Food fermentation has been practised since ancient times to improve sensory properties and food preservation. This review discusses the process of fermentation, which has undergone remarkable improvement over the years, from relying on natural microbes and spontaneous fermentation to back-slopping and the use of starter cultures. Modern biotechnological approaches, including genome editing using CRISPR/Cas9, have been investigated and hold promise for improving the fermentation process. The invention of next-generation sequencing techniques and the rise of meta-omics tools have advanced our knowledge on the characterisation of microbiomes involved in food fermentation and their functional roles. The contribution and potential advantages of meta-omics technologies in understanding the process of fermentation and examples of recent studies utilising multi-omics approaches for studying food-fermentation microbiomes are reviewed. Recent technological advances in studying food fermentation have provided insights into the ancient wisdom in the practice of food fermentation, such as the choice of substrates and fermentation conditions leading to desirable properties. This review aims to stimulate research on the process of fermentation and the associated microbiomes to produce fermented food efficiently and sustainably. Prospects and the usefulness of recent advances in molecular tools and integrated multi-omics approaches are highlighted. The art of fermentation is as old as the human civilisation on earth, as it was traditionally developed by ancient societies for food preservation during harsh seasons, for ritual feasts, and to enhance the sensory quality of food . HistoriFermentation was an integral part of other ancient civilisations; examples include beer brewing in Babylonia, soy sauce production in East Asia, and fruit fermentation in Greece (Greeks attributed ‘Dionysos’ as the god of fruit fermentation) . The mosFermentation continues to be practised due to the evidence of extended shelf life and improved organoleptic properties of fermented foods. There is a wide variation in fermented foods and drinks prepared and consumed worldwide, although we lack detailed knowledge of the microbial properties underlying such variation . In the Aspergillus oryzae in the preparation of koji was discovered by the German scientist Korschelt in 1878 . In. In12].  vinegar . Organicmicrobes . The lacve group .Bacillus spp. and coagulase-negative Staphylococcus, which can produce extracellular proteinase for protein hydrolysis [In alkali fermentation, the proteins in the substate are hydrolysed into amino acids and peptides, releasing ammonia, which elevates the pH (8–9), inhibiting spoilage-associated microbes. Ammonia produced during alkaline fermentation (involved in the preparation of Japanese nattu and African fermented legumes and eggs) is responsible for a strong umami flavour and aroma. Microbes responsible for alkaline fermentation mainly belong to drolysis ,18. Unlidrolysis .Steinkraus  proposedFermented foods and beverages have traditionally been produced by relying on the microbiota naturally occurring on the food substrate. Spontaneous fermentation dependent on autochthonous microbes was the main method for producing fermented food and beverages throughout history, and it remains a mainstay method in domestic, small-scale, and household settings . In thisThe start of the fermentation process may involve transferring a small amount of a previously successful fermented batch into fresh ingredients as an inoculum to facilitate the initial phase of fermentation of the next batch, even without the knowledge of the types of active microbes; this process is called back-slopping . In sponWith progress in the microbiological techniques of isolation, identification, and microbial preservation, specific starter cultures have been isolated and characterised from fermented foods. These cultures are currently used, especially on the industrial scale, to ensure that the process is controlled and the fermentation outcome is stable for quality and properties. The use of well-defined starter cultures was first adopted to produce beer, alcohol, vinegar, and bread, followed by dairy and meat products . The maiLactobacillus, Leuconostoc, Enterococcus, Streptococcus, Oenococcus, and Pediococcus, and some of them may exert direct beneficial effects on health as live probiotic microbes [Bacillus, Micrococcaceae, Bifidobacterium, Propionibacterium, and Brachybacterium, act as a secondary group of microorganisms in the fermentation process [Debaryomyces, Kluyveromyces, Saccharomyces, Aspergillus, Mucor, Penicillium, and Rhizopus species, represent an important part of starter cultures in a variety of fermented foods and beverages, such as cheese and coffee, in which the microbiota significantly affects the appearance and organoleptic properties [Saccharomyces cerevisiae is traditionally used in the fermentation process. In addition, there is an increasing awareness about the enological characteristics of other non-Saccharomyces yeast in imparting the wine with particular flavour and aroma [The starter cultures that are mostly used to produce fermented foods and beverages, particularly acidic fermented products, belong to LAB . Such bamicrobes . Additio process . Along woperties ,31. For nd aroma .Starter cultures do not always contain a single strain; in many cases, a consortium of different organisms and strains is involved. The model example for the fermented beverages to be covered in this review is kombucha, with a starter culture consortium of multiple species that are well-adapted to co-existence. In the case of kombucha, a fermented, sweetened, black tea-derived beverage, which originated in China thousands of years ago and is currently gaining popularity worldwide for its health-promoting and therapeutic effects, a symbiotic culture of bacteria and yeast (SCOBY) is used to initiate fermentation . KombuchSaccharomyces cerevisiae) into fructose and glucose, which are then used for the growth of bacteria in the consortium  producing various organic acids, such as acetic, gluconic, and glucuronic acids [2; ethanol is subsequently oxidised into acetic acid by acetic acid bacteria. These organic acids, along with the alcohols produced by the yeast, act as antimicrobial agents that inhibit the growth of undesirable microbes in kombucha [In this case, the microbes are well-adapted to the substrate and co-exist with other microbes constituting the SCOBY. They act in harmony; the substrate contains sucrose that is first broken down by the action of yeast  is the base for forming the floating biofilm as the solid phase of kombucha. Formation of this biofilm enhances the association between bacteria and yeast and plays a role in adjusting the fermentation condition to support the survival of important microbial groups by retaining bacteria and yeast on the surface of the liquid to ensure adequate oxygen supply and nutrient diffusion (by forming reticulation) to inhabiting bacteria [The microbial cellulose produced by bacteria . The rolbacteria . This phbacteria ,39. FiguThe fermentation process has further evolved as the use of starter cultures has undergone significant improvement with the advancement of molecular biology techniques. Previously, the selection of starter cultures was based on the screening of many isolates, and those that performed well in fermentation on an industrial scale, yielding end products with acceptable organoleptic characteristics, were selected . RecentlLactococcus lactis (formerly Streptococcus lactis), an important microorganism for dairy fermentation, using recombinant DNA techniques in 1982, was considered a turning point for using genetic engineering to improve starter cultures for preparing fermented food [Streptococcus thermophilus and members of the Leuconostoc genus, have been genetic modified to improve traits linked to metabolism, efficiency of proteolysis, and defence against bacteriophages [L. lactis, facilitating understanding of the process of bacteriophage infection and bacterial defence mechanisms. This discovery was translated into constructing strains with bacteriophage components that inhibited phage proliferation and offered significant protection to L. lactis [The successful plasmid transformation of ted food . Followiiophages . Infectiiophages . Progres. lactis . Moreove. lactis .Saccharomyces cerevisiae yeast strain generated to reduce the content of ethyl carbamate, a potent carcinogen in wine; the strain reduces ethyl carbamate in wine by 89.1% and was patented and registered for producing alcoholic beverages [Starter cultures have been generated using recombinant DNA technology for decades and may provide improved fermentation processes and offer better-quality products with desired properties. Despite such potential, none of the developed strains are being used in the industry due to strict governmental regulations and the lack of consumer acceptance of genetically modified food ingredients . Therefoeverages .The revolutionary novel technology, CRISPR/Cas9 , is an extremely precise method of gene editing; it has taken genetic engineering to another level with a wide range of biotechnological applications in many fields, and its discoverers were selected for the Nobel Prize in Chemistry in 2020 ,53. BrieSaccharomyces cerevisiae, an industrially important yeast and starter strain for several fermented products [S. cerevisiae have followed. Recently, engineering yeast for the reduction of production of urea, the precursor of ethyl carbamate, and the modulation of glycerol production in wine have been successfully implemented [Leuconostoc citreum was engineered using the CRISPR/Cas9 toolbox for elimination of cryptic plasmids and this process was suggested as a food-grade method to develop a safe lactic acid bacterial strain without residual antibiotic markers [A. oryzae, which is an industrially important filamentous fungus used in Japanese and Korean traditional fermentation [The technology was first applied in 2013 for genome engineering of products . Since tlemented ,57,58. T markers . Katayam markers  utilisedentation .The novel CRISPR technology for gene editing is promising for food-grade applications; it is highly precise, stable, and should be considered outside the scope of genetically modified organisms (GMO) as the modification occurs in nature . HoweverMicrobial activity in fermented foods and beverages is the main factor responsible for product quality and safety. Hence, the manipulation of the microbial composition, particularly at the initiation of fermentation, is key for controlling the process and shaping the properties of the product. Recent advances in biotechnological tools and the rise of CRISPR-based technologies may not only be involved