefit assessments of newer therapies will potentially speed up approval processes of effective therapies and reduce costs of drug development. Newer research such as the implementation of registry data and various types of DHTs will grow in the near future, reflecting the dynamic state in this field. By funding RWE projects and by involving stakeholders in the development of future guidelines the trust, credibility, reproducibility, and transparency of RWE studies may further be promoted. This will help in speeding up and reducing costs of the delivery of patient-oriented effective therapies, adding beneficial value to public health.


The vaccine hesitancy is a matter of global concern with inadequate global uptake postponing the moment of reaching herd immunity and bringing the COVID-19 pandemics under control. Countries in the Western Balkans struggle with vaccine hesitancy, trying to bring vaccine acceptance and ways to improve it into the focus.A cross-sectional study on vaccine hesitancy was conducted from July to September 2021 and included adult population from Albania, Bosnia and Herzegovina, North Macedonia, Montenegro and Serbia . Convenience sampling was applied using anonymized online questionnaire  measuring, among others, trust in societal factors, social responsibility and, the credibility of information sources about COVID-19 vaccines.The highest degree of trust in societal factors was found in North Macedonia , followed by Montenegro  and Serbia . In Albania 44.7% respondents believed in reluctance of pharmaceutical companies to publish detailed research reports on the risks of adverse reactions to COVID-19 vaccines. The view that the health authorities when they encourage vaccination do so with the best intentions supported 66,3% respondents in North Macedonia and 49% in Albania and Serbia. The highest level of social responsibility  was revealed in North Macedonia. Primary care physicians, health professionals in media, webpages of public health institutions, and scientific literature are the most trusted sources of information about COVID-19 in all countries.The study demonstrated moderate trust in societal factors and moderately high level of social responsibility in all countries. The health professionals enjoy the greatest trust, which implies that medical doctors, especially physicians in primary health care should have a pivotal role in promoting vaccination and educating the general public in the Western Balkans.


Progressive loss of telomere repeats limits the proliferation of pre-malignant human cells but also contributes to aging by inducing apoptosis and senescence in normal cells. Despite enormous progress in understanding distinct pathways that result in loss and gain of telomeric DNA in different cell types, many questions remain. Further studies are needed to delineate the role of damage to telomeric DNA, replication errors, chromatin structure, liquid-liquid phase transition, telomeric transcripts (TERRA) and secondary DNA structures such as guanine quadruplex structures, R-loops and T-loops in inducing gains and losses of telomere repeats in different cell types. Limitations of current telomere length measurements techniques and differences in telomere biology between species and different cell types complicate generalizations about the role of telomeres in aging and cancer. Here some of the factors regulating the telomere length in embryonic and adult cells in mammals are discussed from a mechanistic and evolutionary perspective.The number of (TTAGGG) As aDrosophila melanogaster uses a transposition mechanism to replenish the DNA lost with each DNA replication cycle [reviewed in  documented heterogeneous telomere length distributions as well as sporadic loss of telomeric DNA . LagginThe switch between a protected telomere end and a chromosome end that signals DNA damage has many levels and components. Many of these components themselves have switch-like characteristics in that two alternate states can be present or selected. For example, it is possible that telomere damage signaling involves a liquid-liquid phase separation in the nucleus between heterochromatin and euchromatin  differ from Mus spretus mice with relatively short telomeres (~10 Kb) in RTEL1, the Regulator of Telomere Length helicase predicted to unwind guanine G4 structures at telomeres . First in utero . The twoin utero , 58. At in utero . Perhapsin utero . Telomerelomeres , 36, 61.elomeres , primer elomeres , 64 in telomeres , RAP1  alpha  alpha . The reliewed in . Cultureiewed in . More gein utero growth and differentiation to limit the number of subsequent cell divisions. Both alternative splicing and transcriptional regulation of the telomerase transcriptase gene (hTERT) have been implicated in the silencing of telomerase activity  from the Terry Fox Research Institute, a Project Grant (#PJT-159787) from the Canadian Institutes of Health Research, and a grant (#40044) from the Canadian Foundation for Innovation and the Government of British Columbia.The author is a founder and shareholder of Repeat Diagnostics Inc., a company specializing in clinical telomere length measurements.