Objective: To assess the longitudinal metabolic patterns during the evolution of bronchopulmonary dysplasia (BPD) development. Methods: A case-control dataset of preterm infants (<32-week gestation) was obtained from a multicenter database, including 355 BPD cases and 395 controls. A total of 72 amino acid (AA) and acylcarnitine (AC) variables, along with infants’ calorie intake and growth outcomes, were measured on day of life 1, 7, 28, and 42. Logistic regression, clustering methods, and random forest statistical modeling were utilized to identify metabolic variables significantly associated with BPD development and to investigate their longitudinal patterns that are associated with BPD development. Results: A panel of 27 metabolic variables were observed to be longitudinally associated with BPD development. The involved metabolites increased from 1 predominant different AC by day 7 to 19 associated AA and AC compounds by day 28 and 16 metabolic features by day 42. Citrulline, alanine, glutamate, tyrosine, propionylcarnitine, free carnitine, acetylcarnitine, hydroxybutyrylcarnitine, and most median-chain ACs (C5:C10) were the most associated metabolites down-regulated in BPD babies over the early days of life, whereas phenylalanine, methionine, and hydroxypalmitoylcarnitine were observed to be up-regulated in BPD babies. Most calorie intake and growth outcomes revealed similar longitudinal patterns between BPD cases and controls over the first 6 weeks of life, after gestational adjustment. When combining with birth weight, the derived metabolic-based discriminative model observed some differences between those with and without BPD development, with c-statistics of 0.869 and 0.841 at day 7 and 28 of life on the test data. Conclusions: The metabolic panel we describe identified some metabolic differences in the blood associated with BPD pathogenesis. Further work is needed to determine whether these compounds could facilitate the monitoring and/or investigation of early-life metabolic status in the lung and other tissues for the prevention and management of BPD. Bronchopulmonary dysplasia (BPD), the most common acquired morbidity of preterm infants, is a severe form of chronic lung disease and is generally characterized by the presence of chronic respiratory insufficiency and a supplemental oxygen requirement at 28 days of life with consistent radiologic changes or continuing need for oxygen therapy at 36 weeks’ postmenstrual age . The incIn recent studies, metabolic signatures of acylcarnitines (ACs) and amino acids (AAs) have been identified in newborn screen metabolic panels in association with metabolic disorders and critical illnesses in mature/premature infants, including fatty acid oxidation disorders, carnitine cycle disorders, cardiac malformation/malfunction, gastrointestinal disorders, and respiratory distress ,9,10,11.To identify disruptions in metabolism involving protein and fatty acid metabolism that are linked to acute or chronic disorders, our group has previously identified associations between either at-birth levels or the serial progression of specific ACs and AAs and the development of necrotizing enterocolitis (NEC) in preterm newborns ,19. FollA total of 995 preterm infants were derived from a prospective clinical study that evaluated metabolic profiles in premature infants. Twenty-three sites in 17 states managed by the Pediatrix Medical Group enrolled patients in this study between April 2009 and September 2012, and the original protocol was approved by both the Western Institutional Board and/or each site’s local Institutional Board ,20. The In this study, the infants were all born <32 weeks and the diagnosis of BPD was defined as meeting BPD criteria of oxygen dependency at 28 days of life. A total of 355 infants were recognized as candidates of cases. Control newborns were selected if they meet the following criteria: (1) no diagnosis of any type of intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP), NEC, or BPD during the first six weeks of life; and (2) survival at the end of the study (day 42 of life). Specifically, IVH was diagnosed if an infant had any of subependymal (grade 1), intraventricular (grade 2), intraventricular with dilation (grade 3) or intraparenchymal (grade 4), while ROP was determined as either ROP 1, 2, 3, or ROP surgery. NEC was indicated if an infant had a NEC-medical or NEC-surgical record. A NEC-medical record was present if an infant had at least one of the following clinical signs: bilious gastric aspirate or emesis, abdominal distention, or blood in stool without evidence of a rectal fissure and had one or more of the following radiographic findings: pneumatosis intestinalis, hepatobiliary gas, or pneumoperitoneum, while a NEC-surgical record indicated that a surgery was carried out following the NEC diagnosis . As a ret-test for continuous variables and a chi-square test or Fisher’s exact test for categorical variables. In the first 42 days of life, loess curves were drawn for each nutrition-related variable in the BPD and control sub-cohorts, respectively, to reveal different longitudinal patterns. Among these nutrition-related measurements, the infant’s weight-adjusted daily calorie intake was calculated as calories received per kg per day from all nutrition on a certain time interval , denoted as calories . Similarly, the infant’s weight (g) variable was assessed on each indicated day of measurement.All statistical analysis was conducted using R software. Demographic and clinical characteristics were statistically compared between BPD newborns and controls using a Logistic regressions were applied at each time point to capture the subset of metabolic variables significantly associated with BPD development, using the GA-matched sub-cohort, and the global FDR was used to adjust for multiple comparison testing. Next, fold-changes between cases and controls were further calculated over time for those identified significant metabolites, and a hierarchical clustering heatmap was generated accordingly using R pheatmap and corrplot packages. Lastly, we investigated whether the captured metabolic panel could be used to describe metabolic disparities between infants with and without BPD over the first several weeks of life. The R randomForest package was used for modeling where most parameters were set by default and the number of trees was optimized as 400. Herein, we used the entire dataset of 355 BPD cases and 395 control infants regardless of their GA and randomly and evenly separated the dataset into construction and validation cohorts with a ratio of 2:1 and further constructed the discriminative models using random forest, by considering the subset of significant metabolites, as well as other leading characteristics at birth . These discriminative models’ performance was evaluated and compared using c-statistics.After excluding preterm infants who had been diagnosed with NEC, ROP, or IVH, a total of 355 BPD cases and 395 control infants were enrolled in the primary cohort, whose characteristics are summarized in The longitudinal patterns of body weight and weight-adjusted calories received were measured on day of life 1, 7, 28, and 42 and compared between newborns who developed BPD and controls for both the primary cohort without GA matching A and theIn order to identify metabolic indicators associated with BPD development, while controlling the confounders related to malnutrition and growth faltering, a focal analysis on the 27–28-week GA sub-cohort was pursued using logistic regression by treating birth