in genetic improvement of specific strains in starter cultures, as explained earlier, but the technology also has great potential in microbiome engineering . This suEscherichia coli as a model microbe by targeting specific sequences [CRISPR-mediated microbiome manipulation has been investigated in several recent studies. The main priority for fermented food and beverage microbiome manipulation is to selectively control the undesirable spoilage-associated microbes or microbes competing with fermentation-desirable microbes to enhance product quality and extend shelf life . Specifiequences .Such specific targeting of individual strains in mixed cultures and the differentiation between pathogenic and beneficial microbes was nearly impossible using tailored growth conditions or traditional antibiotics; such targeting allows for further applications by selectively clearing contaminating microorganisms and quantitively controlling the environmental or industrial microbial community composition . The speCRISPR-based microbiome engineering represents the most recent advancement in the evolution of the food fermentation process, starting with traditional spontaneous fermentation through the use of starter cultures and genetic engineering. The evolution of the food fermentation process is summarised in The continuously interacting microbiota of food fermentation ecosystems, encompassing different types of bacteria, yeast, and fungi, plays a major role in shaping the quality and safety of fermented foods and beverages . PreviouOne of the most extensively applied techniques in recent studies to profile the microbiota within food fermentation is the use of high-throughput sequencing (HTS)-based metabarcoding, by analysing the collective genomic markers by employing universal primers, such as the 16S rRNA and internal transcribed spacer (ITS) regions for bacteria and fungi, respectively . The limA more comprehensive approach to study the fermented food microbiome is the application of metagenomic shotgun DNA-seq, which provides more accurate taxonomic information on the microbial communities of high-complexity samples, allowing for profiling the functional potential by detection of the global gene content and identification of unknown species . The appLeuconostoc, Lactobacillus, and Weissella genera. During the initial fermentation stage, Leuconostoc mesenteroides usually dominates the microbial community, and Lactobacillus sakei and Weissella koreensis begin to dominate at the optimum-ripening and over-ripening fermentation stages; although the latter are important for kimchi fermentation, their rapid growth and activity promote acidic deterioration, decrease the fresh flavour, and reduce the shelf life [Among the model examples for studies involving HTS-based metabarcoding approach to profile the microbial community structure and evaluate the influence of fermentation ingredients on the microbial community composition and functional potential that will be covered in this review are kimchi, fermented soy products, and kombucha. Kimchi is the most famous Korean fermented food that has gained worldwide popularity owing to its health-promoting properties . Severalelf life ,83,84,85Konosirus punctatus) during kimchi fermentation on the microbial and chemical composition. The purpose of this study was to evaluate a practice that is traditionally adopted in the coastal cities of Korea, where fish is added during kimchi fermentation, and this type of kimchi has a refreshing taste and a relatively extended shelf life [Lactobacillus sakei, which is linked to the rapid acidic deterioration of kimchi, and by promoting the growth of Leuconostoc rapi, which is known for its health-promoting and taste-improving properties owing to the production of the antioxidant mannitol, contributing to the refreshing flavour and desirable characteristics of kimchi [Mannaa et al.  utilisedelf life ,87. Thisf kimchi ,88,89. Tf kimchi ,91,92. UIn other common Korean fermented products, Korean fermented soy paste (doenjang) and soy sauce (gangjang), manipulating the ingredients during fermentation can cause significant changes in the microbial composition and lead to changes in the properties of the product. Fermentation of doenjang and gangjang is based on two steps, starting with meju, whereby dried soybean blocks are fermented spontaneously using naturally occurring populations of fungi and bacteria. The fermented dry mouldy blocks are then subjected to a second long-term fermentation to produce the solid paste, doenjang, and liquid gangjang . The proChromohalobacter beijerinckii, which dominated the microbial community in the control group. Reduction in C. beijerinckii is considered beneficial for gangjang as it is responsible for the increase in the levels of biogenic amines, such as histamine, putrescine, and tyramine, which are considered potential health risk factors in fermented salty products and should be minimised. This study combined the metabolomic analysis using 1H-NMR to evaluate the content of biogenic amines produced in the gangjang, which revealed a significant reduction in the levels of these biogenic amines. This study demonstrates the advantage of integrating multi-omics tools to evaluate the effect of ingredients on the fermentation process and the end product, by suggesting that adding coriander during fermentation has a positive influence on the quality and health of the product.Mannaa et al.  investigSphingobacterium and Pantoea, were significantly reduced, while potentially beneficial bioactive metabolite-producing microbes, such as Saccharopolyspora and Buttiauxella, were present at significantly higher levels [Similarly, the effect of adding different types of herbs during fermentation for the production of doenjang was investigated using HTS-based 16S rRNA metabarcoding and metabolomic studies ,97. The r levels .Komagataeibacter and Zygosaccharomyces were dominant at different fermentation times. Moreover, functional complementarity was observed between both microbial groups which explains the sustainability of the kombucha ecosystem by ensuring microbial metabolic cross talks [Metagenomic approaches have been applied to study the microbial composition of kombucha by combining whole metagenome sequencing, 16S rRNA and internal transcribed spacer-1 amplicon analysis. The results indicated that ss talks . Such muss talks .Lactobacillus [Several studies have combined meta-transcriptomic analysis, which targets actively expressed genes under specific conditions, with meta-barcoding to decipher the core functions of the detected microbiota that are associated with metabolomic changes affecting the quality properties of fermented foods . The twobacillus . De Filibacillus  used a cAlthough meta-transcriptomic analysis can provide valuable knowledge about the gene expression and potential functional activity, it might fail to establish direct associations between the microbiota and the environment, since the mRNA expression might not be directly associated with protein expression, and cell activity regulation occurs at the protein level. Therefore, the direct analysis of the proteins are essential as a complementary approach along with meta-transcriptomics to study the functional activity of the microbial community . The metThe fermentation process has undergone significant improvement over the years. With technological advancement, it has become possible to manipulate the fermentation starter culture and the associated microbiome to standardise the product stability, improve sensory properties of the food, and ensure safety. Genome editing and microbiome engineering tools based on the CRISPR technology are evolving rapidly and are becoming highly efficient in improving microbial functionality. This modern technology is mostly applied in human cell research, and CRISPR/Cas9-based gene therapy has clinical potential. However, this technology still holds great promise for a wide range of applications, including in the food fermentation industry. The industry of fermented food and beverages may benefit significantly from the application of the revolutionary tools of CRISPR-based genome editing and microbiome manipulation, leading to improved control of the process and the properties of the end product by promoting the desired features linked to the sensory aspects, quality, shelf life, nutritional content, and safety of fermented food. This improvement would be coupled with increasing consumer acceptance and fewer governmental restrictions on the use of CRISPR/Cas9 gene editing in microbe manipulation, especially when no insertion of foreign DNA is involved. Scientific research is continuously proving the safety and precision of modern genomic manipulation tools, with no use of resistance markers and genetic stability of generated strains, thereby becoming accepted as a food-grade process.However, it is important to highlight the worldwide popularity and acceptance of fermented food and its natural and artisanal nature. Therefore, spontaneous and traditional methods of preparation will continue to be a major part of the practice. Hence, the precise characterisation of the whole process and hygienic aspects are important to understand traditional fermentation and ensure the safety of consumption of fermented food. The available tools based on NGS technology and the rise of pioneering integrated multi-omics approaches have allowed deep understanding and high-resolution analysis of the fermentation process with many novel insight into the fermented-food microbiome and the role in the physicochemical and sensory properties of fermented food.The advancement in tools available to study the fermentation process has revealed the inherited wisdom of ancient societies. The selection of suitable fermentation substrates to combine and set up the conditions of traditional fermentation led to the preparation of healthy products with improved sensory properties. The combined multi-omics approaches have provided cutting-edge discoveries in different microbiological research fields, while their application in studying fermentation is limited. Most studies use a single approach to study a particular aspect, especially because of the high cost of combining different omics approaches and the need for sophisticated bioinformatics and biostatistics skills for the analysis of such large datasets. A combined multi-omics approach would facilitate understanding the process, provide new systems-biology perspectives, and decipher the interaction among the fermentation microbiota, the substrate, and the environment. Overall, the near future will bring a greater range of applications of multi-omics in studying food fermentation, leading to detailed characterisation and efficient and sustainable production of fermented food and beverages.


