Extracellular vesicles (EVs) are lipid membrane particles carrying proteins, lipids, DNA, and various types of RNA that are involved in intercellular communication. EVs derived from mesenchymal stem cells (MSCs) have been investigated extensively in many different fields due to their crucial role as regeneration drivers, but research for their use in degenerative diseases of the intervertebral disc (IVD) has only started recently. MSC-derived EVs not only promote extracellular matrix synthesis and proliferation in IVD cells, but also reduce apoptosis and inflammation, hence having multifunctional beneficial effects that seem to be mediated by specific miRNAs (such as miR-233 and miR-21) within the EVs. Aside from MSC-derived EVs, IVD-derived EVs  also have important functions in IVD health and disease. This article will summarize the current knowledge on MSC-derived and IVD-derived EVs and will highlight areas of future research, including the isolation and analysis of EV subpopulations or exposure of MSCs to cues that may enhance the therapeutic potential of released EVs. Degenerative disc disease (DDD) is a major origin of low back pain, which is the leading cause of activity limitation and work absence and results in a high economic burden . In the As in numerous other fields, great hopes were pinned on MSCs for the treatment of DDD due to their regenerative and anti-inflammatory potential that would allow simultaneously targeting all primary pathological drivers . FurtherThis mini-review will briefly summarize both established and more advanced techniques for the collection and size-sorting of EVs before presenting evidence on the content and function of MSC-derived EVs on cells from the inner region of the IVD, the nucleus pulposus (NP). Furthermore, knowledge on the characterization of EVs released from IVD cells are briefly summarized and future areas of research are highlighted.With a rapid increase in EV-related research over the past years, one of the major limitations is the use of heterogeneous, often field-specific, nomenclature. In IVD research, the term exosome is commonly used, whereas the terms microvesicles, apoptotic bodies, and microparticles are less common. Importantly, the rather historical classification of these particles according to size is outdated as recent studies have shown significant overlap in particle subtype sizes . KnowledIn order to study the biological roles and potential applications of EVs, the first step is to reliably and efficiently isolate them from biological fluids and conditioned media. Ideal isolation methods purify intact EVs with preserved biological function and without contamination by proteins or non-EV particles. Over the past years, many techniques have been developed to isolate EVs based on properties such as size, density, and surface proteins, including ultracentrifugation, ultrafiltration, precipitation, and affinity-based techniques.The most commonly employed technique was first described by In the IVD field, the majority of studies have used differential centrifugation protocols. Some modified these protocols by adding a filtration step between the centrifugation steps, while others replaced the ultracentrifugation step by the use of commercially available precipitation-based kits . Precipitation-based techniques have a high yield but tend to suffer from low purity. Two IVD studies showed fractionation of conditioned media by ultrafiltration through membranes with different molecular weight cut-offs (MWCO), yielding spherical species with a size range of 50–100 nm. Overall, techniques currently used in IVD research for collection and purification of EVs are widespread and common, but could be replaced by recent technical advancements. By incorporating newer approaches and more sophisticated techniques such as tangential flow filtration (TFF) and size exclusion chromatography , reliabiThe lipid bilayer membrane of EVs plays an important role in their stability by protecting their bioactive cargo against degradation . In ordeThe presence of EVs in a purified sample should be confirmed by multiple methods. Western blot (WB) has been employed to characterize the isolated samples by evaluating the presence of EV-enriched markers and the absence of negative markers. Alix, TSG101 and surface associated markers such as Tetraspanins  have been used as positive markers for EVs , while pAlthough systemic delivery of EVs is generally considered the easiest approach, biodistribution patterns indicate accumulation in the liver, spleen, and lungs . EspeciaWhile defining optimal doses has been a crucial aspect in MSC-based IVD therapies due to the limited nutritional supply within the tissue , EV dosiin vivo tracking studies of EVs   , howevernt dyes) , may becnt dyes) .Only few studies have thus far been conducted on IVD-derived EVs, although they most certainly play a crucial role during IVD health and disease. In fact, researchers in other fields have become increasingly interested in the analysis of EVs from resident cells, with the hope to identify specific disease biomarkers . With thConcerning function of IVD-derived EVs, data on notochordal NP cells and chondrocyte-like NP cells have been published. Notochordal cells are a developmental cell type in the NP that, at least in humans and some other species, gets increasingly replaced by chondrocyte-like IVD cells over the course of life; the reduction in notochordal cell number is believed to be associated with the onset of IVD degeneration . NotochoAmple evidence exists from a variety of fields that MSC-derived EVs have therapeutic effects , includiAs the success of MSC cell therapies for IVD diseases has been hampered due to the restricted survival and functionality of the cells in the harsh IVD microenvironment , the usein vitro and during myocardial infarction in vivo  proteins by various means, including MSCs and MSC-derived EVs. In fact, 2O2-induced inflammation in NP cells. Results of this study provide compelling evidence for anti-inflammatory properties of EVs via modulation of the NLRP3 inflammasome and through restoration of damaged mitochondria  is associated with back pain development , many rechondria . Althougchondria . Importaon model . Whetheron model .To further improve the outcome of EV therapy, MSCs can be modified to produce EVs with enhanced or reduced content of certain components with biological activity, such as specific miRNAs. With an increasing understanding of the role of certain miRNAs in IVD health and disease , EV therDespite the promise that initial studies on MSC-derived EVs have sparked, one of the remaining challenges may be the limited distribution of EVs within the IVD due to the densely packed collagen and proteoglycan-rich ECM. However, this dense ECM may also help to retain EVs within the IVD following intradiscal injections. Mathematical models should be employed in the future to further investigate this question and to determine whether specific EV sizes or EV membrane compositions would be best suited for IVD applications. However, these parameters will not only affect tissue retention and distribution, but also biological activity, and both aspects will need to be considered .First research results on MSC-derived EVs for the treatment of DDD clearly demonstrate their therapeutic potential by promoting regeneration and proliferation and reducing inflammation and apoptosis in the IVD, although the exact mechanisms of action are not yet fully elucidated.Ample studies in the past have investigated MSC-based therapies for the IVD, leading to increasing knowledge on the bidirectional crosstalk between MSCs and IVD cells. However, MSC cell therapy for the IVD is hampered by the harsh tissue microenvironment; furthermore, cell therapies face safety concerns and thus approval challenges. Although few studies have investigated the effect of e.g., acidic pH on EV stability, it is conceivable that the negative effects that the harsh IVD microenvironment has on MSCs themselves is not relevant for EV-based therapies. In fact, first studies demonstrated a possible increase of cellular uptake of EVs under IVD like pH conditions. Investigating the effect of the harsh IVD microenvironment on EVs is thus an important topic for future investigation.Despite these advantages of EVs compared to MSC cell therapy, a major limitation lies in their low yields. However, technical advancements related to fast expansion of MSCs as well as enhanced release of EVs that can be induced by commercially available media and additives in combination with new purification strategies will likely help to overcome this restraining factor.in vivo delivery of EVs may further enhance the therapeutic benefits through improved captivity within the IVD tissue, sustained release and/or preservation of function.It has become increasingly evident that exposure of MSCs to biophysical and/or biochemical cues may help to further improve the clinical outcome. Therefore, future investigations should aim to identify which signals can enhance the regenerative and/or the anti-inflammatory capacity of EVs, specifically on IVD cells. Alternatively, studies may identify certain subpopulations of EVs that have increased therapeutic properties and/or enhanced tissue retention, based on size characteristics or membrane composition, for example. The use of a biomaterial-based carrier for the With the development of novel techniques for the production, collection and delivery of EVs or EV subpopulations, ample possibilities for optimization of EV therapies may arise. However, a better understanding and subsequent optimization of the effects that temperature and storage conditions have on EV stability will be required to promote successful translation into the clinical setting.NP, MD, TG, and KW-K contributed to the literature search, the design of the figure, and the writing 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.