weight as a covariate to identify metabolic variables that were associated with BPD development at at least one time point during the period of analysis. The global FDR of 0.05 was adopted for multiple comparison adjustment. As a result, among the 72 longitudinally measured metabolic variables, an intersection of 27 metabolic analytes were captured, which led to a compressed metabolic panel representing the difference between infants that developed BPD and controls over the first 42 days of life. Since both birth weight and GA were carefully controlled, the recognized metabolic panel was independent from confounders of malnutrition and poor growth and may be taken as BPD associators independent of established risk factors. During the first day of life, no significant metabolic analytes were noted to be different between cases and controls. Subsequently, the significant metabolites gradually increased from 1 predominant AC metabolic differentials by day 7 to 19 significant AA and AC differentials by day 28 and reduced to 16 metabolic features by day 42 .Since the identified metabolic panel contains several combined variables from different metabolites, we further extracted and generated a subset of 25 distinct metabolites  involvedFinally, we treated this identified metabolic panel of 27 analytes from day 1 to day 42 of life as input and built discriminative models together with birth weight to examine whether the panel could be used to describe certain metabolic differences between infants with and without BPD, facilitating the monitoring of BPD development in early life. To increase the sample size and modeling accuracy, the primary cohort consisting of 355 BPD cases and 395 control infants were ultilized at this modeling stage. After randomly splitting the dataset into the development and validation subsets with a ratio of 2:1, we built the models using random forest algorithms. Results  showed tIn this study we identified distinct longitudinal patterns of fatty acid and protein metabolism intermediates associated with infants who develop BPD among premature newborns. The derived metabolic panels revealed the longitudinal progressive metabolic deviation of BPD babies in the blood over the first six weeks of life after birth and thus potentially described early biologic clues of BPD during its development and newborn clinical management.Prior studies have documented the importance of optimized nutrition on lung development in order to achieve normal lung structural morphology ,22. ReduIn this study, 27 distinct intermediates of fatty acid and protein metabolism were identified that were associated with the development of BPD over the early days of life, after adjusting for both GA and birth weight. At the first day of life, 0 metabolic differentials were captured. Subsequently, the number of identified AC and AA differentials progressively increased to 19 at day 28 of life, suggesting postnatal metabolic reprogramming for amino-acids, acylcarnitine, and fatty acids occurring progressively in response to clinical care and disease evolution .The identified abnormal acylcarnitine profiles over the first months of life in our data imply a critical role of mitochondrial fatty acid β-oxidation in moderating lung injury and maintaining pulmonary function, where carnitine is a key molecule for infant energy utilization as it transports long chain fatty acids across the mitochondrial membrane . In our Regarding protein metabolism, it has been previously shown that citrulline and tyrosine levels were noted to be significantly lower in infants with pulmonary hypertension secondary to BPD, while phenylalanine was increased in infants with persistent pulmonary hypertension ,17,38. IA limitation of this study is that metabolic samples and clinical variables were exclusively collected at four time points over the first six weeks of life; thus it is possible that an element of time-dependent insight and subtle metabolic and clinical alterations may be attenuated or masked in this study. Second, in addition to the 72 targeted metabolic intermediates based upon a standard newborn screen at the time of collection in this study, additional metabolites and nutritional parameters should be captured in future studies to more deeply evaluate metabolic pathways discussed in this study, such as the urea cycle and fatty acid β-oxidation for an evaluation of their potential critical roles in the BPD pathogenesis. Thirdly, in this study, these metabolites are measured in the blood, while it is still unclear how the changes of these metabolites in the blood could reflect their levels in the lung. Fourthly, in this study, the metabolic indicators associated with BPD development were determined from infants with a lower GA ; thus our findings may only be applicable to extremely premature babies.In this study, we identified a panel of fatty acid and protein metabolites associated with newborns who develop BPD. This panel described distinct longitudinal metabolic differences between infants with and without BPD in the blood from birth to day of life 42, independent from GA and birth weight. Several underlying metabolic pathways were discussed, implying their potential association with BPD development for extremely premature infants. However, it is still unclear whether there are similar changes of these metabolites in tracheal aspirates from infants with BPD. Therefore, we hope that our results could provide clues for early-life metabolic status in the blood during BPD pathogenesis and promote metabolic monitoring or investigation in the lung and other relevant tissues to benefit postnatal nutritional administration for future BPD prevention or management.


Inflammatory bowel disease (IBD), mainly including Crohn’s disease and ulcerative colitis, seriously affects human health and causes substantial social and economic burden. The pathogenesis of IBD is still not fully elucidated, whereas recent studies have demonstrated that its development is associated with the dysfunction of intestinal immune system. Accumulating evidence have proven that inflammasomes such as NLRP3 and NLRP6 play a prominent role in the pathogenesis of IBD. Thus, regulating the activation of inflammasomes have been considered to be a promising strategy in IBD treatment. A number of recent studies have provided evidence that blocking inflammasome related cytokine IL-1β can benefit a group of IBD patients with overactivation of NLRP3 inflammasome. However, therapies for targeting inflammasomes with high efficacy and safety are rare. Traditional medical practice provides numerous medical compounds that may have a role in treatment of various human diseases including IBD. Recent studies demonstrated that numerous medicinal herb derived compounds can efficiently prevent colon inflammation in animal models by targeting inflammasomes. Herein, we summarize the main findings of these studies focusing on the effects of traditional medicine derived compounds on colitis treatment and the underlying mechanisms in regulating the inflammasomes. On this basis, we provide a perspective for future studies regarding strategies to improve the efficacy, specificity and safety of available herbal compounds, and to discover new compounds using the emerging new technologies, which will improve our understanding about the roles and mechanisms of herbal compounds in the regulation of inflammasomes and treatment of IBD. Inflammatory bowel disease (IBD), mainly including Crohn’s disease and ulcerative colitis, is a term to describe a group of chronic relapsing inflammatory disorders occurred in the gastrointestinal (GI) tract –3. Thesevia modulating inflammasomes and have been shown to be effective in an increasing number of studies , which is a medicinal plant wildly used in Chinese medicine for treatment of many human diseases (via inducing the expression of thioredoxin-interacting protein (TXNIP), an inhibitory regulator of NF-kB pathway    also hasEvodia Rutaecarpa (ER), which is a herbal plant that has been used clinically in treating some human inflammatory diseases, especially for headache and abdominal pain (Another compound that may play anti-colitis role through regulating NF-κB and the NLRP3 inflammasome is Evodiamine (EVO), a bioactive component with anti-inflammatory role  derived nal pain . To studnal pain  used murDendrobium officinale (DO) on colonic inflammation induced by DSS. DO is a widely used herb in traditional Chinese medical practice to treat gastrointestinal disorders. The data showed that polysaccharides from DO (DOPS) can inhibit β-arrestin1 expression and suppress activation of the NLRP3 inflammasome in both in vivo and in vitro experiments.