Dietary intake was collected using two 24-h recall interviews and a nonquantitative food frequency questionnaire. Macronutrient intakes were calculated using an updated version of the Greek FCT. Results: Only 28.3% of the adult population had high adherence to the Greek traditional Mediterranean diet, with a higher percentage (39.7%) observed for participants over 65 years compared to those under 65 years (25.5%). Differences in adherence to the MD were observed among the four geographical regions in Greece. Younger adults had a higher intake of meat, cereals, alcoholic and nonalcoholic beverages, and sugar products than older individuals who consumed more vegetables, fruits, legumes, dairy, fish, and lipids (mainly from olive oil). Adults do not meet the international dietary recommendations for the intake of several foods and macronutrients. Conclusions: The adult Greek population, especially younger people, has headed away from the Greek traditional Mediterranean diet. These observations indicate potential detrimental consequences in terms of morbidity and mortality.Background: This study aimed to investigate the food and macronutrient intake of the population in Greece and evaluate its adherence to the Greek traditional Mediterranean diet. Methods: Adults over 18 years old ( National health and nutrition surveys are designed to assess the health and nutritional status of a population and are considered important. The World Health Organization (WHO) encourages countries to collect dietary data at an individual level using nationally representative health and nutrition surveys . A revieThe nutritional status of population can be assessed either through the estimation of individual food and nutrient intakes and/or through the adherence to dietary patterns. The last study assessed of the changes in the nutritional behaviour of Greeks at a population level was the Greek EPIC (European Prospective Investigation into Cancer and Nutrition) cohort, which was held for 14 years (1997–2011). The findings of 23,505 participants reported an increase in the consumption of vegetables, fruits, and fish/seafood over the years, and a decrease in dairy products, nuts, cereals, and meat consumption, while no prevailing trend was found or observed for legume consumption . Some stTo the best of our knowledge, there are no other studies that described the usual food consumption for a large variety of food groups and nutrients and that are directly comparable to those of other European countries, as well as the adherence to the MD in a representative sample of the adult population in Greece. The HYDRIA survey collected representative comparable data across EU Member States using standardized procedures in order to provide conclusions and useful observations on population health and related key risk factors, such as the nutritional status of a population as a whole or of specific population groups, and at the same time to fill the gap in providing data on reports requested by international organizations. The present paper aims to describe the dietary habits, including usual energy, food, and macronutrient intakes, of adult men and women living in Greece and the population’s adherence to the Greek traditional Mediterranean diet overall and by age groups.The study design for the HYDRIA survey followed the recommendations of the European Health Examination Survey (EHES)  and the Eligible individuals were men and women aged 18 years and over residing permanently in Greece based on the latest Greek general census (2011). A two-stage stratified random sampling scheme was applied, with the primary sampling unit being the municipality/local community (first stage) and the final unit being the individual (second stage) for to select the HYDRIA representative survey sample. The HYDRIA survey sampling frame covered all the 51 prefectures of the 13 regions of the country [The data collection included interviewer-administered questionnaires on sociodemographic and lifestyle characteristics, and medical history, while measurements of blood pressure, somatometric characteristics, and blood samples were collected by health professionals. Study centres, mainly including health care facilities close to the residences of individuals, were used for the data collection. The data collection started in June 2013 and was completed in December 2014. Overall, 1873 men and 2138 women, aged 18 to 94 years old, participated in the HYDRIA survey, with a 50% response rate. Details for the design, methodology, and data collection of the HYDRIA survey have been described elsewhere [Dietary intake was collected using two nonconsecutive 24 h dietary recalls (24-HDR) per participant and a nonquantitative food frequency questionnaire . The two 24-HDRs were distributed over seasons (in group level) and days of the week to control variations in food consumption. The first 24-HDR (face-to-face interview) was carried out on the first day of the participants’ examination, while the second 24-HDR (by telephone) was performed approximately 15 to 30 days after the first recall. During a 24-HDR, participants were asked to report in detail all the food and beverages they had consumed the previous day. In addition, questions were asked about food preparation practices, types of foods , and the amounts consumed. The 24-HDR data were collected by trained interviewers using a specially designed application, the HHF Nutrition Tool. The HHF Nutrition Tool included a food list, facets, and descriptors of food items that were in line with both the LanguaL™ Food Description Thesaurus  and EFSAIn addition, participants were asked to report the frequency of consumption  of eighty-eight foods and dietary supplements in the previous year through an interviewer-administered, nonquantitative food frequency questionnaire (FPQ). The FPQ for the HYDRIA survey was developed and pilot-tested in the context of the PANEU project .www.dpa.gr, accessed on 9 January 2022) for the establishment and operation of a file registry of sensitive personal data under law N.2472/1997.All the collected data are inputted and stored electronically on the HHF central server anonymously and securely to protect participants’ privacy. The HYDRIA survey data are protected under applicable national law and by approval from the Hellenic Data Protection Authority  were excluded from the analyses. For calculating the energy intake and macronutrients, the foods consumed were linked through the HHF nutrition tool to the HYDRIA Food Composition Table (H-FCT), which was created to accommodate the recorded consumption data. For this purpose, the existing FCT [Individuals who did not participate in any of the two 24-HDRs , monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and alcohol) for the following thirteen food groups: vegetables (excluding potatoes), potatoes, legumes, fruits (without fresh fruit juices), cereals , dairy (including dairy products), meat (including meat products), fish (including seafood and fish products), eggs, fats and oils, nonalcoholic beverages, sugar and products, and alcoholic beverages, and eleven food subgroups: bread, pasta, rice, milk, yoghurt, cheese, poultry, red meat, olive oil, wine, and beer.The usual mean intake of energy, food groups and subgroups, and macronutrients was estimated using the mean of the two individual corresponding 24-HDRs under the assumption that two or more 24-HDRs per person give unbiased estimates of the single-day intakes . The usuTo estimate the distribution  of the usual intake of energy, food groups and subgroups, and macronutrients, we used the statistical method published by the National Cancer Institute  ,31,32. TThe adherence to the Greek traditional Mediterranean diet was assessed with a nine-point score for the consumption of vegetables, legumes, fruit and nuts, cereal, fish, meat, dairy, alcohol, and lipids (expressed as the ratio of monounsaturated to saturated lipids ) incorpoTo estimate the distribution of usual dietary intakes and Mediterranean diet score, all models were stratified by gender and included covariates for ten-year age groups , education level , and geographic area . In addition to the estimation of the usual distribution intake of food groups and subgroups, the corresponding frequency food variables of FPQ were included in the models to supplement the food intake obtained from the two 24-HDRs. Weighting factors were applied in all models to achieve nationally representative results in the usual intakes. The weighting factors were calculated using the 2011 Greek census, considering the complex sampling study design and the response rate of participants by sex, age group, geographical region, and urbanization level.The distribution of study participants according to sex, age, geographic region, and categories of Mediterranean diet score is shown in The HYDRIA study is the first national health and nutrition survey conducted on a large representative sample of the adult population in Greece, providing evidence for the dietary and health status of the population . Our resThe above results are in line with previous findings from the Greek EPIC cohort  and the About one out of three adults (28.3%) was found to have high adherence to the Greek traditional Mediterranean diet. The percentage was lower among younger participants (25.5%) than the older ones 39.7%). These results could point to negative consequences in terms of morbidity and mortality among younger people. Specifically, several national ,38,39,40.7%. ThesThe higher energy intakes reported by the male population in the HYDRIA survey, which decreased with age, were consistent with those reported by other European national nutrition surveys ,48. The To improve overall health and reduce the risk of cardiovascular diseases and certain types of cancer, WHO dietary guidelines recommend more than 400 g of fruit and vegetables per day  for adulIt is essential to mention that the HYDRIA survey implemented a harmonized protocol among EU Member States that generated data directly comparable to that of other European countries. Among the main strengths of the HYDRIA survey were the national representative sample and the standardized data collection. To collect dietary data, a specially designed dietary software application, the HHF Nutrition Tool, and a food composition table specifically designed for this project, the H-FCT, were used. The current study examined the usual intake of a wide range of food groups and selected subgroups. The differentiation between refined and whole grains in the cereal and products food groups was not possible for the current analysis. We only estimated the usual intake of adult men and women and not the dietary intake of children or adolescents, which was later collected in two additional studies. Overall, dietary and other health-related data were collected by interviewer-administered questionnaires. However, the HYDRIA survey has a cross-sectional design and thus cannot be used to prove causal relations, but it does contribute to identifying problems with a significant impact on the health of the population in the country.In conclusion, the HYDRIA survey included a large national representative sample of the population in Greece that provided insights into the dietary behaviours of adults. Younger adults consume more meat, dairy, and alcohol, which are traditionally consumed at moderate-to-low levels in the Mediterranean diet, than fruit, legumes, and vegetables, which were consumed more by older individuals. Furthermore, adults did not meet international dietary recommendations for the consumption of red meat, fruit, and vegetables. Younger generations in Greece seem to move away from food choices included in the beneficial components of the Greek traditional Mediterranean diet at a time when other countries are modifying their diets to support the Mediterranean dietary pattern.