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background/Introduction: Plasma protein binding (PPB) continues to be a key aspect of antibiotic development and clinical use. PPB is essential to understand several properties of drug candidates, including antimicrobial activity, drug-drug interaction, drug clearance, volume of distribution, and therapeutic index. Focus areas of the review: In this review, we discuss the basics of PPB, including the main drug binding proteins i.e., Albumin and α-1-acid glycoprotein (AAG). Furthermore, we present the effects of PPB on the antimicrobial activity of antibiotics and the current role of PPB in in vitro pharmacodynamic (PD) models of antibiotics. Moreover, the effect of PPB on the PK/PD of antibiotics has been discussed in this review. A key aspect of this paper is a concise evaluation of PPB between animal species  and humans. Our statistical analysis of the data available in the literature suggests a significant difference between antibiotic binding in humans and that of dogs or mice, with the majority of measurements from the pre-clinical species falling within five-fold of the human plasma value. Conversely, no significant difference in binding was found between humans and rats, rabbits, or monkeys. This information may be helpful for drug researchers to select the most relevant animal species in which the metabolism of a compound can be studied for extrapolating the results to humans. Furthermore, state-of-the-art methods for determining PPB such as equilibrium dialysis, ultracentrifugation, microdialysis, gel filtration, chromatographic methods and fluorescence spectroscopy are highlighted with their advantages and disadvantages. Plasma protein binding (PPB) is essential for predicting and understanding several important properties of novel antibiotics, including volume of distribution, clearance, therapeutic index and drug-drug interaction ,2,3. PlaThe pharmacological effect of a certain drug is dependent on both its pharmacokinetic (PK) and pharmacodynamic (PD) properties. The effects of PPB on the PK of antibiotics have been studied extensively , but wheHere we provide an overview of the effects of PPB on the antimicrobial activity of antibiotics and the role of PPB in in vitro PD models of antibiotics. By comparing human PPB data for a variety of drugs with PPB data from different animal species, we gain insight into the potential of extrapolating PPB and PK/PD data from animals to humans.Several proteins are involved in the protein binding of antibiotics including plasma proteins, extracellular tissue proteins and/or intracellular tissue proteins. In vivo, drug molecules are either bound to plasma or tissue proteins, or in an unbound/free state. Moreover, PPB properties greatly vary between drugs ,16. SincOnly the free/unbound fraction of small molecules diffuses into the extravascular space where it exerts pharmacological activity and may cause side effects ,23,24. TThe effect of PPB on PD of antibiotics can be assessed in various settings. For example, studies assessing the impact of PPB on the efficacy of an antibiotic against a particular pathogen might reveal variations in the corresponding MIC . AlthougEquilibrium dialysis, ultracentrifugation, microdialysis and ultrafiltration are the most common methods for separating bound and unbound fractions of a drug ,54. TablEquilibrium dialysis is considered the reference method ,54,55,56Another common method for quantifying the free concentration of a drug in plasma is ultrafiltration ,58. An uIn vivo microdialysis allows estimation of unbound drug fractions in the interstitial fluid of various tissues, and quantification of PPB in blood . MicrodiUltracentrifugation is commonly used to estimate the free fraction of lipophilic compounds, by dividing human plasma into three layers by ultracentrifugation, which are then analyzed individually  Figure .In gel filtration , after passing through a dextran gel column, the drug-protein solution is divided into two sections, one containing the free drug and the other containing the protein and protein-bound drug . HoweverSeveral chromatographic methods are available for the separation of different proteins, including high-performance liquid chromatography (HPLC) and gel permeation chromatography (GPC). Chromatographic separation through an HSA-immobilized column allows relative ranking by percent binding. Moreover, equilibrium gel filtration (EGF) with liquid chromatography-tandem mass spectrometry detection has been suggested to be used earlier in the drug discovery process to measure the free concentrations of highly bound drugs without the need for radiolabeled compounds ,76. EGF For preclinical findings to be translated into clinical applications, the differences in physiological processes between animals and humans are important and must first be understood ,78,79. TPPB has received little attention in the discovery and development of antimicrobial agents in recent decades. Equilibrium dialysis and microdialysis are common methods used for protein binding studies. Designing studies to investigate PPB from a PK or PD perspective and generating adequate PPB data in the preclinical setting must be carefully planned, especially when a new drug project enters the investigational phase.The present review provides information on whether animal models are adequate predictors of human PPB by analyzing data from the literature. Rats, rabbits, and monkeys exhibited similar PPB characteristics to humans, whereas significant differences were found in dogs and mice compared with humans. Depending on the drug and drug delivery platform, species-specific physiological differences may result in incorrect extrapolation of drug PK, safety and efficacy. On the other hand, interspecies differences in protein binding as a result of PK variations could indicate potential variations in drug effects when used in human patients. Therefore, we advocate the standardization of experimental settings to study PPB and its effect on PK/PD of novel antibiotics. In addition, we recognize the relevance of interspecies differences in PPB but appreciate the potential of extrapolating from preclinical pharmacology and safety studies in different species to estimate outcomes in humans.