β-arrestin1 is a ubiquitously expressed protein that is identified initially as an inhibitor and adaptor of the G protein-coupled receptors with the purpose of regulating many immunological pathways and cell death , 64. It Fructus Arctii (Great Burdock Achene), which is a herbal plant widely used in traditional Chinese medicine. It has been reported that Arctigenin has extensive pharmacological effects in a variety of diseases such as diabetes  . SIRT1 cdiabetes . Regardidiabetes  found thvia multiple mechanisms  in colonic inflammation. TiO2 is a natural oxide of titanium that widely used in food products as additives or in pharmaceutical formulations, although a minor human health risk of TiO2 nanoparticles has been proposed  extracts, which has been proven to be effective in murine colitis model , which is a medicinal plant used for treatment of ulcerative colitis (UC) in Chinese medicine , which is traditional herbal medicine used as parasiticide of digestive tract and agricultural insecticide in China , a triterpenoid and natural insecticide with anti-inflammatory activity derived from in China . For itsin China  reported2S)  , 96and adiseases , 98. Thein et al  showing Lindera strychnifolia, traditional Chinese medicine used in treating various diseases. The researchers showed that NOR alleviated colitis and reduced the levels of NLRP3, cleaved caspase-1 and IL-1β in colons of the mice. In in vitro studies, NOR was shown to activate AhR, which then enhanced Nrf2 level and reduced production of ROS. Thus, the anti-colitis role of NOR is mediated by inhibiting the NLRP3 inflammasome activation via modulating AhR/Nrf2/ROS pathway.AhR is a universally expressed transcription factor that can bind to a variety of structurally diverse ligands and regulates a range of biological processes such as metabolism of xenobiotic chemicals, immunity and stem cell differentiation . It has Cardamonin is a chalcone derived from cardamon with anti-cancer activity, it also plays a role in anti-infection immunity and the development of inflammatory diseases. A recent study has shown that cardamonin can inhibit the NLRP3 inflammasome activation and thus attenuate experimental colitis induced by both DSS and TNBS . In thisvia modulating PINK1/Parkin regulated mitophagy.Mitophagy is an autophagic pathway that keeps the cell healthy by promoting the mitochondria turnover and preventing the accumulation of damaged mitochondria . It reguGarcinia mangostana L.) that has anti-cancer activity , a natural xanthonoid derived from the fruit hull of mangosteen , 2-(S)-protopanaxatriol, Rh1, F1, and 20(S)-protopanaxadiol. Seong et al  showed tIndigo naturalis (IN) is herbal medicine extracted from leaves and stems of plants and is a component of crude drugs used in treating many diseases including IBD . According to this guidance, the estimation of HED requires studies to determine the No Observed Adverse Effect Level (NOAEL) of a compound in multiple tested animal species. This requires data on systemic toxicology and others, such as bioavailability, metabolite profile and plasma level of the compound. With the availability of all these data, the conversion of NOAEL to HED needs to employ a scaling system based on body weight (mg/kg) or body surface area (mg/m2), a process that needs to consider multiple factors, such as the method of compound administration and NOAELs in different animal species. However, the in vivo data in majority of studies mentioned in this review did not provide sufficient details to estimate the MRSD of the tested compounds. Only four out of twenty-two studies provided in vivo toxicity data that obtained from IBD model induced in one species of animal , Natural Science Foundation of Jiangsu province (BK20201442), Jiangsu Specially-Appointed Professor and Start-up funds for young scientists of Nantong University (03083051).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Ferroptosis, a recently discovered form of cell death, whose role in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) has not been well disclosed. To improve our understanding of the differences in tumor progression and therapeutic effects between BCC and SCC, and to find potential therapeutic targets, this study systematically analyzed ferroptosis-related genes (FRGs) and their associated local immune cytolytic activity (LICA) and tumor microenvironment (TME) metabolic function differences.Methods: Two bulk RNA-seq datasets, GSE7553 and GSE125285, from the Gene Expression Omnibus database were compared within and between groups to screen for common differentially expressed genes (DEGs) for enrichment analysis. The currently recognized FRGs in DEGs gene set were selected as the targets to analyze their correlation and difference in LICA and TME metabolic functions. And validated using immune cell populations from another single-cell RNA-seq (scRNA-seq) dataset (GSE123813) to accurately understand the difference in LICA. All of the gene sets for functional enrichment analysis comes from published results and MSigDB database.Results: Ten FRGs were used to further analyze the differences in LICA and TME metabolic functions between BCC and SCC. In the SCC samples, LICA  and TME metabolic functions  were significantly related to ferroptosis genes , and the ferroptosis potential index were also significantly higher than that in the BCC samples. Finally, based on these ten FRGs and related enrichment results, we postulated a model of NQO1 homeostasis regulated by FRGs during induction of ferroptosis in SCC.Conclusions: The results showed that three FRGs, SLC1A5, CD44 and NQO1, have significant potential in targeted therapies for SCC chemotherapy resistance. And two FRGs, STEAP3 and HMOX1, formed a synergistic effect on the occurrence of ferroptosis in tumor cells. Our findings can be used as the main research materials for metastasis and chemotherapy resistance in SCC patients. Research on tumor cell death has been the main goal of cancer treatment, and the mechanism of acquired or intrinsic resistance to apoptosis and necrosis of cancer cells has become a key research object in the field of tumor treatment. After more than 30 years of exploration of the mechanism of programmed death of cancer cells, necrotic apoptosis has been confirmed to be a typical form of cancer cell necrosis, which is mediated by receptor interacting protein kinase 3 (RIPK3) and mixed-lineage kinase domain-like synergism , 