Both age-related hearing loss (ARHL) and age-related loss in vestibular function (ARVL) are prevalent conditions with deleterious consequences on the health and quality of life. Age-related changes in the inner ear are key contributors to both conditions. The auditory and vestibular systems rely on a shared sensory organ – the inner ear – and, like other sensory organs, the inner ear is susceptible to the effects of aging. Despite involvement of the same sensory structure, ARHL and ARVL are often considered separately. Insight essential for the development of improved diagnostics and treatments for both ARHL and ARVL can be gained by careful examination of their shared and unique pathophysiology in the auditory and vestibular end organs of the inner ear. To this end, this review begins by comparing the prevalence patterns of ARHL and ARVL. Next, the normal and age-related changes in the structure and function of the auditory and vestibular end organs are compared. Then, the contributions of various molecular mechanisms, notably inflammaging, oxidative stress, and genetic factors, are evaluated as possible common culprits that interrelate pathophysiology in the cochlea and vestibular end organs as part of ARHL and ARVL. A careful comparison of these changes reveals that the patterns of pathophysiology show similarities but also differences both between the cochlea and vestibular end organs and among the vestibular end organs. Future progress will depend on the development and application of new research strategies and the integrated investigation of ARHL and ARVL using both clinical and animal models. Aging causes the function of multiple organ systems to decline , and, liThe cochlea provides the brain with acoustic information, which is essential for communication and personal safety. ARHL is typified by progressive, bilateral, symmetric hearing loss, especially in the high frequencies, and accompanied with difficulty understanding speech, especially in noise. ARHL is the most prevalent sensory deficit in the elderly  and is aThe end organs of the vestibular system provide the brain with information necessary for detection of self-motion, spatial orientation, and postural control . ARVL caLike other multifactorial conditions, ARHL and ARVL show substantial individual variation and result from a combination of both intrinsic factors, such as genetics, as well as external factors, like environmental conditions , lifestyle factors , medications , and other comorbidities . ARHL  and low concentration of sodium ions (Na+). This concentration gradient means that influx of K+ is depolarizing  and less commonly measurements of auditory brainstem responses (ABRs) . SimilarIn animal models, peripheral vestibular function is often assessed by examination of reflex responses e.g.,  or measuVarious studies have used functional assessments to characterize ARHL and ARVL . These sTogether and despite the caveats highlighted above, the functional studies available indicate that in humans ARHL precedes ARVL. In many regards, studies directly comparing peripheral ARHL and ARVL using equivalent assessments of evoked potentials (ABR and VsEP) in various strains of inbred mice show similar findings to observations in humans. Specifically, age-related decline in cochlear function generally precedes decline of vestibular function , and theTurning attention to age-related structural changes in the in the inner ear , a varieStructural changes to the vestibular system with increasing age have been investigated in various human histological studies  are common in patients with auditory neuropathy when a peripheral neuropathy is also present . The linInflammation is a hallmark of aging, and other coexisting comorbidities linked to aging, such as cardiovascular disease, type 2 diabetes, and Alzheimer’s . InflammAlthough inflammatory and immune responses have been characterized in various forms of acquired hearing loss , attentiThe involvement of inflammaging in ARVL has not been investigated. Nevertheless, the molecular players required for inflammaging are present in the vestibular end organs, although their association with ARVL requires further investigation. Macrophages and microglia have been identified immunohistologically in the tissue underlying the saccule, utricle, and all three ampullae of the semicircular canals, among Scarpa’s ganglion cells, and within the endolymphatic duct and sac in human adults with no history of hearing or balance disorders . StudiesMore work is needed to investigate the role of inflammaging in the cochlea and especially vestibular end organs . BecauseOxidative stress is implicated in various age-related conditions, such as cardiovascular diseases, chronic obstructive pulmonary disease, chronic kidney disease, neurodegenerative diseases, and cancer  shows accelerated ARHL associated with indicators of oxidative stress, inflammation, altered proteostasis, and apoptosis , consistFinally, additional insights into the mechanisms interrelating age-related pathology in the cochlea and vestibular end organs come from the long-recognized oto- and nephrotoxicity of some drugs, especially aminoglycoside antibiotics but also chemotherapeutics like cisplatin . Like boThe central auditory and vestibular systems also show changes associated with age-related decline in hearing and balance perception that may occur as part of brain aging and/or be driven by age-related changes in the cochlea and vestibular end organs. These changes include both anatomical and functional alterations of the brain  input is itself and regardless of aging a potent trigger of changes in brain structure and function and involves various synaptic mechanisms, ascending and descending auditory pathways, and neuromodulatory circuits . Thus, aBoth ARHL and ARVL are prevalent conditions associated with changes in the structure and function of the cochlea and vestibular end organs. A careful comparison of these changes reveals that the patterns of pathophysiology show similarities but also differences both between the cochlea and vestibular end organs and among the vestibular end organs. This review uncovers gaps in the current research landscape and identifies specific, high impact research objectives that should be prioritized. Specifically:•Assessments of inner ear function in both humans and animal models using a combination of tests, which provide complementary rather than redundant information, are needed to identify and examine correlations between ARHL and ARVL.•Epidemiological studies of the prevalence of ARVL and associated risk factors, including genetic risk factors, for ARVL that utilize standardized, quantitative, clinical assessments of vestibular end organ function are needed.•Analogous and robust assessments of peripheral vestibular function in humans and animal models need to be developed and applied.•Animal models, which have been developed to examine other age-related conditions, should be better leveraged to identify the mechanisms contributing to ARHL and especially ARVL. The background strain of mouse models should be considered carefully since there is considerable strain-dependent variation in the susceptibility to age-related hearing loss and audiogenic seizures.•The relationship between age-related changes in the peripheral and central auditory and vestibular systems need to be examined, especially when considering the complex, multisystem complications, like falls, depression, and cognitive decline, shared by these conditions.Addressing these gaps will require integrated investigation of the auditory and vestibular structures of the inner ear using both clinical and animal models. Ultimately, because both ARHL and ARVL are associated with and likely mechanistically linked to other age-related conditions, treatments to prevent ARHL and ARVL are essential to strategies promoting healthy aging overall.All authors contributed to data collection, analysis and interpretation of data, manuscript preparation, reviewed, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


A significant number of women receive a cancer diagnosis before their age of natural menopause. Among these patients, the most frequent neoplasms are breast cancer, gynecological, and hematological malignancies. Premature ovarian insufficiency and infertility are among the most feared short- to long-term consequences of anticancer treatments in premenopausal patients. Both patient- and treatment-related characteristics are key factors in influencing the risk of gonadotoxicity with the use of chemotherapy. The cryopreservation of oocytes/embryos is a standard strategy for fertility preservations offered to young women interested in future family planning, but it does not allow gonadal function protection during chemotherapy. Ovarian suppression with gonadotropin-releasing hormone agonist (GnRHa) during chemotherapy is now recommended as an option to reduce the risk of gonadotoxicity in order to avoid the negative consequences of premature ovarian insufficiency in premenopausal women receiving cytotoxic therapy, including those not interested in fertility preservation. This review summarizes the risk of treatment-induced gonadotoxicity in premenopausal patients and the evidence available on the protective role of administering GnRHa during chemotherapy to preserve ovarian function. Thanks to improvements in cancer prognosis, increased attention should be paid to the future quality of life for patients exposed to anticancer treatments . The risAs recommended by current guidelines, proper oncofertility counseling is recommended in all premenopausal women irrespective of the type of their disease and stage ,6. DurinThe cryopreservation of oocytes/embryos and/or ovarian tissue are standard strategies for fertility preservation in young women who are interested in future family planning ,6. TheseAs recently shown in the prospective PREFER study, while less than 20% of young women diagnosed with breast cancer before the age of 40 years decided to undergo cryopreservation strategies for fertility preservation, more than 90% of them accepted the use of GnRHa during chemotherapy . MoreoveThis review summarizes the risk of treatment-induced gonadotoxicity in premenopausal patients and the available evidence on the protective role of administering GnRHa during chemotherapy to preserve ovarian function.To estimate the risk of treatment-induced gonadotoxicity, both patient- and treatment-related factors should be considered during counseling ,6. RegarIn terms of treatment-related factors, the type and dose of chemotherapy have a major impact on the risk of gonadotoxicity ,6.In general, different mechanisms are associated with the ovarian toxicity of chemotherapy. The germ cells pool can be damaged through a direct effect on double-strand DNA or acceleration in follicular activation , but alsp = 0.006)  and luteinizing hormone [LH]), regulated by the secretion of GnRH by the hypothalamus . ChemothThe protective action of GnRHa was initially attributed to the reaching of a pre-pubertal hormonal state . The admIn addition, proliferating follicles also release anti-müllerian hormone (AMH), which can negatively regulate the primordial follicles recruiting. During chemotherapy, AMH levels are usually dramatically lowered, causing a recruitment of primordial follicles and exposing them to chemo-induced damage. It has been observed that the addition of GnRHa can raise AMH levels and prevent this effect ,36.Finally, despite a limited number of data, another potential indirect protective mechanism of GnRHa is the reduction of utero-ovarian perfusion, which would also reduce the exposure of follicles to the gonadotoxic effect of treatments .It has been observed that GnRH receptors are expressed on the surface of the ovarian cells and that their activation may result in an anti-apoptotic effect; this effect is currently poorly understood ,38.Furthermore, GnRHa may be involved in the upregulation of the anti-apoptotic molecule sphingosine-1-phosphate (S1P), which inhibits the ceramide pathway involved in chemo-induced apoptosis of the ovarian cells. Moreover, S1P improves neo-angiogenesis in primordial ovarian follicles, producing a potential further protective effect in the ovaries . It has Recently, Scaruffi et al. evaluated the effect of chemotherapy  and GnRHa alone, or in combination with chemotherapy on a culture system of ex vivo human immature cumulus cell-oocyte complexes. The effects of these treatments were evaluated on GnRH receptors, ceramide, and apoptosis pathways and glutathione synthesis. This study demonstrated that cyclophosphamide was mainly detrimental to the cumulus cells compartment through the activation of apoptosis molecular signals. Furthermore, it was found that GnRHa co-administration downregulated the expression of some pro-apoptotic genes and up-regulated transcription of the anti-apoptotic gene only in cumulus cells with an indirect protective effect on oocytes . These rMost of the evidence supporting the use of GnRHa in the prevention of chemotherapy-induced POI exists in premenopausal women with early breast cancer . A totalIn this regard, most studies have reported results at such a short follow-up that an appropriate assessment of fertility preservation potential was not possible, especially in patients who are often