Total knee arthroplasty (TKA) is widely used in clinical practice as an effective treatment for end-stage knee joint lesions. It can effectively correct joint deformities, relieve painful symptoms, and improve joint function. The reconstruction of lower extremity joint lines and soft tissue balance are important factors related to the durability of the implant; therefore, it is especially important to measure the joint lines and associated angles before TKA. In this article, we review the technological progress in the preoperative measurement of TKA. Total knee arthroplasty (TKA) is the most conclusive treatment for severe knee osteoarthritis, and its efficacy relies on the reconstruction of joint lines and soft tissue balance in the lower extremity. The reconstruction of the knee joint, among various structures in the human skeletal and soft tissue systems, is the most complex, for it requires the integration of anatomy, kinesiology, biomechanics, and even tissue engineering. In the reconstructed knee, it is important to take into account the reconstruction of joint lines, the soft tissue balance, and patellar tracking so as to achieve the “forgotten knee” status. The ultimate goal of accurate reconstruction is pursued by orthopaedic surgeons of all ages. In order to achieve more accurate alignment, more perfect soft tissue balance, and better prosthesis matching so as to obtain longer prosthesis life, better postoperative function, and higher patient satisfaction, AI-assisted TKA preoperative design can accurately carry out preoperative planning, artificial intelligence segmentation, and key point recognition of joint CT data and establish standardized measurement model by adjusting the joint orientation. After introducing the open/closed wedge parameters or prosthesis parameters, one must accurately judge the shape parameters, such as the height and inclination of the osteotomy surface, and finally form a highly reliable surgical prediction model and simulate the postoperative image.Therefore, thorough preoperative planning, that is, the measurement of joint lines and angles, helps guide the surgeon during the procedure. Preoperative measurements taken for TKA include traditional plain film radiographic measurements, digital templating, computer navigation, and artificial intelligence (AI) measurements. AI has multiple definitions and is largely agreed upon as the intelligence demonstrated by machines. In the field of computer science, AI research is defined as the study of “intelligent agents”, which are “devices that perceive the environment and act to maximize the achievement of a goal”. In a broad sense, process digitization, computer navigation, and robotics all fall into the category of AI. Existing AI-assisted technologies have enabled modern medicine to make great strides toward precision, intelligence, and individualization. This article reviews the advances in AI-based preoperative measurement for TKA and the problems associated with various measurement techniques.In the past, preoperative measurements for TKA relied on traditional plain film radiographic measurements. Preoperatively, a full-length film of the patient’s bilateral lower extremities was taken in the standing position along with the anteroposterior (AP) and lateral views of the affected knee joint and an axial film of the patella. Further, the surgeons needed to manually trace the femoral anatomical axis, tibial axis, and lower limb joint lines on the full-length film of bilateral lower limbs to determine the valgus angle. The disadvantage of this type of film template measurement based on X-ray films is that the magnification of X-ray images varies among different hospitals, thus affecting the accuracy of measurements made using the same film template. The basis for testing the practical value of preoperative template measurements is that the results of different surveyors are consistent under different conditions ,2. StudiThe initial digital measurement is based on the measurement function that comes with each hospital’s medical imaging information system. These measurements require full-length films of both lower extremities, AP, and lateral knee films. Instead of traditional film measurements, computed tomography (CT) images of lower limbs need to be taken. The joint line and valgus angle of the lower extremity are measured in the same way as in conventional film measurements. The difference is that the medical imaging information system allows accurate labelling of the trans-epicondylar axis (TEA) and the Whiteside’s line based on CT images of the lower extremity at the selected knee level, and the external rotation angle in osteotomy can be determined by the posterior condylar line (PCL) of the knee. However, in the past, based on the preoperative CT markings of the TEA and the Whiteside’s line or the default 3° of external rotation for osteotomy, the intraoperative accuracy was 90%, 83%, and 70%, respectively. The medical imaging information system allows only preoperative measurements of all angles but does not simulate the intraoperative knee osteotomy and the post-osteotomy prosthesis placement. In TKA, according to the