2. FerrFerroptosis-related genes (FRGs) in a variety of tumors  program was used to normalize the data matrices in the two studies respectively. And the differentially expressed genes  in BCC and SCC samples were selected as the DEGs gene set. Genes function enrichment analysis was performed using gene set variation analysis , including differences in correlation between LICA or TME metabolic function and FRGs. In addition, the DEGs gene set was used to compare between the two studies with the gene expression matrix of normal or adjacent normal tissues and the genes enrichment analysis results of BCC and SCC.The LICA related gene set used for single sample gene set enrichment analysis (ssGSEA) comes from the attached files of the cytolytic activity of 18 TCGA tumors by Rooney et al. , includiThe overall score share (OSS) of all samples is a score calculated based on sample size and gene expression levels in selected gene sets to assess the activity of biological processes in different sample types. OSS was calculated by formula The ferroptosis potential index (FPI), proposed by Liu et al.  to explop53 and microRNA alteration. Based on the RNA expression pattern and the co-expression score of protein co-regulation, As shown in In the gene expression DEGs gene set of BCC and SCC, some typical biological signal pathways showed obvious differences . For exaAs shown in Furthermore, by comparing the LICA enrichment results of BCC and SCC tissue samples , only thSubsequently, the TME metabolic function differences between the BCC and SCC groups, and between the high versus low expression groups of FRGs were compared. As shown in Finally, a ccOSS network model was constructed to compare the shared scores of each enrichment result, including the immune cells or function of LICA and the metabolic function of TME, in the overall sample set of BCC and SCC. As shown in Similarly, in the TME functional network diagram , in addiAll of the above, LICA showed significant enrichment differences between BCC and SCC tissue samples. However, the disadvantage was that it is difficult to accurately described the important role of the cytolytic activity of immune cells on the TME due to the mixed cell types in tissue samples. Therefore, this study extracted the T cell populations (include 28554 cells) of BCC and SCC from a public scRNA-seq dataset (GSE123813). Then we classified T cell into 7 subsets according to the annotation results of the data provider , includiAfterwards, two key cytolytic effector genes, GZMA and RPF1, were found significantly higher in SCC than in BCC in T cell community , and alsTo our knowledge, this study systematically investigated the differences in ferroptosis gene-related LICA and metabolic functions of TME in BCC and SCC for the first time. As listed above, this report not only obtained LICA (immune cell or function) and TME metabolic functions that were closely related to FRGs, and confirmed that the LICA of immune T cell in SCC was significantly higher than that in BCC.The comparative results of ccOSS in this study showed that the metabolism of nucleotides, lipids and amino acids were more important in the SCC microenvironment. Among the 10 FRGs analyzed, the genes SLC1A5 and ACSF2 controlled amino acid transport  and fattIn addition, we noticed that energy metabolism was weakened in SCC. Therefore, we hypothesized a model of NQO1 homeostasis that regulates ROS function in the intracellular environment . NQO1 plAs we found above, two FRGs, HMOX1 and STEAP3, were significantly upregulated in SCC tissues, both of which could promote Fe3+ to Fe2+ transformation , 34 Fig. And FPITumor cell metabolic dysfunction also alters the immune and inflammatory responses. CD44, an upregulated FRG in SCC, is highly correlated with immune function in this study, and plays an important role in biological processes such as inflammation response, extracellular matrix remodeling, and tumor cell invasion and metastasis, and EMT –40. At pSignificantly different from SCC, the results of LICA, TME and ROS function enrichment in BCC tissues were very close to those in adjacent/normal tissues , 4B, 4D,In vitro and preclinical trials should be carried out in the future to further evaluate the therapeutic effect of these gene targets.In conclusion, under the protective effects of cellular immune surveillance, NQO1 homeostasis and iron homeostasis, most damaged cells undergo natural apoptosis or necrosis, but those “super tumor cells” must be cleared in time through reasonable treatments to avoid deterioration and metastasis. Therefore, the potential target FRGs, SLC1A5, CD44 and NQO1, for tumor resistance treatment have important research value in the treatment of SCC drug resistance. https://gitee.com/plainTHS/bscc).The data that support the findings of this study are available from the Gene Expression Omnibus database. Bulk RNA-seq datasets (GSE7553 and GSE125285) and single-cell RNA dataset (GSE123813). Complete analysis code was deposited in the platform of gitee (Supplementary FiguresSupplementary File 1


Excitatory-inhibitory (E-I) imbalance has been shown to contribute to the pathogenesis of a wide range of neurodevelopmental disorders including autism spectrum disorders, epilepsy, and schizophrenia. GABA neurotransmission, the principal inhibitory signal in the mature brain, is critically coupled to proper regulation of chloride homeostasis. During brain maturation, changes in the transport of chloride ions across neuronal cell membranes act to gradually change the majority of GABA signaling from excitatory to inhibitory for neuronal activation, and dysregulation of this GABA-shift likely contributes to multiple neurodevelopmental abnormalities that are associated with circuit dysfunction. Whilst traditionally viewed as a phenomenon which occurs during brain development, recent evidence suggests that this GABA-shift may also be involved in neuropsychiatric disorders due to the “dematuration” of affected neurons. In this review, we will discuss the cell signaling and regulatory mechanisms underlying the GABA-shift phenomenon in the context of the latest findings in the field, in particular the role of chloride cotransporters NKCC1 and KCC2, and furthermore how these regulatory processes are altered in neurodevelopmental and neuropsychiatric disorders. We will also explore the interactions between GABAergic interneurons and other cell types in the developing brain that may influence the GABA-shift. Finally, with a greater understanding of how the GABA-shift is altered in pathological conditions, we will briefly outline recent progress on targeting NKCC1 and KCC2 as a therapeutic strategy against neurodevelopmental and neuropsychiatric disorders associated with improper chloride homeostasis and GABA-shift abnormalities. For any particular ion, the direction of flow is dictated by the electrochemical gradient, and the difference in concentrations of ions across the membrane results in a membrane potential. In the adult brain, the opening of ion channels permeable to cations such as Na+ and Ca2+ drives the membrane potential toward the threshold potential (depolarization) through their entry into the cell. Increased permeability for Cl– anions and their cell entry, in contrast, hyperpolarizes the cell to drive the membrane potential further away from the threshold potential, and reduces the chance of the cell firing an action potential.The predominant form of neurotransmission in the brain occurs at chemical synapses where presynaptic neurons release neurotransmitters that bind to receptors on postsynaptic neurons. A major type of postsynaptic receptors are ion channels which allow the selective influx or efflux of monovalent  is crucial for effective GABAergic neurotransmission. The intracellular chloride concentration ([Cl–]i) relative to its extracellular concentration is generally lower in the mature brain than in the immature brain, and thus the opening of GABAA receptors results in the influx of chloride ions into the postsynaptic neuron and hyperpolarizes it , γ-aminobutyric acid (GABA), activates ionotropic GABArizes it . Since drizes it , and inh+-Cl– cotransporter-2 (KCC2) (+-K+-2Cl– cotransporter-1 (NKCC1)  against its concentration gradient in order to maintain a low [Cl–]i   and Na+- (NKCC1) , which aw [Cl–]i . Similarthe cell .