Sex manipulation technologies allow predetermination of the sex of animal offspring by altering the normal reproductive process. In livestock production, the difference in type and gender can translate into significant economic benefits, including alleviation of severe food shortages. In livestock, however, the commercial application of sex manipulation technologies is currently available for cattle only. In this review, we described the brief history of sex manipulation, and the research progresses of common methods used in sex manipulation thus far. Information presented in this review can inform future studies on expanding the scope and use of sex manipulation technologies in livestock. By 2050, the world's population is expected to increase by 9.6 billion, and the demand for livestock products, by 70 percent . Most ofIn ancient times, boys were also preferred to girls since besides being more likely to appease the wrath of the gods, they were regarded to be a strong support for the elderly . The preTheories of what determined the sex of an individual transited from myths to science in the 17th century. In 1651,William Harvey compared the reproductive organs of several animals and stated that “all animals whatsoever, even viviparous creatures, man himself, are all engendered from an egg”  in humans. In 1990, researchers found a 35-kb region from Y chromosome in four masculinized XX patients. Southern blotting revealed that the Y-fragment was the likely carrier of the TDF gene (SRY in humans) (SRY up-regulated the expression of SOX9 in somatic (pre-Sertoli) cells of the genital ridge , transcription-activator-like endonucleases (TALENs), and the clustered regularly interspaced short palindromic repeats/Cas endonucleases (CRISPR/Cas) system has offered great potential in the selection of sex at the gene level .Sry knockout mouse via oocyte injection using TALEN  is a biological process that uses small interfering RNA (siRNA) to induce sequence-specific gene silencing through mRNA degradation . Since iSry mRNA by direct injection of siRNA into pregnant mice through the tail vein generated mouse embryos with feminized gonads  in 1986 , 64. SinGenerally, contemporary sperm-sorting procedures yield a skewed offspring sex ratio of between 85 and 95% accuracy . Flow cyInitial studies focused on identifying the steps in flow cytometry that caused sperm damage. It was earlier thought that optimization of sperm dilution rate –74, as wIn a study that aimed to describe the effects of flow cytometry on the structure of the glycocalyx of bovine sperm by using lectin histochemistry, both flow cytometry and capacitation had similar effects on bovine sperm, which may account for the low conception rates observed after flow cytometry . AnotherIn conclusion, flow cytometry is the most used and mature technology for sperm sexing in livestock production, with a separation accuracy of above 90%. However, due to the high sperm requirement and susceptibility of other species, this strategy has only reached commercial application in cattle production. Future studies should therefore focus on reducing sperm damage caused by flow cytometry, develop related technologies, and optimize artificial insemination technology.Immunology sexing is achieved through the identification and separation of sex-related proteins. This is mainly done by detecting the H-Y antigen on the plasma membrane of Y-bearing sperm by using the H-Y antibody. Thus, far, sperm from many species has been successfully separated including mice , rabbitsin vitro fertilization and embryo transfer. In the results, upstream sperm produced 77 blastocyst embryos, among which 68 were XY embryos and 9 were XX embryos, while downstream sperm produced 83 blastocyst embryos, among which 58 were XY embryos and 25 were XX embryos  as a result of Toll-like receptor 7/8 binding R848. However, the motility of the Y sperm is not affected. Thus, the sperm is selectively separated by using the swim-up method, in which Y sperm with high motility swims to the upper layer of the medium, while the X sperm sinks to the lower layer of the medium. Besides, R848 had no effect on the acrosome of both X sperm and Y sperm, indicating that sperm fertility is not affected even after incubating with R848. In addition, the motility of X sperm returns to normal once R848 has been washed off. The sperm separated by this method was later used for  embryos .However, most immunology sexing strategies affect the motility of sperm and reduce the conception rate due to the long processing time. Although this strategy is presently at the laboratory stage, advances in molecular biology may provide more breakthroughs. Therefore, more efforts should be concerted on the discovery of new sex-related proteins and the blending of other strategies to reduce sperm damage.Millions or billions of sperm are deposited into the reproductive tract after mating, but only a few sperm reach the ampulla or the site of fertilization and only one fertilizes an oocyte. The journey that sperm take to reach an oocyte is long and perilous. The female environment contributes toward this successful transit by providing a vehicle for sperm transport, aiding the removal of dead sperm and other pathogens and applying strict selection pressures to ensure only those cells with the highest quality reach the site of fertilization. Besides, in many locations of the female reproductive tract, sperm interact with the epithelium and the luminal fluid, which can affect sperm motility and function , 91. Altin vitro , polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), can therefore be used to identify the male-specific gene.The blastomere of mammalian embryos is omnipotent and can develop into a complete animal. Thus, each blastomere contains all the genes required for the development of an animal, including genes involved in sex determination such as Amg , Zfy  for precipitation were optimized to improve sensitivity, and multiplex LAMP was used to improve accuracy  is a method of DNA amplification that was developed by Eiken Chemical Co. Ltd. in 2000 and works under isothermal conditions. This method of DNA amplification is highly specific, efficient, and rapid . Besidesaccuracy . The fir 8 weeks .Although PCR and LAMP have made significant progress in embryo sexing owing to their high sensitivity, the impact of the pollution in the external environment on the accuracy of the results remains inevitable. In addition, these two methods are difficult to use in livestock production due to the large sample size and the poor transfer pregnancy rate of recent embryo transfer technology. Therefore, future research should focus on improving tolerance to external environment and the efficiency of these technologies. Besides, these technologies should be applied to identify the accuracy of other sex manipulation strategies, rather than as a sex manipulation strategy.Sex manipulation technologies are valuable biotechnologies that have the potential to revolutionize cattle production. With the development of molecular biology and cell biology, these emerging sex manipulation strategies significantly expand the ability to bias the sex ratio of animal offspring and would be instrumental in correcting clinical male-to-female sex reversal syndrome in humans. However, the knowledge of sex-determining mechanisms, whether sex-determining gene or female reproductive tract, is still very limited. Since different sex manipulation strategies have their individual advantages and disadvantages, the selection of a specific system is dependent on the expertise of the individual researcher rather than on the weaknesses of one of these strategies. Collectively, sex determination is a complex process that is influenced by a variety of internal and external factors. Further exploration of sex-determining mechanisms; continuous improvement of the accuracy, sensitivity, and economic benefits of sex manipulation technologies; and the establishment of a new a sex manipulation system that integrates knowledge from various fields can provide great impetus in livestock production and solve the problem of food shortage.YX and ZX: conceived the idea, conducted the literature review, and drafted the manuscript. ZW and LH: project investigator, decided a review topic, and revised the manuscript. All the authors have read 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.


To review the literature on oral and enteral nutrition therapy andinvestigate the evidence of its efficacy as a treatment, as well as inpreventing relapses and reducing symptoms of inflammatory bowel diseases inthe pediatric population.LiteraturaLatino-Americana e do Caribe em Ciências da Saúde - Lilacs)databases, using the keywords “inflammatory bowel disease,” “diet,” and“diet therapy” in English and Portuguese, with filters for pediatric studiespublished in the previous five years. We performed a bibliographic search in the PubMed, Web of Science, and LatinAmerican and Caribbean Health Sciences Literature  and the Crohn’s Disease exclusion diet (CDED). Thestudies found evaluated the anthropometric profile of patients and theinflammatory profile of diseases in children before and after theintroduction of each specific nutrition therapy. All interventions presentedpositive changes in these parameters; however, the results were inconclusiveregarding the efficacy of SCD and CDED in the treatment and prevention ofrelapses. Exclusive enteral nutrition has proven to be effective in inducing remissionof Crohn’s Disease, and the use of partial enteral nutrition for maintenancetreatment has shown promising results. Other modified oral diets areinconclusive concerning their effectiveness, requiring further randomizedcontrolled clinical trials. The chronicity of the inflammation of the gastrointestinal tract (GIT) interferesdirectly with the quality of life and the nutritional status of individuals withinflammatory bowel diseases (IBD). Eating habits are believed to be involved ininducing inflammation, as well as in its remission.Dietary therapies that involve enteral nutrition (EN), modification of carbohydrates,and dietary fibers have been discussed and prescribed in the pediatric field astherapeutic and preventive proposals for Crohn’s Disease (CD) and ulcerative colitis(UC) inflammatory episodes.This work aimed to review the literature on oral and enteral nutrition therapy andinvestigate the evidence of its efficacy as a treatment, as well as in preventingrelapses and reducing symptoms of IBD in the pediatric population.Literatura Latino-Americana e do Caribe em Ciências da Saúde -Lilacs) databases, using the keywords “inflammatory bowel disease,” “diet,” and“diet therapy” in English and Portuguese, with filters for pediatric studiespublished in the previous five years. We included articles in English, Spanish, andPortuguese.Between May and August 2018, we performed a bibliographic search in the PubMed, Webof Science, and Latin American and Caribbean Health Sciences Literature has been widely studied and recommended asfirst-line therapy for inducing remission of luminal CD. It consists of offeringa nutritionally complete liquid diet, either polymeric or oligomeric, exclusively, for six to eight weeks, through anasogastric tube or orally.,,,,,,Most of the studies evaluated recommended the use of polymeric formulas, savingthe elemental ones for those who do not tolerate diets with intact protein. Oraladministration was preferable, but patients who did not like the taste of theformula or who were unable to feed orally received it through a tube.