isometric osteotomy method, the specific thickness of the osteotomy also affects the choice of the size of the prosthesis. The accuracy of the digital planning software in predicting the size of tibial prosthesis has been reported to be as low as 63%, and that in predicting the size of femoral prosthesis is 69% . SchotanIn the field of orthopaedic surgery, digital technology has made it possible to digitally simulate surgery. The CT scan of the patient’s knee joint, with a slice thickness of 1 mm, is obtained in the DICOM format, and the obtained data are input into the corresponding digital software to reconstruct a three-dimensional (3D) bony model of the knee joint. Further, calibrated weight-bearing full-length AP and lateral radiographs of the lower limb are taken, and the statistical shape model (SSM) technique is used to derive the 3D model of the knee joint. The 3D model is then automatically aligned with the CT-reconstructed bony 3D model of the knee joint to obtain a 3D spatial model of the knee joint and the full-length of bilateral lower limbs. After scanning all models of the selected prosthesis, the installation and alignment of the prosthesis are simulated on the 3D model using the measured joint lines and angles. The application of such digital simulation technology in the preoperative measurement of TKA has enabled the lower extremity joint line measurement and osteotomy and prosthesis installation to be more accurate, which is a great leap forward in joint surgery measurement technology. It has been confirmed ,7 that tIn computer-assisted orthopaedic surgery, information contained in the multimodal imaging data is digitally processed and combined with stereo-navigation systems to identify and display anatomical structures. The computer plans the surgical path and formulates a surgical plan for preoperative surgical simulation under the guidance of appropriate image monitoring and stereo-navigation systems. A certain guidance system is used to perform intraoperative surgical interventions, wherein computers and medical robots assist surgeons to complete the operation. Numerous studies have confirmed that computer navigation-assisted total knee arthroplasty (TKA) can improve the accuracy of lower extremity alignment and prosthesis placement ,9,10,11.In the field of orthopaedics, computer-aided systems were first used in spine surgery, and Saurol et al. first developed and applied a pedicle screw navigation system in 1992. In 1993, research on knee surgical navigation systems began in France, and Sal’agaglia’s  group piComputer navigation systems can be divided into two broad categories: The first is based on imaging data (CT and MRI), wherein preoperative imaging is entered into the computer, and preoperative images based on the recorded anatomical landmarks are compared with the intraoperative morphology. The second type of navigation system does not require imaging data. Instead, it contains a large amount of anatomical data and determines the movable surface of the knee and the alteration of the biomechanical axis of the femur and tibia through passive flexion and extension activities of the knee joint . The dif®, an image-free, infrared-based computer navigation system from Aesculap , VectorVision® from Brainlab, and the navigation system from Stryker . Computer navigation systems can accurately calculate the patient’s hip–knee–ankle angle and clearly mark the lower extremity joint lines and the mechanical axes of the femur and tibia, ensuring accurate osteotomy intraoperatively. As per a meta-analysis by Mason et al. [The main knee joint navigation systems currently used in China include OrthoPilotn et al.  includinn et al. ,16.®, have the advantages of a shorter operation time, shorter learning curve, and lower cost over traditional computer navigation. The perception of spatial position is through precise accelerometers and gyroscopes, and some studies have shown that this system has significant advantages in osteotomy of the femoral condyle and tibial plateau [® navigation system is that it is not able to plan the type of prothesis to be used in advance and requires the surgeon to decide on the type of prothesis during the surgery using traditional surgical methods. The system requires intraoperative rotation of the limb to obtain the 13 femoral positioning points for calculating the centroid of the femoral head, which, together with other intraoperative positioning manoeuvres, takes more time than conventional surgery.Although computer navigation systems can improve the accuracy of joint line estimation and prosthesis placement in TKA, there are no clear studies showing that the application of computer navigation significantly improves lower extremity function after TKA. Accelerometer-based portable navigation systems, such as OrthAlign™ and iASSIST plateau ,18. Iori plateau  performe plateau  also indNotably, the development of novel computer navigation systems has risen markedly in recent years. The new computer navigation system, Knee 3 software, from Brainlab, Germany, was launched in China in 2020. The system comes with the advantage of simple and fast registration. It enables intraoperative