–]i in immature neurons causes a chloride efflux when GABAA receptors are activated and ultimately results in membrane depolarization, thus effectively making GABA signaling excitatory in nature in the immature nervous system. While depolarizing responses are typically excitatory, there are counter-examples of GABA acting to depolarize cells leading to inhibition of network activity  increases the number of glutamatergic synapses formed. However, the authors also assessed the effect of bumetanide inhibition of NKCC1 on excitatory synapse numbers; they found that even under these conditions, GABA inhibition still increased excitatory synaptic density.Finally, the network undergoes a large burst in synapse formation during the final stages of depolarizing GABA, with GABA-dependent excitation acting to facilitate the generation of new synapses, both in the hippocampus and cortex , by helpelopment . On the Altogether, the downstream effects of GABA on aspects of brain organization and function differ across the GABA-shift. Let us next discuss how the beginning and end of this process is controlled.When exactly does the GABA-shift happen? Interestingly, the exact timing of the switch from depolarizing to hyperpolarizing GABA varies substantially across neuronal type, species, brain region, and even sexes. The shift happens earlier in cortex than in hippocampus , earlierA blocker (bicuculine) from birth to P15-18 did not prevent the development of direction-selective circuitry  mice  mice  , both of which are expressed only in neuronal cells   . Thus, telopment  and thatelopment .SLC12A2 gene), but both BDNF and oxytocin have been observed to downregulate its expression , a possible chemical toxicant used in the production of a wide range of consumer products linked to increased incidence of neurodevelopmental disorders  (WNK)-serine/threonine protein kinase 39 (SPAK)/OSR1 oxidative stress-responsive 1 (OSR1) pathway is a principal regulator for both transporters to coordinate their activities based on intracellular chloride concentrations .WNK1 mutations result in an autosomal recessive disease characterized by congenital pain insensitivity  and increase NKCC1 activation (to enhance Cl– import). In contrast, WNK kinases are inhibited by high [Cl–]i, thereby reversing these changes to enhance KCC2-mediated Cl– export while suppressing further import by NKCC1.Of all four WNK isoforms (WNK1-4) in the human genome, WNK1 is believed to be the principal WNK kinase in the human CNS . This issitivity . WNK kinactivity . Active activity , which iactivity . When i present in immature neurons and in turn reduce KCC2 phosphorylation to enhance Cl– export. However, although Wnk1 knockdown was found to reduce [Cl–]i in immature neurons, –]i in immature neurons or that additional kinases are at work during this stage. Further studies comparing the WNK-SPAK/OSR1 pathway between immature and mature neurons are necessarily to fully delineate the importance of this pathway to [Cl–]i regulation and the GABA-shift during neurodevelopment. More recently, two groups independently produced knock-in mice with phosphomimetic mutations in these two phosphorylation sites  to evaluate their physiologic significance. The authors observed reduced postnatal survival of homozygous mutants due to respiratory arrest and epileptic seizures  was found to activate KCC2 activity by enhancing the phosphorylation and dephosphorylation of S940 (to be discussed below) and T1007, respectively, to stabilize its surface expression  located on the C-terminal intracellular domain of KCC2 , which sactivity . Notablyactivity . Specifiseizures , while hseizures , therebypression . It was pression . Moreovepression , while apression . In addipression . Similarpression .–]i and alter KCC2/NKCC1 activities to maintain an optimal intracellular chloride concentration. This control of KCC2/NKCC1 activity by the WNK-SPAK/OSR1 pathway is primarily achieved by influencing the surface expression of these two cotransporters on the cell membrane  and enhances its surface expression to increase overall transporter activity. The physiological importance of this phosphorylation site was revealed as knock-in mice incapable of S940 phosphorylation  exhibit ASD-like deficits in social behaviors and cognitive functions , while aXenopus oocytes without any changes in surface expression of the mutants. It remains unclear, however, how phosphorylated Y1087 could directly impact KCC2 activity. In addition, other groups have also found the Y1087D variant to disrupt KCC2 activity in subsequent studies  and are believed to regulate its membrane targeting . NotablyIt is clear that the correct expression, surface targeting, and phosphorylation status of NKCC1 and KCC2 are critically important for healthy circuit development and function, but how does this impact the myriad of different cell types in the brain? We will discuss this topic next.Changes in the balance of chloride across neuronal membranes are not expected to strongly affect postsynaptic neuronal activity unless there is concomitant release of GABA from presynaptic inhibitory neurons. The development of the inhibitory network has been widely studied across brain regions  challenge  (Cl), thereby potentially reducing GABAAR-mediated hyperpolarization compared to neurons carrying wild-type KCC2 protein. In a separate study  in KCC2 from patients from an Australian family with febrile seizures that similarly disrupted chloride homeostasis by reducing KCC2 surface expression and function  . The loste study , two of function . Anotherfunction . Notablyfunction . More red in ASD . In addiisorders . The patSLC12A5 and SLC12A2 expression in patients. Consistent with their opposing roles in maintaining chloride homeostasis and GABA-shift, whereas NKCC1 protein levels were observed to be increased in patients with temporal lobe epilepsy (TLE) (SLC12A5 (KCC2) mRNA expression was found to be downregulated  , SLC12A5egulated . In addiegulated .MECP2), intellectual disability , epilepsy    , and ASDpatients . At the patients . It willRecent studies have also identified alterations of NKCC1 and/or KCC2 expression in patient-derived brain samples or animal models of neuropsychiatric disorders such as SZ and bipolar disorder (BD). For example, a significant reduction in KCC2 expression and thus an reducing trend for the KCC2/NKCC1 transcript ratio (as NKCC1 mRNA levels were not different between patients and controls) were observed in the hippocampi of SZ patients, suggesting the possibility of an immature GABA-shift . ConsistSLC12A5 and SLC12A2 mutations have also been identified in SZ patients, including the R952H mutation in SLC12A5 described above  patients (ed above . The sampatients . Functiopatients , thus poper se. Furthermore, multiple studies have