candidates for adjuvant endocrine therapy for 5 to 10 years ,67,68. MTwo safety concerns have been highlighted regarding temporary ovarian suppression with GnRHa during chemotherapy, particularly in patients with estrogen receptor-positive breast cancer: a potential antagonism of endocrine therapy with chemotherapy and a possible negative prognostic effect of POI prevention .However, several randomized clinical trials did not demonstrate any difference in the prognosis of patients who received ovarian suppression during chemotherapy ,71,72. TFurthermore, two randomized trials that evaluated the administration of GnRHa during chemotherapy also in patients with estrogen receptor-positive breast cancer and its impact on survival showed no difference in the survival outcomes; in both studies, most of the patients were also treated with GnRHa as part of their adjuvant endocrine therapy ,62. ReceNevertheless, some considerations are needed to better interpret the results of these trials; most of the studies had a small sample size including less than 100 patients, median age of the included patients was close to 40 years in most of them, and the most used chemotherapy regimen was an anthracycline- and cyclophosphamide-based regimen. However, it should be remarked that the definition of chemotherapy-induced POI was based only on menstrual function after treatment in the majority of the studies. Only few studies used a composite endpoint that included both amenorrhea and post-menopausal hormonal levels and the timing for POI evaluation was also different, ranging from 6 months up to more than 5 years after the end of chemotherapy treatment. To date, there is no uniform and accepted definition of chemotherapy-induced POI. This is reflected by the use of different definitions that in most cases included amenorrhea, and in the timing of its assessment. However, amenorrhea is not an optimal surrogate marker to define the gonadotoxicity of anticancer treatments, especially in breast cancer patients who often receive adjuvant endocrine therapies (including tamoxifen), which can further impact menstrual function recovery. Experts recommend empirically to define chemotherapy-induced POI with a composite definition of amenorrhea for ≥2 years and a post-menopausal hormonal profile . NotablyResults from the available randomized trials have been summarized in several meta-analyses in order to better define the efficacy and safety of temporary ovarian suppression with GnRHa during chemotherapy in breast cancer patients  79,80,8,85,86,87The use of GnRHa during chemotherapy as a strategy to preserve ovarian function has been considerably less investigated in women with malignancies other than breast cancer  75,76,7,89,90.Four randomized trials have been conducted in premenopausal women with lymphoma ,76,78,89p = 0.02) [Only one study on the efficacy of temporary ovarian suppression with GnRHa during chemotherapy was conducted in premenopausal women with ovarian cancer. A total of 30 premenopausal women were randomized to receive chemotherapy with or without GnRHa. All women who received GnRHa during chemotherapy had a resumption of menstrual bleeding at 6 months after the end of their cytotoxic treatments, whereas 33% of women who did not receive GnRHa had chemotherapy-induced POI ( = 0.02) ,90.There are potential clinical and methodological reasons for the different results observed in patients with hematological malignancies as compared to those in the breast cancer setting.From a clinical point of view, there are important differences between premenopausal patients with hematological diseases, particularly lymphomas, and those with breast cancer. In general, patients with hematological malignancies are characterized by a younger age at diagnosis and are treated with different chemotherapy regimens ranging from a low- to very high-risk of gonadotoxicity. Younger patients generally have a higher ovarian reserve and acute POI is usually observed only after treatment with high-risk chemotherapy regimens, whereas low- or medium-risk chemotherapy are less likely associated with POI in these patients.. On the contrary, premenopausal breast cancer patients have an average older age at diagnosis and often receive chemotherapy regimens characterized by an intermediate risk of gonadotoxicity. Therefore, the protective effect of GnRHa may become visible only in this situation and not in the case of very low or high gonadotoxicity risk.It should be also mentioned that lymphoma patients may have a reduced ovarian reserve due to their disease even before starting anticancer therapies ,92, wherFrom a methodological point of view, only four small studies with a total of 154 patients affected by lymphoma have been conducted, while to date 15 studies involving 1743 patients are available in the breast cancer setting, four of which randomized more than 200 women. The lack of power in trials concerning malignancies other than breast cancer must be considered as an important reason for the controversial results.Several meta-analyses were performed to gather stronger data in favor of this technique in different types of cancers, and not only breast cancer  32,59,9,103,104.Among these meta-analyses, all but one showed that the use of GnRHa during chemotherapy has a protective effect on the risk of POI, while only one of them showed that this technique could increase the probability of spontaneous pregnancy in the short-term after the end of treatments . HoweverSeveral limitations are present in these meta-analyses due to differences in the included trials. First, as mentioned above, different definitions of POI were used in the study and the limited duration of the follow-up of the trials precluded the ability to determine the long-term impact of GnRHa on the preservation of ovarian function and fertility. In addition, a moderate heterogeneity among the studies is also due to the different chemotherapy regimens administered that have been modified in clinical practice over the years and may have partially affected the outcomes of the trials included in the meta-analyses.Despite continuous research efforts in this field, many physicians and patients are still concerned about discussing preservation of ovarian function and fertility for the fear that the proposed strategies may have a negative effect on oncological outcomes . Thanks POI development is potentially a direct effect of gonadotoxic therapies in all premenopausal cancer patients; the use of adjuvant endocrine treatments for many years after diagnosis can further amplify this issue . To dateHowever, it must be highlighted that most of the available randomized trials assessing the use of GnRHa during chemotherapy have been conducted in breast cancer patients, with limited and mostly negative evidence in women with hematological malignancies. Nevertheless, also in the setting of hematological malignancies, GnRHa during chemotherapy could be taken into consideration to prevent menometrorrhagia by controlling the menstrual cycle . The maiImportantly, it should be remembered that, for patients interested in fertility preservation, temporary ovarian suppression with a GnRHa during chemotherapy is not an alternative to cryopreservation techniques ,6.The main limitation of the current evidence, in addition to the limited data outside the breast cancer field, is the short follow-up of most of the randomized trials without the possibility of evaluating the long-term outcomes, including post-treatment pregnancies and age at menopause. Moreover, collecting information on ovarian reserve markers during treatment and follow-up should be planned in all new trials that want to investigate the effect of cancer treatments on ovarian reserve and the impact on POI.Besides GnRHa during chemotherapy, other pharmacological and biomedical techniques that can prevent POI in these patients are under constant development , but the


Polyelectrolyte polymers have been widely used in the pharmaceutical field as excipients to facilitate various drug delivery systems. Polyelectrolytes have been used to modulate the electrostatic environment and enhance favorable interactions between the drug and the polymer in amorphous solid dispersions (ASDs) prepared mainly by hot-melt extrusion. Polyelectrolytes have been used alone, or in combination with nonionic polymers as interpolyelectrolyte complexes, or after the addition of small molecular additives. They were found to enhance physical stability by favoring stabilizing intermolecular interactions, as well as to exert an antiplasticizing effect. Moreover, they not only enhance drug dissolution, but they have also been used for maintaining supersaturation, especially in the case of weakly basic drugs that tend to precipitate in the intestine. Additional uses include controlled and/or targeted drug release with enhanced physical stability and ease of preparation via novel continuous processes. Polyelectrolyte matrices, used along with scalable manufacturing methods in accordance with green chemistry principles, emerge as an attractive viable alternative for the preparation of ASDs with improved physical stability and biopharmaceutic performance. Over the past decades, novel drug delivery systems have been a key area of research into overcoming the limitations resulting from the development of poorly water-soluble drugs. The number of active pharmaceutical ingredients (APIs) having high therapeutic potential, but low water solubility, is constantly increasing. The low dissolution and bioavailability of APIs limit the available formulation approaches, their clinical application, and their marketability ,2. VarioASDs are usually prepared with the aid of fusion- or solvent-based methods. The most applicable manufacturing methods for the formation of stable ASDs are, nowadays, hot-melt extrusion (HME), and spray drying (SD), as they provide the potential for easily scalable production . HME is,Specific and nonspecific intermolecular interactions between the drug and the polymer in ASDs stabilize the preparation, and include ionic interaction, hydrogen bonding, dipole–dipole interaction, or the van der waals interaction. The presence of strong drug-polymer interactions in the system has been extensively investigated in order to promote ASD formation featuring good miscibility, drug loading, physical stability, supersaturation capability, and dissolution rate, and it is thought to facilitate the manufacturing process by fine-tuning key product parameters ,16. The The aim of this review, therefore, is to discuss the potential of ASD-based formulations, and to highlight the potential advantages of the use of polyelectrolytes in ASDs in terms of stability, dissolution enhancement, the maintenance of supersaturation levels, and controlled/targeted drug delivery, providing a focused insight into colon-targeting preparations. Furthermore, this review thoroughly presents the applicability of the HME technique in the continuous, efficient, economical, and scalable formulation of ASDs, while modified polyelectrolyte matrices, and recently reported interpolyelectrolyte complexes as potential carriers for ASD formation, are extensively discussed.ASD is a major class of solid dispersions where the drug exists in the amorphous state and is stabilized while molecularly dispersed (dissolved) in an amorphous polymer carrier. Chiou and Riegelman were the first to define the general term, “solid dispersions”, as the “dispersions of one or more active ingredients in an inert carrier in solid form prepared by fusion, solvent or melt-solvent methods” . As a ruNowadays, ASDs are considered to be one of the most advantageous formulation approaches for tailoring, and providing, the superior biopharmaceutical properties of poorly soluble drugs without altering the chemical structure of the API . NumerouHowever, despite the increased research interest, the number of ASDs available on the market is still limited ,36. ASDsOver the years, different polyelectrolytes have been employed as excipients in pharmaceutical technology. Anionic polyelectrolytes are characterized by the presence of a carboxylic, sulfate, or phosphate ionizable group, with the derivatives of acrylic acid , alginic acid, sodium carboxymethylcellulose (NaCMC), and hyaluronic acid. On the contrary, cationic polyelectrolytes possess a protonated nitrogen atom in their structure. Representative examples of this category are chitosan and methacrylate copolymer Eudragit E100 ,47.The applications of polyelectrolyte polymers are wide-ranging and they include hydrogels  and swel®, have a remarkable presence. Eudragits are synthetic polymers obtained by the polymerization of acrylic acid  and methacrylic acid, or their esters. These polymers have been extensively used as excipients for enteric film-coating formulations, sustained release, taste/smell masking, and moisture prevention coatings [In ASD formulation, methacrylate copolymers, marketed under the brand name Eudragitcoatings ,62. Amoncoatings ,64. Polycoatings ,66,67. THot-melt extrusion (HME), as an environment friendly, low-cost, fast and continuous operation, has become a pivotal process in ASD formulation in order to achieve enhancement in drug dissolution rates and drug release modification ,97. AccoSeveral polyelectrolytes have been employed in the HME process for the