- PCDAI).,,,,,,,,,When compared to systemic corticosteroids or anti-TNF (immunomodulatory agent),EEN seems to be as effective as these drugs in inducing clinical remission ofCD, characterized by the reduction in the disease activity index  adjusted for age andheight of a group that received EEN with another that received corticosteroidsas therapy for inducing remission for six to eight weeks, using dual-energyX-ray absorptiometry (DEXA). They analyzed the changes in BMD (at the beginningof the study and after 12 to 18 weeks of follow-up) in both groups, and, despitethe lack of statistical difference, the z-score variation was greater inpatients who received EEN compared to those who received corticosteroids. Theauthors suggested that the result probably has clinical relevance since thechange in BMD was close to 0 in the group that received corticosteroids anddeclared that long-term use of high doses of this medicine could cause growthdamage and bone mass loss, especially in adolescents. In the same study, theremission and relapse rates showed no difference between the groups after a yearof follow-up, but the group that received EEN had an improvement inweight-for-age.In 2014, after patients with CD achieved remission with the use of EEN orcorticosteroids for six to eight weeks, Hojsak et al. analyzed the influence ofa few factors during the remission period and found that, after a year, only EEN was aprotective factor against relapse. Moreover, the duration of remission wassignificantly higher in patients who received EEN as an induction therapycompared to those who used corticosteroids.Regarding the duration of EEN, the European Society for PaediatricGastroenterology, Hepatology and Nutrition (ESPGHAN) recommends at least sixweeks of use, even though the mucosa usually heals after eight weeks.,,Clinical and laboratory aspects showed that EEN significantly improved weight,albumin, erythrocyte sedimentation rate (ESR), hemoglobin, and hematocrit, witha decrease in inflammatory markers such as C-reactive protein (CRP) and fecalcalprotectin levels.Grover et al. found that EEN was also able to promote good endoscopic responsewith reduced rates of relapse, hospitalization, need for anti-TNF, and surgicalresection one year after remission.,,,,Despite the complexity of the pathogenesis of IBD, some studies show a directrelationship with dysbiosis, mainly because of the deregulated induction of theimmune system, which reinforces the need for dietary interventions that act asprotective factors against intestinal inflammation.Another unclear aspect is whether the location of the disease influences theoutcome of nutritional treatment. Currently, EEN is recommended for anytopography of luminal CD.According to ESPGHAN, EEN is not indicated for patients with UC.,Partial enteral nutrition (PEN) consists of providing a nutritionally balancedliquid formula to supplement a diet composed of solid foods (unrestricted orexclusion). PEN is being studied as maintenance therapy to prolong the remissionof CD, but there is still no consensus or recommendation about its use.In 2015, Konno et al. revealed that the intake of at least 30 kcal/kg/day ofelemental diet as maintenance therapy (after remission induction) showed aprotective effect against relapse, controlling complications and delaying theneed for surgery and the use of drugs , with no difference regarding location or phenotype of thedisease. Nevertheless, the diet was combined with aminosalicylates, whichmodulate the secretion of pro-inflammatory cytokines, leading to a bias for theoutcome.In 2014, Duncan et al. used EEN to induce remission of CD, and after eight weeks,they encouraged the patients to consume 25% of the initial volume of the diet until the end ofone year of follow-up. After six months, clinical remission was more significantin patients who received PEN alone as maintenance treatment compared to thegroup that received no treatment . Thegroup that received PEN+azathioprine presented a remission rate three timeshigher than the group without treatment. At the end of a year of follow-up, theuse of PEN was comparable to that of azathioprine. Despite the promisingresults, the low adherence to PEN (31% of participants) shows the limitingfactor of palatability and the monotony of taste in this modality oftreatment.In 2018, Gavin et al. demonstrated that the use of PEN as maintenance treatmentfor four months after remission led to a relapse rate similar to that inpatients on a conventional diet. Besides, EN increased the risk of overweight inpatients.A prospective cohort study conducted in Canada and the United States divided 90patients into three groups according to remission induction therapy: PEN, EEN,and anti-TNF. At the end of eight weeks, the results were positive for EEN andanti-TNF regarding the mucosal healing, and EEN has proven superior to PEN as tothe quality of life and reduction in intestinal inflammation, evaluated by thedecrease in fecal calprotectin levels, even though the energy intake was higherin the group that received PEN.Despite the promising results concerning the relapse time, further studies arenecessary to ratify the indication of PEN and establish the optimal dosage andduration for maintenance of remission. For now, this type of treatment is anoption to help maintain the remission in patients with mild diseases and lowrisk of relapse, but the evidence is limited as to its use in Pediatrics, and itis not indicated as a monotherapy in the maintenance of remission of CD.Most of the studies found about EN presented limitations, such as retrospectivedesign, single-center research, and possible data loss, as the information wasgathered from medical records. In addition, the sample population washeterogeneous, and a large part of the participants received concomitant drugtreatment, which suggests the need for prospective multicenter studies, such ascontrolled clinical trials, with stratified randomization (according to the useof medication).The specific carbohydrate diet (SCD) was first described in 1920 by Hass as atherapy for celiac disease and was later studied for IBD.Some studies showed a positive association between SCD and IBD, with reducedsymptoms and inflammation and changes in anthropometric patterns. We found fivepediatric studies that described the use of SCD as dietary treatment and itspossible outcomes.In 2016, Obih et al. reviewed the medical records of 26 children with IBD whoreceived SCD concomitantly with drug treatment with immunosuppressive agents;among these records, only six had UC as the diagnosis. The children weremonitored for 24 months on their biochemical parameters of ESR, CRP, albumin (asinflammatory markers), and hematocrit (as a marker for anemia), in addition tothe anthropometric assessment, including body mass index (BMI) and growthvelocity (GV). Patients diagnosed with CD or UC presented an improvement intheir serum inflammatory markers, with a reduction in ESR and CRP and anincrease in albumin. Their anemia (with increased hematocrit) and nutritionalstatus  also improved. However, out of the sixpatients with UC, only three responded positively to SCD . Although SCD has proven to be positivefor both pathologies, the sample size of UC was relatively small when comparedwith CD.Another study analyzed the exclusive use of SCD in seven patients with CD,adopting the same biochemical and anthropometric parameters. The authors alsoidentified an improvement in the inflammatory response, with decreased CRP andincreased albumin and hematocrit. Three months after the beginning of the diet,they detected weight gain and a reduction in clinical symptoms. The GV of thepatients remained normal.nd week, only seven patients remainedin the study, and they presented a reduction in the CD activity index; however,four patients had a higher Lewis score for mucosal inflammation than in the12th week. Among the other patients, two presented mucosalhealing, and one continued to improve.Besides the biochemical, anthropometric, and clinical parameters, a studyconducted in Atlanta with nine patients with CD evaluated the mucosal integrity(through endoscopy) and the inflammation (with the Lewis score) in patients whoreceived SCD+drug treatment for 12 to 52 weeks. The anthropometric andbiochemical parameters were similar to those of other studies. The researchersidentified an improvement in the intestinal mucosa, with ulcer healing after 12weeks of treatment. After the 52Since the SCD is a restrictive diet that requires changes in the dietary pattern,making long-term adherence challenging to maintain, some protocols adopt theliberalization of the diet for a certain period  at the request of the patient and based ontheir food preferences. This liberalization usually consists of a modifiedversion of the SCD (mSCD), with the introduction of one or two restricted foodsper week or day. Nevertheless, no consensus was reached about the prescriptionof this protocol due to the lack of evidence of its effectiveness in theremission of inflammation and symptoms of the disease.In 2016, Burgis et al. analyzed the effects of SCD for 12 months and the impactsafter eight months of mSCD in 11 patients. The study showed an improvement inthe levels of albumin, ESR, and hematocrit, as well as in anthropometricparameters, even after the mSCD. The patients gained weight, with a small losswithout statistical significance after the liberalization.In 2017, Wahbeh et al. assessed biochemical and anthropometric parameters, aswell as mucosal healing in the upper and lower GIT (with endoscopy) in sevenpatients who received both SCD and mSCD. They found that, in the two diets, mostpatients maintained the CRP within the normal range, with slightly abnormallevels of albumin, hematocrit, and fecal calprotectin. BMI did not change, andthe authors did not detect mucosal healing in the patients.Despite the positive results of the presented studies, the authors highlight thatthey are not conclusive regarding whether these diets ensure the remission ofIBD. Factors such as the small sample size, the retrospective design of moststudies, and the combination of diet and drug treatment prevent theirrecommendation. These limitations led ESPGHAN not to indicate this type ofintervention for children with these diseases.,,Crohn’s Disease exclusion diet (CDED) consists of offering fruits and vegetables,some types of meat, and carbohydrates and restricting or excluding theconsumption of animal fat, processed meat (including fish), gluten, dairy,emulsifiers, canned foods, and some monosaccharides. Its use is recommended incombination with EN, which consists of a polymeric formula that provides 50% ofthe daily energy intake.,The mechanisms by which this therapy works are not known, but one assumption isthat the exclusion of some foods might decrease the bacterial translocation andprevent the pro-inflammatory action of some dietary components, facilitating theeffect of medicines.We found two recent pediatric studies on the use of CDED and EN performed by thesame authors in Israel. In 2014, Sigall-Boneh et al. analyzed the use of CDED+ENfor six weeks and obtained positive responses, such as most pediatric patientsreaching clinical remission and improvement in the PCDAI, CRP, ESR, albumin, andweight after 12 weeks of follow-up in individuals with mild or moderatediseases. The institutional standard demanded that all patients usedimmunomodulatory agents; therefore, whether the diets led to remission inductionremains unclear.In 2017, Sigall-Boneh et al. evaluated the same nutrition therapy for the sameperiod and found that this strategy can induce remission of CD or, at least,favor clinical response, as evidenced by the drop in the Harvey-Bradshaw index(HBI) and inflammatory markers. According to the authors, the positive outcomesresulted from a reduction in intestinal exposure to inflammatory triggeringfactors. However, as in the other study, these findings have a bias, as allpatients used anti-TNF since the start of treatment.,,,The small number of longitudinal studies on the use of CDED is a limiting factorfor any kind of indication. Nevertheless, some studies have shown associationsbetween low risk of developing IBD and a diet with a high intake of fiber,fruits, and vegetables and between high risk of IBD and a diet rich in linoleicacid, animal fat and protein, and refined sugar. Since the Western diets haverisk characteristics for the development of IBD, we can assume that diets whichexclude or restrict certain types of food, such as CDED or semi-vegetariandiets, can help reduce symptoms and prolong remission, although more studies arenecessary to validate their use.,The discussion about fermentable oligo-, di-, monosaccharides, and polyols(FODMAP) in IBD Escherichia coli Nissle, 1917, and the VSL#3 (mixture oflactic acid bacteria and bifidobacteria). These probiotics can be considered inthe maintenance of remission in patients with UC.,Experts claim that no specific diet exits for the remission stage of IBD;however, some strategies can help, such as the use of the strain of,In addition, ESPEN states that probiotics are effective in inducing remission ofmild to moderate UC. ESPGHAN indicates that the evidence is limited butfavorable to the use of probiotics combined with the standard treatment forinducing remission in pediatric patients with UC. None of these societiesindicates their use in CD.,There is no recommendation for the use of prebiotics, synbiotics, or omega-3 inIBD. On the other hand, the intake of dietary fiber is good for improvinggastrointestinal functions and might be effective in UC; when associated withthe standard therapy, it can help the maintenance treatment of the disease.Fiber restriction should not be recommended for patients with IBD, except forthose who have the stenotic phenotype of the disease.,,The literature has described the supplementation of curcumin as a strategy forthe treatment of IBD. Its anti-inflammatory and antioxidant action might lead toa remission of UC,We found no recent studies about supplementation in IBD.The study of oral and enteral nutrition therapy in IBD is complex and has beenincreasingly discussed. EEN continues to be as effective as drugs for remissioninduction in CD among pediatric patients, and the use of PEN as maintenancetreatment has become more relevant, particularly due to the possibility ofdecreasing the use of medicines at this stage of the disease.In this review, we found few original and recent articles, which presented smallsamples and some methodological biases, as discussed throughout the work. Theselimitations did not allow us to recommend oral diets for inducing remission orpreventing relapses in patients with IBD, mainly because of the concomitant useof medication during the treatment. These interventions might assist in thecontrol of IBD symptoms, but randomized controlled clinical trials are necessaryto establish safe and effective recommendations for the pediatricpopulation.