also observed immature morphological and functional properties in neurons of SZ animal models or mutant animals exhibiting behavioral deficits relevant to neuropsychiatric disorders (To this end, it is of interest that several studies have recently identified transcriptomic signatures of “immaturity” in the dentate gyri and prefrontal cortices of adult patients with ASD, SZ, or BD , suggestisorders . Given tisorders . In paraisorders  and may isorders . An indeisorders . InteresIn addition to the above study that demonstrated the beneficial effects of NKCC1-specific inhibitor bumetanide, commonly used clinically as a diuretic, it has been shown to be a useful treatment in mouse models of SZ , Down Syin vivo pharmacokinetic profile in mice as compared to bumetanide (Thus far, clinical trials have provided mixed results for the use of the NKCC1 blocker bumetanide to help reduce ASD symptoms in patients. While there have been reports hinting at successes in correcting for some symptoms in open-label pilot studies  and randmetanide , thus ofmetanide  could be– across the neuronal membrane, and regulators of those proteins, such as WNK kinases. The balance of Cl– across the membrane is primarily regulated by KCC2 and NKCC1, and as such, they are prime targets for pharmacological modulation. However, the widespread expression of NKCC1 in other tissues of the body complicates the utility of the pharmacological approach for therapy. Furthermore, our understanding of disturbances in the normal GABA-shift is hampered by the high variability: it happens at different times, in different cell types, that may be intermingled within a particular brain region. Moreover, in human cases with a mutated or missing chloride transporter, it is not clear how much of a patient’s symptoms may be attributable to an abnormal GABA-shift, and how much is related to ongoing network dysfunction. Further research is required to understand the pathological consequences of a mis-timed or incomplete GABA-shift, and treatment options, whilst currently available, are limited.This review has attempted to lay out the changes that occur in GABAergic signaling during early development, with a particular focus on the GABA-shift, both in how it proceeds in healthy tissue, and how it is disrupted in various pathological states. Abnormalities in the progression of the GABA-shift have been linked to several neurodevelopmental and neuropsychiatric disorders. Potential therapeutic targets typically focus on a handful of proteins: the transporters that move ClA better grasp of the factors that control the GABA-shift during development may provide a greater understanding of what happens in the process of dematuration within the adult network. What are the exact triggers for the onset and successful completion of the GABA-shift? The impact and participation of different cell types in the GABA-shift, and the ongoing maintenance of GABAergic signaling, and both intra- and extracellular chloride regulation is an extremely important topic, and the extent of the contribution of glial cells remains a topic of much interest.KH and TC conceptualized and wrote the manuscript. YG and MT provided intellectual input and edited 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is a chronic disease of the gastrointestinal (GI) tract; its burden has significantly increased in recent decades, with 6.8 million cases of IBD reported in 2017 according to the Global Burden of Disease study [Mucosal inflammation, immune cell infiltration and increased leukocytes are regularly observed in IBD patients, always associated with immune system malfunctions, including T helper (Th)2 and Th17 responses and abnormal levels of cytokines . For exaThe gut microbiome is essential in regulating the immune system in IBD patients, with studies having shown varying microbial diversities in IBD patients and healthy individuals. The maintenance of the balance of the gut microbiota is a novel insight in the treatment of IBD . For exaInterestingly, IBD patients have presented with similar microbial changes to patients with COVID-19, characterised by gut issues, including diarrhea, vomiting and abdominal pain. This could potentially indicate a link between COVID-19 and IBD, but recent studies have shown that IBD patients have no increased risk of contracting COVID-19 . The relThe rapid ageing of the population is a global phenomenon that entails the elderly people to be more prone to developing IBD. Biological aging results in cellular senescence that leads to immune dysfunction and chronic inflammation , a proceDiet has been widely recognised as an important factor in the prevention and management of noncommunicable diseases (NCDs) ,13,14,15Accumulating epidemiological and experimental studies have reported that diets rich in fibre or particular dietary patterns  might help in the management of IBD . HoweverIn this Special Issue, manuscripts highlight creative and novel findings linking diet and IBD and present research to help advance the knowledge of how dietary fibre affects IBD in biomedical science, the population’s health and clinical practice.


Carulli et al.; Wingert and Sorg). Pathological alterations in PNNs have been reported across several neuropsychiatric conditions including, but not limited to, post-traumatic stress disorder (PTSD), multiple sclerosis (MS), epilepsy, substance use disorders (SUDs), and schizophrenia. Thus, targeting the restoration of normal PNN function has emerged as a potential avenue for the development of therapeutics for the remediation of several diseases/disorders.Dysregulated synaptic plasticity and excitatory inhibitory (E/I) imbalance are molecular hallmarks of multiple neuropsychiatric disorders. Perineuronal nets (PNNs) are condensed portions of the extracellular matrix (ECM) that stabilize homeostatic synaptic plasticity, facilitate neuronal-glia communication, and provide a physical barrier protecting neurons from toxic molecules within the ECM  to a hyaluronan backbone on a neuronal cell. Hyaluronan-CSPG binding is stabilized by link proteins, such as hyaluronan and proteoglycan link protein 1 (HAPLN1) and HAPLN4, and CSPGs have an overall negative charge. PNNs typically contain the lectican aggrecan, with varying levels of neurocan, versican and brevican . The fundamental role of PV-containing interneurons is to inhibit firing of local pyramidal neurons (Wingert and Sorg). Depending on the firing patterns of the inhibitory circuit, PV positive interneurons have the capacity to shape network responses to external stimuli. Wingert and Sorg critically review the impact of PNN removal from these circuits to determine the electrophysiological impact across multiple brain regions implicated in psychiatric disorders. Overall, it has been established that PV interneurons are highly excitable and most active during awake sensory processing. A single action potential efficiently recruits PV cell-mediated feedback inhibition on firing pyramidal neurons. That is, a single evoked or spontaneous action potential in a pyramidal cell will induce excitatory post-synaptic potentials (EPSPs) that generate an action potential in a connected PV positive interneuron. These properties allow PV interneurons to synchronize principal neuron output, but the exact motif of inhibition produced by PVs within local brain regions and circuits is still largely undefined. Summarizing the most consistent findings from the field, the evidence indicates higher action potential thresholds and a decrease in firing rate of fast spiking PV positive