preparation of stable ASDs showing an enhanced dissolution rate for various poorly soluble APIs, such as Carbopol , NaCMC , and HPMAn amorphous single-phase supersaturated system comprised of the poorly soluble acidic drugs naproxen and Eudragit EPO, as well as a furosemide-Eudragit EPO polyelectrolyte complex, were successfully produced by HME . The selw/w, respectively, at low temperatures and preoptimized conditions. Stronger drug-polymer interactions were obtained in the melt in comparison to the quench-cooling method, and no apparent recrystallization of the drugs was detected after storage at 95% relative humidity [Eudragit EPO has also been investigated for the development of high drug loading ASDs with the model drugs indomethacin, ibuprofen, and naproxen, in maximum drug concentrations of 65, 70, and 60% humidity . Howeverhumidity .Propranolol HCL and diphenhydramine HCL, as cationic model drugs, were successfully processed via HME with Eudragit L100 and Eudragit L100-55 as polyelectrolyte carriers, while thermal analysis confirmed the amorphous state of the complexes. Results from an X-ray photon spectroscopy (XPS) advanced surface analysis of the extrudates revealed the existence of hydrogen bonds between the amide groups of the drugs and the carboxyl group of polyelectrolytes, in complete agreement with the results of molecular modeling, Except for the different Eudragit grades, anionic polymers HPMCAS, and HPMCP of various types have been reported as suitable matrices for stable ASDs produced by HME ,79,101. Nifedipine-HPMCAS and efavirenz-HPMCAS amorphous extrudates were effectively developed, while FT-IR data confirmed the existence of strong intermolecular interactions. Among the different grades of the polymer, HPMCAS-MG and HPMCAS-LG for nifedipine and efavirenz, respectively, were proven to be the optimal formulations due to their capacity to achieve the higher solubility rate and the extended maintenance of supersaturation . ASDs ofExtrudates of nitrendipine with HPMCP and Carbopol as carriers were produced via twin screw extrusion. Powder X-ray diffraction and DSC analysis confirmed the amorphous state of the drug in the solid dispersions, and results from FT-IR analysis revealed the presence of hydrogen bonding between the secondary amine groups of nitrendipine, the hydroxyl groups of HPMCP, and the carboxyl groups of Carbopol, respectively. Τhe electrostatic interactions between nitrendipine and HPMCP, although proven to be stronger, decelerated the dissolution profile of the drug, making Carbopol a more suitable carrier to enhance the dissolution of nitrendipine . Moreove® L100 or Eudragit® S100 with hypromellose (HPMC) was investigated via co-spray drying, The concept of combining polyelectrolytes with other polymers to optimize the dissolution profile and the resistance toward the crystallization of amorphous drug formulations has been reported in several studies. In many studies, polyelectrolytes have been combined effectively with other polymers in order to optimize the HME processability or the stability of the extruded ASDs ,106. A pThe incorporation of Carbopol 974P as a stabilizing agent into a Eudragit L100-55 HME-matrix containing itraconazole had a positive effect on supersaturation levels. The strong association of the drug and the polymers, and the maintenance of this electrostatic interaction in the solution, were apparently prolonged by increasing the viscosity of the acidic polymer, resulting in delayed precipitation and, thus, a prolongation of supersaturation . In anotThe use of interpolyelectrolyte complexes (IPECs), obtained as precipitates upon mixing polyelectrolytes with an opposite charge in aqueous media, has been reported as an effective approach to broaden the applicable polymers in oral drug delivery systems, and they could possibly be used as matrices in ASDs ,110,111,An IPEC between cationic Eudragit EPO and anionic Eudragit L100 was successfully developed via the solvent evaporation method at a pH of 6.0, in which both polyelectrolytes were soluble and partially ionized, Following the same synthesis process of mixing polyelectrolyte solutions, Mustafine et al.  formed iIn their study, Ngwuluka et al.  elucidatAs suitable pharmaceutical polymers for the development of ASDs are limited, research has focused on the development of modified polyelectrolyte matrices combining the already used polymers with small molecular additives ,126. FroAccordingly, the addition of saccharin to the phenytoin-Eudragit EPO amorphous system accelerated the dissolution rate and improved the supersaturation level of the drug, due to the ionic interactions between the polyelectrolyte and the additive molecule. The molecular interactions between Eudragit EPO and saccharin were proven to be responsible for the promotion of the dissolution of EPO from the gel layer, resulting in the enhancement of the dissolution and the supersaturation rate of the drug . At the Ditzinger et al.  designedThe physical stability of the amorphous drug remains one of the most challenging issues during the formulation, development, and storage of an ASD . Among oWegiel et al.  investigStability studies of the ASDs of lapatinib with acidic HPMCAS and HPMCP revealed that the drug remained in the amorphous state for over six months, even after storage at accelerated conditions. The enhanced physical stability of the two systems was attributed to the drug-polymer intermolecular interactions. In the case of lapatinib-HPMCP, remarkably higher Tg values than those predicted indicated a strong intermolecular interaction. Solid-state NMR analysis elucidated the formation of ionic interactions between protonated lapatinib within the phthalate groups of the polymer, while the stabilization of lapatinib-HPMCAS was possibly due to hydrogen bonding. In the same study, PVPVA and nonionic Soluplus were characterized as poor crystallization inhibitors since they failed to maintain the stability of the amorphous dispersions of lapatinib . The preIn another study, an unusual instance of the enhancement of electrostatic interactions under appropriate temperature and humidity conditions was observed in the amorphous extrudates of indomethacin, Eudragit EPO and itraconazole, and HPMCAS-LF, after three months of storage. Sarode et al.  suggesteThe antiplasticization phenomenon, known as the reduction in the plasticity of a material, is evident by the increase in the Tg of the drug in an ASD while the free energy for its recrystallization increases . The antThe extent and maintenance of supersaturation achieved by polyelectrolyte ASDs are crucial parameters to be considered, as they directly affect the dissolution and absorption properties of active pharmaceutical ingredients that suffer from poor bioavailability ,132. TheStudies on a supersaturated solution produced by mefenamic acid-Eudragit EPO amorphous solid dispersion revealed that drug-polyelectrolyte intermolecular interactions are responsible for the high stabilization of this system. The maintenance of supersaturation was attributed to both the hydrophilic and hydrophobic interactions between the carboxyl group of mefenamic acid and the tertiary amine of the EPO, and the two aromatic groups of the drug with the backbone of the polymer, respectively, as demonstrated in the 2D1H/1H NOESY and RFDR MAS NMR analyses . Results0→24 h and Cmax of dutasteride increased with the supersaturation concentration. Similarly, the ASDs of sirolimus with Eudragit E were efficiently manufactured by the spray drying method. The evaluation of the dissolution studies showed that the dispersion significantly inhibits the degradation and precipitation of sirolimus in a dose-dependent manner. The addition of a nonionic polymeric surfactant, such as D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), to the formulation enhanced the stability and degree of supersaturation, as shown by the dissolution and solubility tests in simulated gastric fluid, while pharmacokinetic studies in rats indicated a significant improvement in the oral absorption of sirolimus [Beak and Kim  investigirolimus .On the contrary, the dissolution studies of the ASDs of ezetimibe monohydrate with PAA in different biorelevant media revealed that crystallization from the solid matrix occurred rapidly when the ASDs came into contact with either the sodium phosphate buffer or the fed state simulated intestinal fluid medium, incapable of achieving supersaturation, while only moderate supersaturation was maintained for a longer time period in a biorelevant lipid-rich medium . SimilarRecently, extensive research has been conducted assessing the performance of pH-responsive, water-insoluble, and modified polyelectrolytes in controlled-release studies ,137. ConIn their study, Balogh et al.,  producedA novel approach to targeting the colon involves the use of the anionic polymer Eudragit S100 to form IPECs with cationic Eudragit EPO. The results of the FT-IR spectroscopy, and the modulated-temperature differential scanning calorimetry, confirmed that the new IPEC was stabilized by the cooperative intermacromolecular ionic bonds between ionized EPO dimethylamines and the S100 carboxyl groups. The investigation of the swelling capacity in media imitating passage through the gastrointestinal tract pointed out the increasing swelling of the polycomplex structural fragments, and the subsequent compaction in the final medium with a pH = 7.4, corresponding to the characteristics for a carrier providing delivery to the colon . Additio® Retard, making this particular IPEC promising as a candidate for colon-specific delivery [Similarly, a new ionic-bonded polycomplex between the soluble cationic polyelectrolyte Eudragit EPO and Carbopol 940, with high swelling capacity, was synthesized and examined as a controlled-release drug delivery system . Dissoludelivery .As our understanding of the drug-polymer interactions responsible for the stabilization of the amorphous state is increasing, the natural next step in the development of ASDs is the modulation of the aforementioned interactions by the clever choice of materials possessing chemical moieties that are expected to interact favorably. This rationale is of particular interest in the case of weakly basic drugs that tend to precipitate in the intestine, losing their solubility advantage. In this perspective, the use of polyelectrolyte polymers has gained much attention in the pharmaceutical industry in the attempt to extend the polymeric excipient landscape for oral drug formulations. The application of polyelectrolytes and novel polyelectrolyte complexes as matrices for ASDs could lead to more effective delivery systems with controlled and targeted drug release, while the intermolecular interactions could enhance the bioperformance and the physical stability of the ASDs. ASDs based on polyelectrolyte polymers and their complexes constitute a promising enabling technology for the maintenance of supersaturation in biological fluids, especially in the case of weakly basic drugs. Scalable manufacturing methods, such as hot-melt extrusion, could be utilized in accordance with green chemistry principles to investigate innovative interpolyelectrolyte complexes and modified polyelectrolyte matrices for further improvement in drug delivery applications.On the downside, only a limited number of polyelectrolyte polymers has proven suitable for processing by hot-melt extrusion, a fact that is limiting the available choices for the formulation scientist. Newly investigated polymers, such as NaCMC, or novel materials, such as interpolyelectrolyte complexes, cannot be easily formulated into ASD matrices. One additional limitation regarding their physical stability enhancement and supersaturation-maintaining effect, is that they are mainly effective in the case of weakly acidic/basic APIs, where the electrostatic and polarization interactions are more prominent.In our opinion, future research should focus on the rational model-based selection of suitable polyelectrolye polymers. Both thermodynamic , and atomistic modeling (molecular dynamics simulations), have a lot to offer as a means for the interpretation of experimental findings and the prediction of suitable polymers for API categories. As our understanding of the main types of interactions responsible for API crystallization advances, atomistic simulations, especially, can be of grave importance to ASD formulation design, in general. Another field that needs to be more intensively researched relates to the HME development process relevant to polyelectrolyte polymers. As already discussed, many promising polyelectrolyte polymers have shown poor processibility alone. Therefore, there is a need for research on suitable additives that will not impair, but rather enhance, their beneficial action on supersaturation maintenance, in addition to the enhancement of their processibility.In conclusion, although the use of polyelectrolytes is not a general solution to ASD stability and biopharmaceutical performance problems considering its limitations, nevertheless, despite the aforementioned limitations, it represents yet another step forward in our efforts to control critical factors affecting physical stability and drug release and could, eventually, lead to ASD-based pharmaceutical products of enhanced safety and efficacy.