visualization of osteotomy positioning and dynamic real-time display of knee flexion and extension gaps throughout the procedure, which helps the surgeon create individualized lower extremity alignment and soft tissue balance in patients. Compared with conventional computer navigation systems, Knee 3 software not only ensures accurate osteotomy but also improves the soft tissue balance in the knee joint. The Knee 3 system provides real-time intraoperative display of the joint gap in the form of gap values and gap graphs, allowing the operator to visually assess the knee flexion and extension gap balance. When planning the surgery, the surgeon can adjust the angle and position of the femoral and tibial osteotomy according to the patient’s lower extremity joint lines, and the system can also display the impact of the corresponding osteotomy changes on the joint space in real time. The disadvantage of the Knee 3 system is that it includes system setup, tracker fixation, and registration steps. Moreover, since the Knee 3 software dynamically displays the lower limb joints line and knee gap information in real time, the operator needs to quickly interpret the relevant information and devise a suitable surgical plan, which increases the operation time. In addition, because computer navigation-assisted surgery requires the installation of fixation pins to fix the tracker, there is a risk of fracture ,22,23,24On 19 July 2022, Enovis announced the launch of ARVIS  and successfully completed more than 200 clinical cases in the United States. This FDA-approved system is the only proprietary real-time hands-free augmented reality (AR) technology designed for surgeons, allowing them to conduct real-time visualization and precise guidance of hip and knee joints with the support of AR. AR technology is a form of surgical guidance technology, which can help surgeons place and align implants during surgery. Unlike other traditional robot systems that require additional personnel, ARVIS is an independent wearable surgical guidance device controlled by surgeons, which can be worn on the ARVIS headband and is compatible with helmets that can be worn during surgery, thus ensuring that surgeons can focus on their patients through the surgical view. ARVIS is the first system with proprietary hardware designed to help surgeons accurately place hip and knee replacements to help improve joint replacement recovery results. ARVIS is more sustainable and environmentally friendly than other technologies because it eliminates disposable plastic instruments and consumables that need to be handled and is approved in the United States for navigation of total hip, total knee, and single-space knee arthroplasty. ARVIS can provide AR navigation support for orthopaedic doctors, such as visual navigation during knee surgery.Some scholars do not support the inclusion of patient-specific instrumentation (PSI) under the category of artificial intelligence, though the authors do not agree with this view. PSI also requires data on the hip joint, knee joint, and ankle joint of the patient to be obtained through CT imaging, which are then digitally processed. The two-dimensional data are reconstructed into a 3D model, and then the corresponding osteotomy volume, angle, and prosthesis type are determined. After planning, the PSI is automatically generated, and the personalized osteotomy surgical guide is obtained via 3D printing technology . Several studies have shown that the use of 3D-printed osteotomy guides can achieve favourable lower limb force lines during TKA ,27, shorBefore robot-assisted total knee arthroplasty, the AIHIP/KNEE software system was used in the preoperative planning of hip arthroplasty or knee arthroplasty. The system applies computer 3D data analysis based on CT or MRI, applies computer depth learning algorithm to segment joint CT data and identify key points, establishes a standardized measurement model, and then introduces prosthesis parameters to accurately judge parameters, such as osteotomy plane height and posterior inclination angle.Institutions performing robotic research in China and abroad have explored the introduction of robotic surgery in arthroplasty, and the use of robot-assisted TKA systems is an historic moment. Robot-assisted TKA is currently the highest-level embodiment of artificial intelligence (AI) in knee surgery.AI technology was once limited by basic hardware, barren databases, deficits of algorithms, and so on.Alan Turing first invented artificial intelligence in 1950. He established the Turing test, which is the earliest prototype of AI. Turing proposed that the concept of AI technology is a computer algorithm similar to human brain intelligence, but at the same time, its complexity and efficiency are no less than or even higher than human brain intelligence. At that time, due to the backwardness of basic hardware equipment, poor databases, lack of algorithms, and other limitations, the development of AI technology was basically at a standstill. However, in recent years, the rapid development of computer technology and Internet technology has brought about the improvement of computer hardware, computing