The relevance of experimentally gained information represents a long-term debating issue in the field of molecular biology research. The loss of original conditions in the in vitro environment affects various biological mechanisms and cellular interactions. Consequently, some biochemical mechanisms are lost or critically altered. Analyses in these modified conditions could, therefore, distort the relevancy of experimentally gained information. In some cases, the similarities with original conditions are so small that utilization of simpler in vitro models seems impossible, or could occur in a very limited way. To conclude, the study of more complex phenomena places higher demands on the complexity of the experimental model. The latest information highlights the fact that the tumor angiogenesis mechanism has very complex features. This complexity can be associated with a wide range of angiogenic factors expressed by a variety of malignant and non-malignant cells. Our article summarizes the results from various experimental models that were utilized to analyze a photodynamic therapy effect on tumor angiogenic mechanisms. Additionally, based on the latest information, we present the most important attributes and limitations of utilized experimental models. We also evaluate the essential problems associated with angiogenic mechanism induction after photodynamic therapy application. The era of modern photodynamic therapy (PDT) was begun by Lipson and Baldez in the 1960s, when fluorescence of neoplastic lesions after injection of hematoporphyrin (Hem) was observed . In the Despite a general effort to minimize side effects occurrence, there is evidence of its constant presence. The results of a huge amount of analyses showed that side effects evoked by PDT utilization belong to the major reasons not only for lowering therapeutic efficiency, but, moreover, they are associated with some pathological conditions’ development in PDT-affected areas. A wide range of analyses point to a tumor angiogenic mechanism induction as one of the most serious pathological conditions evoked specifically by PDT treatment see . MoreoveBased on these facts, we assume that the targeting potential of PDT represents a fundamental condition for the extension of applied potential for a wide range of tumors of different histological origins. Simultaneously, the development of novel experimental models convenient for angiogenic research now represents a very interesting and examined area in the field of tumor angiogenesis research ,21,22. MBiological experiments focused on the analysis of a huge spectrum of molecular mechanisms are possible due to the use of experimental models, such as culture systems and animal models see  69]. So. So69]. Original cell phenotype maintenance in cells utilized for laboratory experiments represents the key effort in the field of cellular biology analyses ,76. NowaThe surviving cell fraction represents one of the most serious problems after PDT observation. Chen assumed that PDT with hypericin (HY) could probably induce immediate cell death only in 30% of fibrosarcoma tumor mass . On the The observed surviving cell fraction after PDT could be connected with fundamental components for PDT realization, which means light, oxygen, and PS presentation in tumor tissue . In thisThe spatial characteristics of the 2D cell model represent a major restriction for its utilization, as this model could not consider any of the above-mentioned factors. In 2D cell models, light and oxygen have relatively homogenous distribution through a total cell population. Moreover, the impossibility of mimicking the tumor vasculature effect on PS permeability into the tumor leads to its inadequate accessibility associated with a significantly higher cell accumulation. Finally, comparative analyses in relation to PDT showed that the same treatment regimens cause significant differences in cytotoxic effects. Within the given frame of references, the cells cultivated in the 2D cell model exhibit higher sensitivity to PDT, and therefore, the rate of surviving cells fraction in equal treatment conditions is minimalized. As was mentioned, a PS accumulation level is associated with a total cytotoxic effect, subsequently affecting angiogenic factor expression. Therefore, the consideration of the third dimension seems to be a very convenient approach for mimicking spatial tumor characteristics ,88,89,90The history of 3D cell cultures started in the 1970s . Until nIn the context of the above-mentioned information, there is no doubt that the presence of spatial characteristics in the experimental model utilized for PDT concerning tumor angiogenesis mechanism research is fundamentally needed. The geometry of the experimental model is sufficient enough for alternative splicing angiogenic factor alterations, like vascular endothelial growth factor (VEGF) in malignant cells . NonetheConcerning novel methods and experimental model utilization in cancer research, we must abandon a simplistic description of solid tumors as complex structures of genetically modified cells with high clonogenic capabilities ,107.The last decade is characteristic of the growing interest in tumor environment study, as there have been many potential antitumor targets identified . FinallyThe above-mentioned facts are important not only for our better understanding of tumor stroma effect on tumor phenotype characteristics, but they also show that, in 3D cell models, these interactions are developed ,113,114.Besides that, it was shown that the tumor angiogenic mechanism is directly modulated by the presence of the tumor microenvironment. This mechanism is associated with complexity and reciprocal interaction of multiple tumors, creating cell types significantly influencing the angiogenic expression profile of tumors, as was proven by multicultural spheroids utilization. Data showed that tumor-associated fibroblasts were not just the major producer of mentioned growth factors. However, if they were co-cultivated with cancer cells, a significant increase of VEGF and FGF-2 secretion by analyzed spheroids was observed. The mentioned information means that a non-malignant section of tumor mass has a key impact on the level of expression of angiogenic factors in malignant cells. Consequently, a significant upregulation of the expression of angiogenic factors in malignant cells was induced . NeverthAlthough the fact that there is a massive effort to develop novel 3D in vitro experimental models and that novel information proved its possible utilization in a huge range of cancer types, its optimization has occurred mainly with lung adenocarcinoma . Based oThe CAM of an avian embryo is a structurally simple, highly vascularized extraembryonic membrane. Except for a respiration function, allantois also serves as a reservoir of waste products of the embryo. The CAM also functionally participates in sodium and chloride transport from the allantois cavity and calcium transports from the egg shell to start the bone mineralization . For resStructurally, the CAM consists of two epithelial sheets. The upper epithelium is of ectodermal origin, while the stroma and the lower epithelium are of mesodermal and endodermal origin, respectively. Blood and lymphatic vasculature are located in a stromal part, so every compound delivered on the CAM surface has to pass across the surface epithelial layer, and reach vessels in the stroma .Moreover, the CAM is simple to handle, its utilization is low-cost, and ethics committee approval is free to obtain, as an avian embryo is not considered as a living animal until day 17 of its development in most countries . The CAMHowever, quick rearrangement of the vascular system limits the precision of the detection of novel vessels . MoreoveThe extensive vascular potential of the CAM makes it possible to analyze the effect of a wide spectrum of substances on an angiogenic mechanism. The analyzed substances are topically applied on the CAM surface , and theThe CAM vasculature provides extensive possibilities for PDT analyses. PDT induces reactive oxygen species production that subsequently activates a cascade of chemical and physiological processes, initiating the creation of occlusions on the CAM . The PS As mentioned above, the majority of experimental analyses focused on PDT concerning tumor angiogenesis were performed with in vivo mice or rat experimental model utilization. In this context, the fundamental requirements, such as the multicultural character of experimental tumors, the possibility of vascular application of PS, and the long-term effect observation, could be considered as the major advantages of these experimental models for tumor angiogenesis research after PDT. The therapeutic effect of PDT with a wide range of PSs utilized was associated mainly with the induction of the tumor angiogenesis mechanism see . FerrariAs a possible solution for the expression of angiogenic factor lowering in cancer cells, PDT targeted mainly on tumor vasculature with HY ,49,50 or2). Nevertheless, significantly increased ECs proliferation and VEGF upregulation were also detected, in comparison to the untreated contralateral brain region. Subsequently, immunohistochemical analyses with PT-PDT utilization showed that the brain adjacent to tumor tissue also represents a rich source of VEGF, and neither vascular endothelial growth factor receptor-1 (VEGFR-1), MF1, nor vascular endothelial growth factor receptor 2 (VEGFR-2), DC101 antibody application could decrease VEGF expression to non-treated control levels [In this context, Bhuvaneswari et al.  observedl levels . Similarl levels , and in l levels .In addition, combination treatment analyses with ALA-PDT were also performed on Walker carcinosarcoma with chitosan  and bladHumanized anti-VEGF monoclonal antibody bevacizumab, in combination with mTHPC-PDT, decreased VEGF expression in correlation to the mTHPC-PDT group, but on the contrary to chitosan and deferoxamine utilization in ALA-PDT ,25 the rA combined therapy utilized in relation to HY-PDT seems to be very promising for angiogenesis inhibition. In comparison to non-treated tumors, a significant downregulation of VEGF expression on mRNA  and protThe above-mentioned information has shown that PDT, with the huge scale of PS utilized, induces an angiogenic mechanism in a wide range of tumor types see . In addiConcerning that, a higher level of cancer cell-specific accumulation of PS is fundamental because non-malignant cells could potentiate tumor growth by angiogenic factor expression after PDT ,18,19,20Induction of angiogenic mechanism after PDT represents a serious problem associated with a tumor regrowth development in affected tumors. Besides that, the analyses have shown that there are many factors regulating tumor angiogenesis, and the tumor stroma represents the key regulator of this mechanism in many aspects. The tumor stroma fundamentally affects the behavior and phenotypical characteristics of malignant and non-malignant cells. Additionally, there is evidence that angiogenesis stimulation in cancer is not only associated with malignant cells, but also that the non-malignant cell fraction represents an important source of angiogenesis stimulating factors. Moreover, PDT could also induce angiogenic mechanism in these normal cells. Therefore, the preservation of tumor microenvironment conditions and multicultural characteristics in experimental models represents an essential requirement for PDT research about angiogenesis. Simultaneously, the improvement of PS accumulation in targeted tumor cells associated with novel carriers and angiogenesis inhibitors development represents a cardinal challenge in the field of angiogenesis research about PDT.