Hereditary sensory and autonomic neuropathies (HSAN) are rare genetic disorders that often manifest during childhood in the form of absence of pain sensation or self-mutilation. Patients often present significant oral self-mutilation manifestations, and biting of the lips, tongue, and cheeks have been frequently reported. This case report describes a case of hereditary sensory and autonomic neuropathy with oral and cutaneous ulcers.Our patient was a 14-month-old girl with the chief complaint of a tongue ulcer, as stated by her parents, who were referred to our private dental clinic. Clinical examination revealed severe ulcers due to biting (Riga-Fede disease) on the ventral surface of the tongue and superficial ulcers on the dorsal surface of the tongue caused by the anterior maxillary teeth, along with some sores on fingers. The parents were healthy, with no congenital disease or familial history of a similar condition. The electrodiagnostic test revealed the absence of sensory nerve action potential response. However, the electromyographic findings and the compound muscle action potential of the tibial and ulnar nerves were normal. Oral ulcers such as trauma to the lips and tongue, and self-mutilation trauma to the fingers can be used for early detection of Hereditary sensory and autonomic neuropathies. A multidisciplinary approach involving a professional dental team and a regular treatment protocol are imperative to prevent complications of Hereditary sensory and autonomic neuropathies. Hereditary sensory and autonomic neuropathies (HSANs) are a group of rare genetic disorders caused by different mutations that can affect the function, development, and survival of sensory and autonomic neurons. Previously, the diagnosis and classification of HSANs were based on the age of onset, the pattern of inheritance, and clinical manifestations. However, at present, the diagnosis is based on the detection of pathogenic mutations by the whole-exome sequencing technique. While the phenotypic presentation of the disease may be variable, the gene penetrance is 100%. The impaired sensation of pain and heat are among the prominent symptoms ,3. This Herein, we report a patient with HSAN, and the methods administered to prevent oral self-mutilation are also explained. Parents of a 14-month-old girl presented to our private dental clinic in June 2018 complaining of a large ulcer on the ventral surface of the tongue of their child . AccordiIn the electrodiagnostic test, the compound muscle action potential (CMAP) was normal. However, the sensory nerve action potential (SNAP) was absent, and severe sensory neuropathies were reported in all extremities. Further examinations revealed that the patient was sensitive to thermal alterations; however, she was less sensitive to pain stimuli. Extraoral clinical examinations revealed self-injuries to the fingers that were healed after bandaging  and 4. IGrinding of the teeth significantly decreased the frequency of oral ulcers. Also, preservation of the teeth as much as possible helped preserve the alveolar ridge for future appliance therapy. On the other hand, preserving the four maxillary incisors improved the patient's appearance and satisfied the parents. The time intervals between the grinding sessions were gradually increased. At the 6-month follow-up, all teeth and the oral mucosa were sound, and grinding was no longer required. At the 38-month follow-up, the patient's mandibular right canine tooth was missing. According to her mother, she was playing with the tooth for 2-3 days until she could finally take it out .HSAN is a rare syndrome with an unknown etiology. Patients often have oral manifestations related to self-mutilation ,2. At prIn some cases, the parents had a third-degree of consanguinity . HoweverIn Conclusion, Patients with HSANs may present a variety of clinical symptoms, among which oral manifestations such as tongue biting can significantly help in early diagnosis. HSANs are rare genetic disorders with no specific treatment. Thus, education of the parents, regular follow-ups, selective grinding, and other supportive treatments are recommended for such patients to preserve their oral and dental health.All authors have read and approved the final manuscript. The authors gave their contribution to the drafting and critical review of the article.The authors declare that they have no conflict of interest.


There has been significant scientific progress in skin care and skin damage repair, but the complete understanding of skin homeostasis is still beyond our reach. With an increase in environmental stress factors, the incidence rates of skin cancer and skin disorders are on the rise. Taken together with the incidence of scar- and burn-related morbidities, there is an urgent need to understand interactions between skin cells to develop novel therapies for the regeneration of healthy skin. One of the recurrent stress factors affecting the skin are the harmful free radicals, also referred to as oxidative stress. This study aimed to address the influence of oxidative stress on the interaction between two types of skin cells, keratinocytes and fibroblasts. The study utilized cold atmospheric plasma (CAP) to induce oxidative stress in cells and to assess the interactions between the two cell types. We showed that CAP can stimulate cells to enhance their proliferation and migration. This study provides a further understanding of skin cell regulation under stress conditions. Such knowledge may help in designing treatment therapies for rapid wound healing and skin repair.KGF, MMP2, GMCSF, IL-6, and IL-8) in fibroblasts, indicating the activation and initiation of the skin repair process. Additionally, enhanced migration was observed under co-culture conditions under the given redox stress conditions, and expression of the upstream regulator and the effectors of the Hippo pathway , which are associated with enhanced migration, were elevated. Overall, this study reinforces the application of CAP and redox stress in skin repair physiology.Although the skin is constantly subjected to endogenous and exogenous stress, it maintains a homeostatic state through wound repair and regeneration pathways. Treatment for skin diseases and injury requires a significant understanding of the various mechanisms and interactions that occur within skin cells. Keratinocytes and fibroblasts interact with each other and act as key players in the repair process. Although fibroblasts and keratinocytes are widely studied in wound healing and skin remodeling under different conditions, the influence of redox stress on keratinocyte-fibroblast crosstalk has not been thoroughly investigated. In this study, we used cold atmospheric plasma (CAP) to generate and deliver oxidative stress to keratinocytes and fibroblasts and to assess its impact on their interactions. To this end, we used a well-established in vitro 3D co-culture model imitating a realistic scenario. Our study shows that low CAP exposure is biocompatible and does not affect the viability or energetics of fibroblasts and keratinocytes. Exposure to low doses of CAP enhanced the proliferation rate of cells and stimulated the expression of key genes ( Human skin, the largest organ of the human body, is involved in the regulation of body temperature and metabolic functions and acts as a barrier against harsh external environment, preventing skin damage ,2,3. TheCYR61 and CTGF in fibroblasts, which are target genes of YAP .,42.KGF, roblasts . While G reports  [37,65,6ex vivo complementing 3D in vitro models such as those used in this study could overcome its limitations and recapitulate the skin repair process. Alternatively, extensive transcriptome analysis of cells derived from healthy skin and oxidative stress damaged skin will scale up our knowledge. Taken together, new approaches with CAP will help in deciphering the subtle mechanisms involved in skin repair.The clinical importance of skin homeostasis is evidenced by the association between cutaneous defects and morbidity. The increasing number of skin-related issues has become a daunting issue that requires novel therapies and research. Current therapy modules and research models are limited to skin grafts for wounds and burns. Additional research using novel biocompatible options, such as CAP, is necessary to use new biocompatible options. In recent years, many clinical studies have investigated the effects of CAP, especially in wound healing and melanomas, with the aim of understanding interactive networks in skin biology. Few studies have been conducted to investigate the role of CAP, ROS, and associated electrical signals in the proliferation and differentiation of epidermal cells, fibroblasts, and myofibroblast transdifferentiation ,68. HoweIn conclusion, CAP exposure can engage with keratinocyte-fibroblast crosstalk at the onset of injury and can modulate it further to enhance the repair process. To this end, CAP may stimulate the cells to proliferate and upregulate redox-induced pathways such as the Hippo signaling cascade, which may facilitate paracrine signaling between cells. Although CAP-mediated biological responses are largely attributed to RONS, additional research is required to understand the synergistic and additive functions of CAP, especially in the skin.