GABAergic interneurons after PNN removal (Wingert and Sorg). This can result in significant alterations at a circuit level, for example reduced hippocampal long-term potentiation (LTP) within the Schaffer Collateral pathway (Wingert and Sorg). At a functional level, this deficit manifests as impaired learning and memory performance, an important facet in multiple disorders and diseases.PNNs predominantly enwrap the cell bodies and proximal dendrites of fast spiking parvalbumin (PV) positive GABAergic interneurons within the central nervous system . Indeed, it has been previously shown in mice that the PNN composition exhibits circadian rhythmicity. This includes elevated densities of Wisteria floribunda agglutinin (WFA)-labeled PNNs during arousal and lower levels of WFA during sleep in the hippocampus . This pattern of decreased PNN composition during sleep coincides with decreases in synaptic downscaling and LTP, which may facilitate memory consolidation, processes which are blunted following sleep deprivation. Of interest are the coincident and entrained alterations in the expression of the transcription factor orthodenticle homeobox 2 (OTX2), glutamatergic NMDA receptors, matrix metalloproteinase 3 (MMP3) and 9 (MMP9) and cathepsin-S that occur with sleep deprivation, reviewed in Gisabella et al. With regards to PTSD, the importance of PNNs in the acquisition/retention of trauma associated memories have led many researchers to evaluate specific PNN components as therapeutic targets.In the context of disease, it is also important to note that PNN levels may be increased and attenuation of PNNs in that context can facilitate learning and cognitive flexibility  activation by CSPGs binding in constraining tyrosine kinase 2 (TrkB) dependent neuronal plasticity. Previous studies have reported that deletion of PTPs increased phosphorylation of TrkB, thereby promoting plasticity within the visual cortex  characterized the molecular profile of enhanced plasticity in haploinsufficient mice. PTPσ+/- mice, generated on a BALB/c background, exhibited a 50% increase in phosphorylated TrkB and an 25% increase in phosphorylated phospholipase C gamma 1 (PLCγ1) expression in the prefrontal cortex, hippocampus, and amygdala relative to wildtype controls, with no significant regional variation in expression . Overall, these molecular alterations should facilitate neuronal plasticity. At a behavioral level these haploinsufficient mice did not show altered behavioral performances when screened for exploratory activity on the elevated plus maze, marble burying behavior, locomotor activity or time spent immobile in the forced swim test . In contrast, long term consolidation of memory was robustly diminished for novel object recognition (discrimination index for the novel/familiar object) and contextual fear memory (% time spend freezing in the context) when assessed for recall at 5 and 10 days following the initial behavioral exposure, respectively. This contrasted with the increase in novel object discrimination of PTPσ+/– mice when tested 4 h following training . Although no direct alterations in the number of synapses were reported in these PTPσ+/– mice, there is evidence in the literature to support the supposition that PTPσ deficiency results in impaired LTP and EPSCs . In particular, microglia produce proteases, including MMP9 and cathepsin-S, which cleave CSPGs . Primary hippocampal astrocyte-neuron co-cultures and mixed glial cultures derived from the cortices of SV129 mice were utilized in medium exchange experiments to evaluate the impact of microglial activation by the toll like receptor 3 (TLR3) ligand Poly I:C on synapse formation and network activity . Single occurring action potentials (spikes) were robustly elevated in all conditions when evaluated following 3 h of incubation. Increases in spike number persisted (up to 48 h) only in neurons treated with microglia conditioned medium relative to their non-microglial conditioned Poly I:C and hippocampal medium controls. The number and duration of burst firing of neurons was also significantly enhanced in microglia conditioned medium. The increased spontaneous network activity of neurons treated with microglia conditioned medium was not dependent on baseline spike frequency or impacted by apoptosis, and no specific subpopulation of neurons was identified as mediating this change in network activity. Wegrzyn et al. proceeded to demonstrate that PNN ensheathed neurons (aggrecan positive cells) treated with microglia conditioned medium exhibited robust disruption of PNN covering relative to the even covering of the soma and proximal dendrites of neurons by PNNs in the control medium groups . These cells exhibited a robust decrease in formation of inhibitory but not excitatory synapses , and a resultant shift toward higher excitatory input overall .Microglia are also known to be critical factors in the acquisition, consolidation and recall of memory  for a range of psychiatric and neurodegenerative disorders. This may involve evaluation of small molecule inhibitors such as morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) which is currently in clinical trials for the treatment of psoriasis.35−55)-induced experimental autoimmune encephalomyelitis (EAE) reported a pronounced loss of both perisomatic PV+ and PNNs in layer 2/3 of the primary somatosensory cortex and a compensatory increase in VGlut1 positive cells  . The eight semaphorin classes, the importance of neuropilin (Npn) and plexin (Plxn) proteins in binding semaphorins and the known functions of Sema3A, 3F, 3G, 4C, 5A, and 7A in the adult brain are presented in a comprehensive review by Carulli et al. In terms of therapeutic development, inhibition of Sema3A  may yield the most compelling results in the treatment of MS. Consistent upregulation in the aforementioned semaphorins, Npn and PlxnA1, in human brains of individuals diagnosed with MS are suggestive of a possible pathophysiology maker of MS. These changes are significant given that semaphorins are known to inhibit axonal regeneration, oligodendrocyte progenitor cell differentiation and oligodendrocyte migration. Utilizing murine models of MS, Biname and colleagues demonstrated that inhibition of Sema3A through local microinfusion of a peptide that antagonized Npn-1-PlxnA1 dimerization, effectively facilitate remyelination and rescue of MS associated motor deficits in murine models of the disease , suggesting an inconsistency across data sets. Overall it is suggested that carrying a mutation in PlxnA2 may confer an added risk of developing this debilitating psychiatric disorder . These data support the need for more comprehensive evaluation of the PNNs and semaphorins in particular in the precipitation and treatment of schizophrenia. Indeed, it would be extremely worthwhile to determine whether alterations in PlxnA2 are associated with either the positive, negative, or cognitive symptoms of schizophrenia.Klimczak et al. extend their previous findings and detail alterations in PV expressing interneurons within the hippocampus and retrosplenial cortex of adult male FVB mice exposed to a “double hit” model (DHM) of schizophrenia. In this case, DHM was comprised of early life NMDA antagonism [MK-801 administered at post-natal day 7 (P7)] paired with social isolation (starting on P21), to mimic the pathophysiological disruptions observed in humans. Previously, the Nacher group has characterized behavioral deficits and altered plasticity in the amygdala of mice exposed to DHM, specifically identifying decreased PV expressing