When we engage in social exchange, we join information with one another by producing actions and simultaneously adapting to the other person's actions via a sharing of mental representation and action within- and between-individuals  and biofeedback, allowed to detect the neural correlates of two or more individuals involved in various social interaction processes  task. The discussion of the groups of participants was video recorded and coded to investigate leadership and communication , more implicated in processes of social mentalization, within the dyads composed of leader-follower (LF) compared to follower-follower (FF) ones. Specifically, a significant difference has emerged regarding the frequency of communication, which is higher for LF dyads than FF ones. On the contrary, no differences have emerged concerning the frequency of leader initiated and follower-initiated communication (Jiang et al., These authors observe that as evidenced by evolutionary theories, which report human and non-human animals' tendency to develop skills useful to compete for survival, human leaders play a fundamental role in maintaining group cohesion (Jiang et al., As interesting demonstrated by different neuroimaging studies, during cooperative situations compared to competitive ones, individuals' neural activity appear to be more synchronized (Cui et al., Furthermore, the INS is connected to the level of interagents' understanding (Stephens et al., Moreover, it has been shown that high-quality communication increases the level of mentalization of individuals, consisting of the ability to understand social situations by adapting their behavior to others. Verbal and non-verbal communication skills, indeed, have been considered as a fundamental competence of the leader (Jiang et al., Also, an increased neural and bodily attunement, demonstrated by the use of gestures, has emerged between leaders and followers assigned a priori during social interactions (Yun et al., These different applications of hyperscanning within the organizational context open the way to other possible uses of this paradigm for the investigation of different processes and behaviors related to work performance, such as proactive and social resources, useful for implementing more functional strategies for corporate performance (Ingusci et al., Indeed, the increase in individuals' proactive behavior and social relationships could enhance job redesigns' strategies, such as job crafting, with positive effects on workers' well-being (Kim and Beehr, Besides observing the mechanisms of brain areas' synchronization and coupling, some studies have shown asymmetric synchronization mechanisms within dyads composed by leader and follower (Dumas et al., Our daily social life is characterized by the performance of joint actions, during which we adjust our actions with that of other people according to what is required by different tasks (Zhou et al., In addition, the cerebral mechanisms underlying leadership have been observed by the implementation of a recent fMRI study that required participants a tapping task, including an adaptive stimulus (Fairhurst et al., In particular, this study wanted to implement a two-brain analysis capable of reporting useful characteristics to follow or conduct the behavior of two individuals involved in a realistic dyadic interaction. The use of body movements, indeed, characterizes social exchanges, as happens in gesture communication in which different types of gestures are associated with the transmission of different information (Balconi et al., Therefore, hyperscanning allows exploring interpersonal brain mechanisms generated by social interactions, such as those generally represented in organizations. The mutual adaptation, as shown by brain-to-brain and body-to-body coupling of two or more interactive subjects, results in brain synchrony, which is considered the basic neurophysiological counterpart of functional mutual inter-relationships.MB wrote the first draft and each section of the manuscript. MB and GF contributed to manuscript final writing and revision, read, and approved the submitted version. Both authors contributed to the article and approved the submitted version.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.


Background: During the last decades, fathers have increasingly participated in prenatal care, birth preparation classes, and childbirth. However, comparably little is known about the prenatal emotional well-being of fathers, particularly content and extent of broader paternal concerns that may arise during pregnancy beyond those focusing on childbirth. Thus, the aims of this study were to investigate the manifestation of paternal pregnancy-related worries in a population-based sample and to identify relevant associated factors.Materials and Methods: As part of a longitudinal pregnancy cohort at the University Medical Center Hamburg-Eppendorf, Germany, N = 129 expectant fathers were assessed once during pregnancy. Pregnancy-related worries centering around medical procedures, childbirth, health of the baby, as well as socioeconomic aspects were assessed with the Cambridge Worry Scale (CWS). Additionally, paternal socioeconomic background and maternal obstetrical history, symptoms of generalized anxiety and depression, and level of hostility were investigated, as well as perceived social support. The cross-sectional data were analyzed based on multiple regression analyses.Results: The level of reported worries was overall low. Some fathers reported major worries for individual aspects like the health of a significant other (10.9%) and the baby (10.1%), as well as the current financial (6.2%) and employment situation (8.5%). Pregnancy-related worries were negatively associated with household income and positively associated with anxious and depressive symptoms and low perceived social support. Associations varied for specific pregnancy-related worries.Limitations: Due to the cross-sectional data examined in this study, a causal interpretation of the results is not possible. The sample was rather homogeneous regarding its socioeconomic background. More research needs to be done in larger, more heterogeneous samples.Conclusion: Though overall worries were rather low in this sample, specific major worries could be identified. Hence, addressing those fathers reporting major worries regarding specific aspects already in prenatal care might support their psychosocial adjustment. Fathers with little income, those with elevated levels of general anxious and depressive symptoms, and those with less social support reported higher pregnancy-related worries. Our results indicate the relevance of concerns beyond health- and birth-related aspects that could be relevant for fathers. Measurements developed specifically for expectant fathers are needed to properly capture their perspective already during pregnancy. Even though most parents perceive the transition to parenthood as a positive experience with excitement and joy, often negative feelings and insecurity are a substantial part of becoming a mother  or fatheDuring the last decade, the focus on mental health in the peripartum period has gained increased attention. Research in mothers showed that prenatal emotional distress is associated with negative consequences for the health of mother and baby and needs to be identified and addressed as early as possible , 37–40. In the literature, a variety of conceptualizations of pregnancy-related anxiety exists –58. In aPrevious studies on pregnancy-related concerns in fathers have moreover often focused specifically on fears related to childbirth. Hanson et al.  describeSince having a child affects many different aspects in one's life, concerns for expectant fathers might also be associated with domains like caregiving competence and important relationships, as well as the living and financial situation. Previous studies frequently described that fathers felt unprepared or even inadequate for their role as a father , 70, 71.However, few studies have explicitly investigated a broader range of paternal pregnancy-related worries. Forsyth et al.  found inFurther, the assessment of fear of childbirth, as one specific aspect of pregnancy-related anxiety, often focuses on the identification of expectant parents with high or even pathologic levels of fear. In contrast, given the little research on different pregnancy-related worries in fathers and the lack of valid father-specific instruments thus far, the investigation of a wider concept and different levels of prenatal worries are needed to contribute to the understanding of the paternal perspective before further differentiation. An assessment of the content and extent of worries closely related to pregnancy as well as general worries in expectant parents during pregnancy that are non-pathologic, but might still substantially increase distress, is important , 76. HenContextual aspects of the socioeconomic background might especially be relevant when having a child. Lower income has been associated with higher levels of prenatal general anxiety in men  and chilStudies on perinatal adjustment indicate that those fathers expecting the first child might feel a higher level of insecurity during pregnancy and in preparation of childbirth than those who already have children . In linePregnancy has been identified as the most stressful period during the transition to fatherhood , 98, whiOn the other hand, perceived social support has been identified as one important protective factor in relation to mental health, also in the context of pregnancy and paternal distress , and less perceived social support to be associated with higher levels of pregnancy-related concerns.The data for this cross-sectional analysis were derived from a collaboration between two related ongoing population-based prospective pregnancy cohorts, carried out at the University Medical Center Hamburg-Eppendorf, Germany , hostilFinally, perceived social support was associated with pregnancy-related worries, which is in line with previous results indicating the protective effect of social support on perinatal mental health and specifically pregnancy-related worries , 108. Thsocioeconomic and relationships and health of the baby/other, the results of the supplementary analyses should be interpreted with caution. While questionnaires assessing fear of childbirth have been used in fathers more often in the past , Hamburg Chamber of Physicians, Hamburg, Germany. The patients/participants provided their written informed consent to participate in this study.All authors contributed substantially to this study. AG developed the research question, conducted the statistical analyses, drafted and revised the manuscript. PA and AD contributed to the final research question and the research design, the drafting and revision of the manuscript. KH and MS-M contributed to the development of the final research question and the research design, the drafting and revision of the manuscript. SM developed the research question, contributed to the research design, drafted and revised the manuscript. All authors approved the final version of this 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.