Bacteroides to Prevotella dominance, and compromises gut barrier integrity, which may promote microbial translocation and consequent systemic inflammation and exacerbation of neuroinflammation. Further, though the use of antiretroviral therapy has been found to partially counteract HIV-related dysbiosis, it may also induce its own dysbiosis patterns, presenting a unique challenge for this research.The microbiome-gut-brain axis, or the various interactions between the gut microbiome and the brain, has been of recent interest in the context of precision medicine research for a variety of disease states. Persons living with human immunodeficiency virus (PLWH) experience higher degrees of neurocognitive decline than the general population, correlating with a disruption of the normal gut microbiome composition (i.e. dysbiosis). While the nature of this correlation remains to be determined, there is the potential that the microbiome-gut-brain axis contributes to the progression of this disease. Previous research has established that the pathology associated with HIV induces alterations in the composition of gut microbiome, including a shift from More recent research has suggested the gut microbiome as a target for therapeutic interventions to improve symptoms associated with a variety of disease states, including HIV. Early findings are promising and warrant further research regarding the gut microbiome as a potential modifiable factor to improve health outcomes for PLWH. This review will discuss the current knowledge concerning the neuropathogenesis of HIV in the brain, role of the gut microbiome in neuroinflammation, and the relationship between HIV-status and the gut microbiome, followed by a conclusion that synthesizes this information within the context of the microbiome-gut-brain axis among PLWH. This review will also highlight the limitations of existing studies and propose future directions of this research. •The microbiome-gut-brain axis has been of recent interest as a potential novel target for precision medicine.•Individuals with HIV experience considerable neurocognitive decline and disruption of the normal gut microbiome composition.•Probiotic supplementation has been found to reduce neuroinflammation and improve neurocognition in individuals with HIV.•Future research controlling for confounders, using diverse samples, and evaluating long-term interventions is needed. Recent research has highlighted the role of the microorganisms living on and inside of human hosts in the development and progression of several disease states, such as inflammatory bowel syndrome, autism, mental health disorders, and neurological conditions, as well as in a number of critical health functions, including for gut , liver , and bra1.1The advent of combined antiretroviral therapy (ART) has vastly improved patient outcomes and revolutionized the field of human immunodeficiency virus (HIV) research. Persons living with HIV (PLWH) who achieve sustained viral suppression on ART now reach near-normal life expectancies, nearly doubling the proportion of PLWH over the age of 50 since 2015 . DespiteOne hypothesis developed to explain this excess morbidity in neurocognitive impairment in PLWH is related to HIV neuropathogenesis. Infection with HIV can lead to neuroinflammation and premature aging of the brain, each contributing to neurocognitive decline . HIV infEstimates report nearly half of all PLWH suffer from HIV-associated neurocognitive decline (HAND) , which iThe neuropathology associated with HIV shares many characteristics and pathways with that of Alzheimer’s disease (AD); therefore, it is currently unclear whether PLWH are truly at greater risk for AD and other similar dementias compared to the general population . As the Another study conducted among PLWH reported that having at least one apolipoprotein E4 (ApoE4) allele, a particular ApoE genotype thought to be associated with late-onset Alzheimer’s disease , was assIn addition to having an increased burden of neurocognitive disorders and dementias, PLWH also tend to have higher levels of depression and substance use than those without HIV . These m1.2The human microbiome is defined as a collection of microorganisms, their genes, and their associated metabolites that occupy diverse anatomical sites both on and within the human body . The micStudies of germ-free mice have enhanced the current understanding of the gut-brain axis. Germ-free mice tend to have elevated levels of serum corticosterone, a measure of hypothalamic-pituitary-adrenal (HPA) axis activation, relative to specific pathogen free (SPF) colonized controls . AdditioHelicobacteraceae and Desulfovibrionaceae and decreased relative abundance of Prevotella species in Alzheimer’s disease transgenic mice when compared to wild type (WT) mice  mice . Numerou disease , however disease . Decreas disease , and has disease  and regu disease  and are  disease .Bifidobacterium breve strain A1 in transgenic mouse models of Alzheimer’s disease – reporting upregulation of the bdnf gene, which is involved in learning and memory processes, reduced latency time and reversed impairment of alternation behavior  and Bifidobacterium bifidum for 12 weeks  and high-sensitivity C-reactive protein (a biomarker of inflammation) were observed in the probiotic supplementation group compared to the control group. Importantly, these studies reported minimal impact of probiotic administration on composition of the gut microbiome, fasting glucose levels, and lipid profiles  and controls   . Taken tumption) .Actinobacteria and Firmicutes and lower proportions of Proteobacteria in the brains of Alzheimer’s patients compared to controls  in vivo  who often suffer fewer neurocognitive dysfunctions and metabolic disturbances, calling into question the representativeness of the sample populations and the generalizability of the results to the general target population of PLWH. Additionally, many microbiome-disease association studies in PLWH fail to control for factors that influence the microbiome separately from HIV infection, including ART use and adherence, dietary habits (including use of probiotics), sexual behaviors, substance use, long-term exposure to certain medications, physical activity, and other comorbidities. For example, one study reported no evidence of an association between HIV infection and gut microbiome dysbiosis after controlling for sexual orientation .Granulicatella and Clostridium cluster XIVa species and decreased presence of Actinomyces, Campylobacter, and Firmicutes classes whereas, receptive anal sex practices were associated with increased presence of Peptostreptococcus and Anaerococcus species, and likewise decreased presence of Firmicutes organisms , or, may simply be a product of HIV-associated physiological alterations in the gastrointestinal tract of the host. Understanding of the causal molecular mechanisms, such as bacterial metabolite production or immune modulation, underlying associations between the microbiome and target health outcomes is essential for the future development of therapeutic interventions intending to modify the gut microbiome.It is important to note that the microbiome studies discussed in this review do not make causal conclusions. While many associations between characteristics of the microbiome and markers of health outcomes have been established, the underlying molecular mechanisms of many of these associations remain unknown. Therefore, the nature of this association, and whether it is truly causal, also remains to be determined. For example, the shift from 2.3As noted extensively in this review, lack of consideration of individual characteristics such as ART adherence, physical activity, other medication or supplement use, diet, substance use, sexual behaviors, and presence of other comorbidities in analyses of the microbiome-gut-brain axis is common and presents a concern with respect to the scientific validity of the presented conclusions. Lack of controlling for these potential confounders may obscure the true nature of the relationship between the microbiome and the brain in the context of HIV; however, the number of participants and level of resources needed to account for these factors makes this approach challenging. Aside from the analytical flaws, ignoring the social and behavioral factors, which may be independently perpetuating gut dysbiosis, also calls into question the sustainability of current intervention approaches. These approaches, which are designed to modify the gut microbiome in the short-term through prebiotic or probiotic supplementation, neglect to consider the effectiveness of these interventions in the long-term, which are dependent upon adherence, especially in the absence of any behavioral changes. Future longitudinal studies which incorporate survey data on behavioral factors, such as medication adherence, diet, sexual practices, and substance use, are necessary to determine the success of current intervention approaches. Consideration for long-term intervention is of great importance given the dynamic nature of the microbiome over time and across the human lifespan .2.4As mentioned previously, the potential cognitive benefits of altering the gut microbiome via dietary or behavioral modification and prebiotic or probiotic supplementation warrant additional intervention studies in this domain, with important considerations. In addition to supplementation, alternative approaches to improving gut health should be considered in this population. For example, findings from a focus group study conducted among PLWH indicated that participants did not have knowledge about the role of diet in reducing inflammation and promoting a healthy gut microbiome . This su


Depression causes individual suffering, loss of productivity, increased health care costs and high suicide risk. Despite the fact that depressive disorders are a serious brain dysfunction, their etiopathogenesis is not yet fully understood. Only 60–70% of patients suffering from Major Depressive Disorders (MDD) respond to the standard antidepressant treatment, which means that Treatment Resistant Depression (TRD) can be as prevalent as 1/3 of patients with clinical depression [There is strong evidence that major depression (MDD) is a neuro-immune and neuro-oxidative disorder with increased levels of pro-inflammatory cytokines ,3.Dysregulation of the immune system is recognized as one of the most important contributory factors to TRD . No wondAccording to the traditional monoamine theory of depression, most currently available antidepressant drugs were designed to inhibit monoamine reuptakes and increase the utilization of serotonin, noradrenaline and dopamine in the central nervous system (CNS). Now we know that monoamine deficiency is the result of more primary abnormalities. Monoamines are formed from tryptophan. However, in a situation of increased activation of the immune system, inflammatory factors cause excessive activation of IDO , an enzyme present in microglia, astrocytes and neurons, catabolizing the transformation of tryptophan into the neurotoxic kynurenine (KYN), thereby reducing the availability of tryptophan for the production of serotonin. Kynurenine, in turn, influences the intensification of neurodegenerative processes .Research on twins, families and adoptive children suggests the influence of genetic factors on the development of depressive disorders. The expression of a given feature is influenced not only by the sequence of DNA nucleotides, but also by epigenetic factors . The expDue to epigenetics, new research highlights the importance of early stages of development , includiRegarding the biological hypotheses , several molecular factors, including those involved in neuroplasticity, might serve as biomarkers of affective disorders ,10. SomeFurther research into the biological basis of depressive disorders is necessary for their targeted, personalized treatment.