power, and large databases; thus, intelligent algorithms, such as machine learning, reinforcement learning and deep learning have gradually emerged, which have ushered in the great-leap-forward development of AI technology and produced several macro technical directions, such as computer vision, speech recognition, natural language recognition, decision planning, and big data analysis. Great breakthroughs have been made in the intelligent identification, understanding, and decision-making of data. During this period, many scholars published important works on AI , and theIn recent years, the rapid development of computer technology and the internet have granted AI intelligent algorithms for machine learning, reinforcement learning, and deep learning, in particular, convolution neural networks, cyclic neural networks, and recurrent neural networks. AI technology has ushered in development by great leaps and is, thus, being widely used in joint surgery.Robot-assisted preoperative planning system includes mainly high-precision 3D reconstruction based on CT and MRI images, image-based determination of preoperative osteotomy area and osteotomy plane, joint prosthesis design and preparation, preoperative joint line measurement, biomechanical analysis of the knee joint after prosthesis implantation, and so on. Current robotic systems can be divided into three categories according to their working modes: active, semi-active, and passive. The commonly used robot systems are shown in The active robot does not require manual operation and operates according to the preoperative plan. However, the system has potential surgical risks, and its working process needs to be monitored by the doctor throughout to avoid accidents. The semi-active robot, also called the haptic robot, is used to perform surgical planning before the operation, and during the operation, the surgeon completes the operation with the help of the robotic arm. Moreover, if the intraoperative osteotomy exceeds the preoperative planning range, the system gives feedback to the surgeon by means of a sound and force feedback, and the robotic arm automatically stops the operation, thus ensuring that the surgical path and the range are consistent with those planned preoperatively ,32.At present, several robotic-assisted arthroplasty surgery systems have been developed in the United States, Japan, Israel, and the United Kingdom, including the MARS surgical system developed by the Research Office of Mechanical Engineering Department in Israel , the 7-dROBODOC robots were first introduced for TKA in 2000, received FDA approval in 2008, and were renamed THINK robots in 2014. In addition to the ROBODOC and MAKO systems, there are also the NAVIO and iBlock systems. NAVIO is a handheld robot that can be manipulated by a surgeon . The sysCurrently, several hospitals in China are collaborating on the development of surgical robots for joint surgery. The computer-assisted orthopaedic surgery system, WATO, developed by the Department of Orthopaedics of Shanghai Changzheng Hospital and Shanghai Jiao Tong University Institute of Image Processing and Pattern Recognition in 2009, provides the surgeon with a surgical plan based on CT images before surgery, albeit with the disadvantage that the surgical incision is larger than that of conventional surgery . The GusThe significance of robot-assisted joint replacement is to greatly improve the accuracy of intraoperative operation, which benefits from three guarantees: (1) accurate measurement and planning of preoperative CT data; (2) the stable assistance of the mechanical arm during the operation; and (3) timely registration and monitoring during operation. The shortcomings and complications of early robotic joint surgery, after years of continuous correction and improvement, have no longer become the limiting factor of surgery.It is generally accepted that excellent long-term postoperative results can be obtained when the postoperative knee varus or valgus is controlled within 3° after TKA surgery, but most of the current coronal lower extremity joint lines are controlled by relying on femoral intramedullary positioning and tibial extramedullary positioning. Further, the operation depends on the surgeon’s experience, which makes it difficult to achieve standardization and reproducibility of surgical outcomes. As can be seen from the above, the gradual deepening of the study of artificial intelligence has brought revolutionary changes in medicine, and concurrently, the preoperative planning of total knee arthroplasty in joint surgery is also moving towards the paradigm of precision medicine.TKA requires accurate force line and soft tissue balance after operation. In order to achieve this goal, the planning of preoperative imaging of patients is particularly important. The application of computer-aided technology and artificial intelligence preoperative measurement can enable surgeons to control the osteotomy plane and angle more accurately. Although these advances have certain risks and disadvantages, in the field of joint surgery, AI assistance will still be the direction of future development and will also bring more breakthroughs.