Gastric cancer is the fifth most diagnosed cancer in the world, affecting more than a million people and causing nearly 783,000 deaths each year. The prognosis of advanced gastric cancer remains extremely poor despite the use of surgery and adjuvant therapy. Therefore, understanding the mechanism of gastric cancer development, and the discovery of novel diagnostic biomarkers and therapeutics are major goals in gastric cancer research. Here, we review recent progress in application of omics technologies in gastric cancer research, with special focus on the utilization of systems biology approaches to integrate multi-omics data. In addition, the association between gastrointestinal microbiota and gastric cancer are discussed, which may offer insights in exploring the novel microbiota-targeted therapeutics. Finally, the application of data-driven systems biology and machine learning approaches could provide a predictive understanding of gastric cancer, and pave the way to the development of novel biomarkers and rational design of cancer therapeutics. Although the incidences and deaths of gastric cancer are declining in Northern America and Western European, gastric cancer still remains as the fifth most common diagnosed cancer worldwide, and is second compared to lung cancer in terms of worldwide cancer deaths . GastricThe vast majority of gastric cancer are adenocarcinomas, which can be classified based on their histological and etiological characteristics. Traditionally, gastric cancer can be divided into two major subtypes: intestinal- and diffuse- types of adenocarcinomas according to the Lauren’s criteria . AdditioSystems biology approaches aim to the transcendence of individual genes/proteins and to the integration of biological system that taking account into the intrinsic interactions. With more and more available omics data, systems biology approaches have developed many new methods and applications in gastric cancer research. In this review, we will briefly summarize the recent progress in “omics” technologies and their applications in gastric cancer research. We will then highlight the use of omics data integration to classify gastric cancer, and the application of systems approaches and machine learning methods to discover novel biomarkers and potential therapies. Furthermore, how the gastric cancer research shift from human omics to human-microbiota omics for current and future applications will be discussed.Next-generation sequencing technologies are mainly based on the massively parallel sequencing of short DNA/RNA fragments, which have been extensively reviewed elsewhere . The advTP53) and structural variations are frequently identified in gastric cancer , gene regulatory network, gene interaction network, and metabolic network], the omics data can be mapped into the biological networks to identify potential functional subnetworks . The actThe applications of machine learning methods, which learn functional relationships from data, had been largely increased in cancer research and drug discovery . One impNot only cancer diagnostics, machine leaning also brings personalized treatment to clinics . SurgeryImmunotherapy has revolutionized both the cancer research and treatment landscape by targeting the host immune system . AntibodThe advances of omics technologies in decades are enabling the parallel measurement of millions of biomolecules at the same time. Omics-wide association studies have been widely applied in gastric cancer research, which revealed strong associations between omics features and the gastric cancer development. With the omics data from genome, transcriptome, proteome, and epigenome levels, gastric cancer have been extensively stratified, and the resulting subtypes show strong correlations with the therapeutic outcomes. Both the TCGA and ACRG classifications revealed four distinct gastric cancer subtypes, and the comparison between these two classification systems showed similarities such as tumors with MSI in both data sets, and the TCGA GS, EBV+, and CIN subtypes were enriched in ACRG dataset . HoweverWith the proteomics and metabolomics data, numerous gastric cancer-specific biomarkers had been identified, which pave ways for the diagnosis of gastric cancer at the early stages. Systems biology based integration of multi-omics data have provided lot of insights into the cancer diagnosis and therapeutics. However, the application of such methods in gastric cancer still lags behind. Moreover, the application of big data and machine learning approach in gastric cancer studies are still limited. With increased omics data generating from the gastric cancer research field, the application of systems biology approach would provide a systematic scenario of gastric cancer in the future.YW and BJ conceived the study. X-JS, YW, and BJ wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


Recent advancements in technology have enabled researchers to probe the brain with the greater region, cell, and receptor specificity. These developments have allowed for a more thorough understanding of how regulation of the neurophysiology within a region is essential for maintaining healthy brain function. Stress has been shown to alter the prefrontal cortex (PFC) functioning, and evidence links functional impairments in PFC brain activity with neuropsychiatric disorders. Moreover, a growing body of literature highlights the importance of neuropeptides in the PFC to modulate neural signaling and to influence behavior. The converging evidence outlined in this review indicates that neuropeptides in the PFC are specifically impacted by stress, and are found to be dysregulated in numerous stress-related neuropsychiatric disorders including substance use disorder, major depressive disorder (MDD), posttraumatic stress disorder, and schizophrenia. This review explores how neuropeptides in the PFC function to regulate the neural activity, and how genetic and environmental factors, such as stress, lead to dysregulation in neuropeptide systems, which may ultimately contribute to the pathology of neuropsychiatric diseases. The prefrontal cortex (PFC), located in the anterior portion of the frontal lobe, is responsible for several higher-order behaviors including executive function and response to emotional stimuli -ergic neurons (which often co-release neuropeptides) in the PFC have received dense focus throughout the decades due to their strong regulation of both glutamatergic inputs to and outputs from the PFC, as well as their ability to modulate each other. This, combined with the extensive role of the PFC in stress and neuropsychiatric disorders, has led to a keen interest in their function , corticotrophin-releasing factor (CRF), somatostatin (SST), dynorphin opioids (DYN), and the endorphin/enkephalin opioid systems. Where possible, each section will explore the peptide expression and known effects, the effects of known receptors, and the role the peptide and receptors play in a variety of neuropsychiatric diseases. Importantly, this review attempts to bridge clinical studies of psychiatric populations with preclinical research investigating the neural circuit actions of PFC neuropeptides and how dysregulation of these systems contributes to specific behaviors associated with diseased states .It is important to note that the profound overlap and complexity in human neuropsychiatric diseases is not well recapitulated in animal models; many animal models represent a single representation of behavior involved in disease but do not fully encompass the actual human manifestation of the disease. Disorders like MDD and substance use disorder each have many criteria for diagnosis, of which an individual only needs to meet a few characteristics. This results in great heterogeneity in the expression of neuropsychiatric disorders, and likely, great heterogeneity in underlying causes. Also, the interpretation of some animal models has evolved  is a 36-amino-acid neuropeptide with potent physiological effects and dense expression in the central and peripheral nervous systems  and a decrease in evoked α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated excitatory postsynaptic currents (EPSCs) onto layer 5 pyramidal cells in the infralimbic cortex. Collectively, this suggests that NPY may function to reduce action potential discharge of pyramidal neurons of the PFC. Moreover, NPY has been shown to increase the release of other neurotransmitters in the PFC such as dopamine  ]. SymptoHuman subjects with PTSD demonstrate decreased NPY in the cerebrospinal fluid  individuals must display at least 2 of 11 symptoms ranging from impaired control over substance use, social impairment, risky behavior, and the development of tolerance and withdrawal [American Psychological Association (APA) ]. SeveraMDD is characterized by a combination of symptoms including, depressed mood and loss of interest or pleasure, present during the same 2-week period [American Psychological Association (APA) ]. ClinicGenes regulating NPY have been shown to interact with environmental factors such as stress to increase susceptibility to negative emotional symptoms associated with anxiety and depression  when compared to controls  also known as Corticotropin-releasing hormone  is a 41-amino acid neuropeptide which belongs to a family of neuropeptides including Urocortin 1  showed no change in PFC CRF mRNA  into the mPFC increased anxiety-like behavior in the elevated plus-maze (EPM) in both acute and chronically stressed rats  signaling  CRF-R1 , also known as somatotropin releasing inhibitory factor , was characterized over 50 years ago as a hypothalamic extract capable of inhibiting the release of growth hormone from the rat anterior pituitary SST in the CNS is highly evolutionarily conserved, and expression has been observed in several different species including humans, non-human primates, and rodents  which are predominately Gi/Go coupled and result in inhibition of adenylyl cyclase  in the PFC and frontal cortex respectively  in the orbitofrontal cortex in subjects with schizophrenia . DNA microarray for expression of GABA-related transcripts in the dorsolateral PFC of schizophrenic subjects (post mortem tissue) and matched controls revealed a robust decrease in SST mRNA in subjects with schizophrenia  with schizophrenia exhibited unchanged SST-R1 but significantly decreased SST-R2 mRNA in the dorsolateral PFC, and this reduction was localized to pyramidal cells in layers 5–6  with schizophrenia, suggesting that BDNF may function to regulate SST expression in the PFC  administration of 3,4-methylenedioxy-N-methylamphetamine (ecstasy) in male Sprague–Dawley rats raised levels of prodynorphin mRNA in the PFC and decreased levels of Dynorphin-A  exhibited downregulated enkephalin mRNA in the PFC /nischarin, a putative I1-imidazoline receptor which regulates MOR trafficking . Also, further research should address interactions between peptide populations in the PFC—both those expressed in different and in the same, GABAerigc neurons. For instance, subpopulations of neurons within the PFC express both NPY and SST, while SST and dynorphin have also been found to co-express here. This overlap is surprising given the general opposing roles of NPY and dynorphin. Also, increasing technological advancements have allowed for the greater investigation of peptide release independent of co-expressed neurotransmitters, which will allow a greater understanding of when same-neuron peptides are released . Greater assessment of the combinatorial role of these peptides will better inform disease models and treatment. Overall, the literature suggests a strong and important role of peptides in the PFC in stress and neuropsychiatric disorders.Future work, in addition to expanding the existing depth of literature, also needs to focus on filling the gap in the literature. Specifically, much of the pre-clinical work above has been conducted in male rodents, with very little focus on females. Others have eloquently expressed elsewhere Shansky, , 2020 thsingle behavioral or biological manipulation are unlikely to fully recapitulate the complexity and range of the human disorder. Therefore, it is no surprise that animal models using a signal manipulation  do not replicate the work of these nuanced psychiatric conditions in humans. Leading researchers in the preclinical field have agreed that animal models thus far are not fulling capturing human disorders but are nevertheless a crucial component of treatment identification and testing. These researchers identified key gaps in the literature needed for the synthesis of human and animal work: “in vivo experiments, precision, innovation, integration and complexity, and leadership setting the tone”  ] includeOverall, though some discrepancies exist in the role of specific neuropeptides in specific disorders, their importance is clear and further investigation for each of them is paramount to understanding complex disorders in humans, and for targeting both preventions and interventions. By both filling in these gaps , and expanding the focus into new questions and domains overall, a greater understanding of the role of peptidergic signaling in the PFC during stress and neuropsychiatric diseases may be obtained.DB and NC wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


The development of novel genome editing tools has unlocked new opportunities that were not previously possible in basic and biomedical research. During the last two decades, several new genome editing methods have been developed that can be customized to modify specific regions of the genome. However, in the past couple of years, many newer and more exciting genome editing techniques have been developed that are more efficient, precise, and easier to use. These genome editing tools have helped to improve our understanding of genetic disorders by modeling them in cells and animal models, in addition to correcting the disease-causing mutations. Among the genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has proven to be the most popular one due to its versatility and has been successfully used in a wide variety of laboratory animal models and plants. In this review, we summarize the customizable nucleases currently used for genome editing and their uses beyond the modification of genome. We also discuss the potential future applications of gene editing tools for both basic research and clinical purposes. The ability to precisely manipulate the genome revolutionized not only molecular biology, but also opened several areas of biotechnology that are useful for research, agriculture, and medicine. Initial gene targeting primarily depended on homologous recombination to insert exogenous DNA sequences in the human cells . The estDouble-strand breaks (DSBs) on DNA are repaired using three different mechanisms, namely non-homology end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). Due to the nature of the repairs, NHEJ and HR are referred to as error-prone and error-free, respectively. NHEJ is active during all the cell cycle phases, whereas HR occurs only during the S-G2 phase of the cell cycle. For this reason, NHEJ is active in all the cell types, both dividing and non-dividing. During the repair of DSBs, the donor template with homology arms can get inserted at the site of repair using homology-directed repair (HDR) and dramatically increases the gene targeting efficiency . Based oThe discovery of customizable nucleases that can be programmed to induce DSBs at desired loci on the genome dramatically increased the efficiency of homologous recombination, leading to another revolution in gene editing with much broader implications in several different fields. This review aims to provide an overview of the recent developments in and applications of engineered nucleases that have helped lay the groundwork for their use not only for genome editing in various animal models, but also to correct the genetic mutations in human cells for clinical use. We also discuss the additional applications of genome editing tools in addition to modifying the genome, such as modulating the expression of genes and live-cell imaging. Finally, we review the use of genome editing tools in human embryos. Fokl endonuclease domain. The DNA-binding zinc fingers can be engineered to bind to specific regions on the genome. Fokl is enzymatically active as a dimer; therefore, two ZFN subunits are designed for each target sequence to facilitate the dimerization and cleavage [Fokl and binding of fingers to the target sequence have increased ZFNs attractiveness as a tool for introducing DSB at desired locations on the genome, which is mostly repaired by NHEJ pathway, resulting in the disruption of the amino acid sequence and function of the gene [CCR5 gene to interrupt the expression of co-receptor and prevent HIV infection [ZFNs are a class of DNA-targeting components with two monomer subunits containing DNA-binding and cleavage domains. Each monomer is composed of three zinc fingers, which recognize nine base pairs and a cleavage . The dimthe gene ,11. ZFNsnfection . Moreovenfection ,14. HoweFokI nuclease domain. The TALE-DNA-binding domain is composed of conserved repeats domains derived from transcription activator-like effectors (TALEs) secreted by Xanthomonas bacteria to alter the transcription in host cells [FokI endonuclease are used to create a DSB. TALENs exhibit higher specificity and efficiency than ZFNs. For this reason, TALENs have been widely used for genetic manipulation in different organisms [Similar to ZFNs, TALENs have a customizable DNA-binding domain and st cells . Each TArganisms . Additiorganisms . Moreoverganisms . The sinThe clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) nuclease is a recently identified system in prokaryotes with adaptive immunity against viruses and plasmids. Cas9 complexes with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to form an endonuclease that can recognize and cleave foreign genetic sequences. The DNA binding occurs using a 20-base pair DNA sequence in the crRNA that complements the targeting region next to protospacer adjacent motif (PAM) that triggers Cas9 to create DSB. During the repair of DSB, primarily by NHEJ, small indels are created at the target site, which results in deletions, insertions or frameshift mutations leading to loss of function of the gene. The sequence of crRNA can be replaced with any synthetic target sequence without modifying the other components. The crRNA and tracrRNA were fused to form a single chimeric RNA (sgRNA) and complexed with Cas9 to induce site-specific DNA cleavage . The appIn an adult mammal, the majority of the cells in the body are post-mitotic, with a few exceptions like cells in the liver. The classical HDR mechanism is generally inefficient in non-dividing cells, which limits the possibility of performing a knock-in in these cells. We developed an NHEJ-based homology independent strategy using CRISPR-Cas9, named homology-independent target integration (HITI), for the integration of transgene in non-dividing cells ,37. UnliIn addition to the use of CRISPR-Cas9 for gene deletion and insertion, Cas9 nickase and catalytically deficient Cas9 (dCas9) are fused with deaminases for the conversion of single nucleotides. The cytosine base editor (CBE) is the first base editor developed that can convert C to T . The comInterestingly, three independent groups have recently reported the development of dual adenine and cytosine base editors by combining both the deaminases with Cas9 nickase ,44,45. UBase editors do not generate DSBs and the chances of undesired changes such as indels are low. However, the base editors sometimes show off-target specificity . MoreoveAnzalone et al. reported a prime editing approach similar to base editors, where Cas9 nickase was fused to an engineered reverse transcriptase, which is guided by a two-part guide RNAs termed “search” and “replace”. The “search” guide localizes the Cas9 to target loci, where it cuts the DNA and the “replace” guide sequence is used by reverse transcriptase to make complementary DNA to integrate at the cut site . Since mEscherichia coli, researchers have hypothesized that transposon encoded CRISPR-Cas promoted this process [Interestingly, the CRISPR-Cas complex was also found to be encoded in a class of bacterial Tn7-like transposons . Due to  process . In a re process . The uni process .In addition to the targeting of DNA, Cas9 is also directed to target the single-stranded RNA (ssRNA) matching the guide RNA sequence when PAM is presented in trans as a separate DNA oligonucleotide (PAMmers). The PAMmers were able to stimulate the Cas9 endonuclease activity on ssRNA, similar to DNA cleavage . This apThe specificity of nucleases used for genome editing is one of the essential criteria for success in both basic and translational research. The customizable nucleases are engineered to target the region of interest on the genome using sequence information; however, the shorter-targeting sequence length used for the identification and few mismatches might allow the binding of nucleases at off-target sites. The concern of off-target applies to all the customizable nucleases in general. For therapeutic use of nucleases, they need to demonstrate higher specificity, activity, and gene modification ability. They should also be easier to deliver. Along this line, re-engineering strategies have been implemented for Zinc finger proteins and TAL effectors to improve the targeting precision ,62,63. SThe traditional approach of genetic modification in the cells is less efficient, especially in performing targeted gene knockout or knock-in of fluorescent reporters. The customizable nucleases allowed the generation of a variety of genetic modifications in mammalian cells to model diseases, including cancer, metabolic and neurodegenerative diseases . Stem ceCCR5 gene in T-cell precursor cells isolated from HIV patients to avoid the infection of HIV-1 [The correction of genetic mutations to treat human diseases has proven an exciting potential application for the genome editing tools. In this line, ZFNs, TALENs, and the CRISPR-Cas system have been used to correct mutations in cells in vitro and transplanting them back to the patients. ZFNs have been successfully employed in mammalian cells to deliver the vectors expressing the ZFN-coding sequences by transfection of DNA or infection with viruses . The higof HIV-1 . Currentof HIV-1 ., Moreovof HIV-1 ,76. Simiof HIV-1 . β-globin gene in patient hematopoietic stem cells [Similar to ZFN and TALEN, T-cells have been genetically edited using CRISPR to knockdown immune checkpoint inhibitor programmed cell death-1 (PD-1), which is upregulated during activation of T-cells to reduce the autoimmune reaction and aid cancer cells in evading the immune system . Moreoveem cells .ZFNs and CRISPR-Cas9 have been used to edit the HIV-1 genome and block its expression in T-cells, microglia and promonocytes ,81,82. CLmna and prevent the expression of mutated copy, which led to the extension of the lifespan of progeria mice [The recent development of the NHEJ based genome-editing approach in non-dividing cells now allows for correcting mutations that cover a wide range of diseases, including Parkinson’s disease and amyotrophic lateral sclerosis. HITI was employed to correct a mutation that causes inherited degenerative eye disease, retinitis pigmentosa, in rats . Likewisria mice . Furtherria mice . Notablyria mice . Likewisria mice . AlthougThe traditional approach for creating gene-edited animals requires a time-consuming method that involves the use of modified ES cells. The generation of modified ES cells is a time and labor-intensive process. Moreover, the low success rate of obtaining the founder mice with a higher contribution of injected ES cells to the germline also hindered the process. Furthermore, the lack of ES cells for certain animals, including rats, also limited the generation of genetically modified animals of different species. However, the development of customizable nucleases has helped to overcome the need for ES cells by performing the gene modifications directly in the zygotes. ZFNs were the first nucleases widely used for targeted mutagenesis and gene replacement, beginning with fruit fly and nematodes and progressing to other organisms such as silkworm and zebrafish, by injecting the ZFN pair at the early stages of zygote or embryo development. Notably, ZFNs opened the door to the possibility of creating genetically modified rats, which was not possible with other approaches . In miceLMNA gene [Currently, the CRISPR-Cas system is the most favored customized nuclease and is widely used for performing in vivo genetic modification. Importantly, the CRISPR-Cas system allows the possibility of the simultaneous manipulation of several different genes, thereby dramatically reducing the amount of time required for the generation of double- or triple- knockout animals . MoreoveMNA gene . HoweverMNA gene .Among the different nucleases, so far, ZFNs and TALENs are the only ones so far that have been successfully used to target the mitochondrial genome. Unlike the nuclear genome, mitochondrial DNA (mtDNA) is mostly maternally inherited. The number of mitochondria and mtDNA can vary between different cell types and tissues. Moreover, multiple copies of mtDNA exist, ranging from a few hundred to thousands in each cell based on their energy demand. The mutations in the mtDNA lead to the degeneration of tissues and organs with high energy demand, including muscle and neurons, resulting in the manifestation of mitochondrial disease phenotypes . Due to However, the CRISPR-Cas system has not been reported to be successfully used for targeting mtDNA. The major hurdle for the application of CRISPR-Cas system to mtDNA editing has been the successful import of gRNAs into the mitochondrial matrix, partially due to the inefficient RNA import mechanism present in the mitochondria of mammalian cells . AlthougAlthough initial applications of nucleases mostly focused on gene editing, they were quickly redirected for other purposes, such as gene regulation. Zinc-fingers and TALEs were fused to transcriptional activators, among the different activators, VP64 and p65 proved the strongest when targeted upstream of the transcription start site and in promoter regions . SimilarWe have shown, for the first time in vivo, that Cas9 and transcription activation complexes can be recruited to the target loci using modified guide RNAs to activate the expression of endogenous genes . Using tAmong the different repurposes, nucleases were also used for labeling specific regions of chromatin using fluorescent proteins. Before the identification of CRISPR-Cas system, ZFN and TALE proteins were used to recruit the fluorescent protein to the regions such as telomeres and centromeres for live imaging in cells ,117. HigThe use of CRISPR for molecular diagnosis has provided another exciting development during the last couple of years. Initially, the promiscuous ribonuclease activity of Cas13a upon target recognition of the surrounding RNA molecules was used to develop a molecular detection platform termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). This platform was used for the detection of the Zika and Dengue viruses . MoreoveInterestingly, the RNA targeting property of CRISPR was also tested to target the SARS-CoV-2 genome. A CRISPR-Cas13-based strategy, PAC-MAN , was shown to target and degrade RNA from SARS-CoV-2 sequences and H1N1 IAV load in respiratory epithelial cells . In thisGenetic mutations in the germline of parents are passed down to the next generation. Some of the mutations can be lethal and affect embryo development that may lead to the early termination of pregnancy. In some less severe cases, the mutation can lead to the development of disease later during life. With the advancements in diagnosis and availability of cutting-edge medical interventions, it is now possible to treat or prevent the progression of many diseases. However, we still lack effective methods for treating the majority of the diseases caused by the genetic mutations inherited from the parents, so the correction of these mutations in the early stage of embryo development has been considered. Nevertheless, genome editing in somatic cells and germline differs greatly, not only in terms of DNA repair mechanism, but also in long-term consequences. Genome editing in somatic cells involves the modification of patient cells to cure a disease, which can be performed by isolating the cells and transplanting them back after correcting the mutation. However, for germline editing, the correction needs to be performed during the early stages of embryo development, and all the cells from the embryo may carry the modification, including the germ cells, which will also impact the future generations. Alternatively, it is possible that only some cells in the embryo undergo correction, which can lead to the generation of a mosaic embryo.In the last few years, several research groups have performed gene editing in human embryos. Until now, more than seven different studies have been carried out using human embryos to test the cleavage efficiency and off-targets using CRISPR-Cas system ,135,136.Interestingly, the development of a long-term in vitro culture system for human embryos (until 14 days) ,141, opeCCR5 gene in the human embryos that were later transferred to a human resulting in the birth of twin babies. This controversial experiment reignited an international debate on the necessity and ethical issues on genome editing in human embryos. Currently, in several different countries, a moratorium exists on genome editing in human embryos for clinical purposes. According to the guidelines developed by the National Academy of Science, clinical trials for heritable genome editing can be permitted when performed adhering to the regulatory framework and fulfilling a list of criteria that includes, among others things, the absence of reasonable alternatives, the prevention of the transmission of serious disease, the restriction of the conversion of genes to the versions already prevalent in the population, plans for long-term and multigenerational follow-up, and oversight to prevent the use for other purposes [Until recently, the goal of genome editing in human embryos was intended to better understand the efficacy of gene correction and early developmental problems without implanting the edited embryos. However, to prevent HIV infection, one researcher in China to prevent HIV infection attempted to modify the purposes ,147. Reppurposes .Customizable nucleases have opened new possibilities in the treatment of genetic mutations. Promising results from in vitro experiments and animal models demonstrate the potential application of CRISPR-Cas system in both basic research and clinical settings. However, before these nucleases can be used in clinics, several improvements must be achieved, including more precise targeting efficiency, lower off-targets, fewer unintended changes and a good delivery vehicle that can target a wide range of tissues when delivered in vivo. Certainly, significant progress on these requirements has been made. Notably, the repair efficiency has been improved by delivering Cas protein, and the availability of different types of Cas nucleases isolated from other prokaryotes and nickases are used to avoid DSBs and reduce the off-target activity . MoreoveIn addition to the application of customizable nucleases for gene editing, their use in modulating the expression of the genes by changing the epigenetic marks on the promoters offers great potential. Interestingly, genetic diseases are not only caused due to the mutations in a gene, but also due to the lower expression of said genes that can affect the function of tissues and organs. Notably, during aging, dysregulation of epigenetic marks leads to the decreased expression of several different genes that are important for the normal function of cells and tissue, which eventually leads to the manifestation of disease phenotypes ,151,152.In conclusion, during the last couple of years, we have not only witnessed the discovery and development of new genome editing approaches, but also their implementation to treat various diseases. Currently, many clinical trials are underway that use the newly developed gene-editing tools, and in the next few years, some of them will be eventually used in clinics not just for the treatments of genetic diseases, but also to prevent or treat viral infections such as SARS-CoV-2.