neurons and PNN expression in amygdala of these mice  relative to isolation and MK801 treatment alone. Of interest was a marked reduction in the number of PNN positive/PV negative somata in this region following isolation alone . Within the retrosplenial cortex, a marked reduction in overall number of PV positive cells was evident in all treatment groups relative to vehicle control socially housed mice. PV positive neurons surrounded by PNN exhibited a marked reduction in the isolation alone group . These regions are important in spatial working memory, a cognitive domain consistently impaired in patients with schizophrenia  are important factors that should be considered when designing future experiments.There are several limitations to our current understanding of PNN physiology and function. Optimization of PNN visualization remains a concern, for example not all aggrecan-containing PNNs are labeled with WFA. A recent review by Hartig and colleagues has outlined the conditions under which WFA staining is compromised, and detail strategies for optimized visualization  and the Generalitat Valenciana (PROMETEU/2020/024). KC is supported by R01NS10881. CB is supported by Transforming Technology for the Warfighter HU0001-20-2-0014 and Congressionally Directed Medical Research Programs PRMRP award W81XWH-21-2-0011.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Some diets, such as high lipid and high glucose diets, are known to increase the risk of colorectal cancer. On the other hand, little is known about diets that prevent colonic carcinogenesis. The ketogenic diet, which is characterized by high fat and very low carbohydrate content, is one such diet. The ketogenic diet decreases the amount of available glucose for tumors and shifts to the production of ketone bodies as an alternative energy source for healthy cells. Cancer cells are unable to use the ketone bodies for energy thus depriving them of the energy needed for progression and survival. Many studies reported the beneficial effects of the ketogenic diet in several types of cancers. Recently, the ketone body β-hydroxybutyrate has been found to possess anti-tumor potential in colorectal cancer. Despite its beneficial effects, the ketogenic diet also has some drawbacks, some of which are related to gastrointestinal disorders and weight loss. Thus, studies are being directed at this time towards finding alternatives to following a strict ketogenic diet and supplementing patients with the ketone bodies responsible for its beneficial effects in the hope of overcoming some potential setbacks. This article discusses the mechanism by which a ketogenic diet influences growth and proliferation of tumor cells, it sheds the light on the most recent trials regarding its use as an adjunctive measure to chemotherapy in patients with metastatic colorectal cancer, and it explains the limitations of its usage in metastatic patients and the promising role of exogenous ketone supplementation in this setting. Colorectal cancer (CRC) ranks third among cancers worldwide but is second in terms of mortality risk . DespiteOn the other hand, paucity of data exists on diets that could prevent or treat intestinal carcinogenesis. While abstinence and calorie deficit show potential in impairing glycolysis and inhibiting proliferation in CRC cells ,18, implIn this review, the process of ketogenesis is explained and the effect of KD as an alternative treatment choice in CRC patients is highlighted. The mechanism by which KD influences tumor growth and proliferation is thoroughly examined with emphasis on the future potentiality of this dietary intervention.The ketogenic diet is distinguished by its very low carbohydrates , moderate protein  and high fat content . The most common adaption of this diet involves a 4:1 ratio of fat to carbohydrate and protein . When caThe anti-tumor potential of KD was demonstrated in some in vitro studies. These studies revealed that tumor cell lines  are not able to employ ketones as their dietary energy source when starved of glucose thus resulting in decreased glycolysis and tumor proliferation ,33,34. Mp < 0.05) [Additionally, a few case reports and pre-clinical studies obtained promising results in cancer patients. The first attempt to treat cancer patients with a long-term controlled KD was reported by Nebeling et al. for two pediatric patients with astrocytoma . Ketosis < 0.05) . No seveκB kinase pathway enhances angiogenesis and metastasis in CRC by stimulating nuclear factor-κB (NFκB) and β-catenin [One feature of cancer metabolism is an increase in glucose uptake, which leads to elevated insulin levels, which in turn elevates the insulin-like growth factor 1 (IGF1). Binding of insulin and IGF-1 to their receptors tyrosine kinases leads to autophosphorylation and subsequent activation of phosphoinositide 3-kinase (PI3K) . PI3K in-catenin   controls the secretion of IL-6 and takes part in the enhancement of the protumor M2 phenotype of macrophage . Tumor-aHOPX gene. βHB acts through the surface receptor (hydroxycarboxylic acid receptor 2) HCARr2 to influence HOPX expression and decrease epithelial growth [HOPX promotes progression in CRC [HOPX, with serum from 41 patients with CRC further indicating that serum levels of βHB correlate with HOPX expression in these patients [In vivo, the KD down-regulated the protein expression of MMP-9 and enhanced the M2 to M1 TAM polarization. Moreover, the levels of HDAC3/PKM2/NF-κB 65/p-Stat3 proteins decreased in the KD group . Additiol growth . Epigenen in CRC . A similpatients . Multiplpatients .KD diminished tumor growth and prolonged survival in mouse models. In mouse xenograft models of colon cancer cell lines, a KD composed of omega3 and medium chain triglycerides and another composed of lard significantly delayed the tumor growth compared to standard diet group . In anotHuman studies on the prognostic effect of low carbohydrate diets in patients with CRC remains low. Stage III CRC patients consuming a diet high in carbohydrates and glycemic load had decreased survival . The effIn addition to the disease itself, the side effects of chemotherapy and radiotherapy can be very incapacitating and severely influence quality of life (QoL). Some of these side effects include nausea, vomiting, fatigue, hair loss, nerve and muscle damage, cognitive impairment and weight loss . Thus, aCachexia and unintentional weight loss are grave complications that can negatively influence the prognosis of CRC patients. The KETOCOMP study which is a prospective trial that studied the effects of a KD on body composition in rectal patients receiving curative radiotherapy revealed that a KD significantly reduced body weight and fat mass averaging 0.5 and 0.65 kg/week . There wRecently, it was reported that ketogenesis regulates the tumor microenvironment (TME) in CRC and has potential to overcome immunosuppression and enhance survival. This study revealed that the products of ketogenesis, the ketogenic enzyme HMGCS2 or the ketone body βHB decreased expression of Kruppel Like Factor 5 (KLF5), which binds the C-X-C Motif Chemokine Ligand 12 (CXCL12) promoter and induces CXCL12 expression in cancer-associated fibroblasts. Low expression of KLF5 improves the immunosuppressive TME, augmented penetration of natural killer and cytotoxic T cells, and improved the antitumor effects of Programmed cell death protein 1 (PD-1) blockade in murine-derived colorectal cancer. This reveals that down-regulation of de novo ketogenesis in the TME is a vital step in CRC progression .So far, and especially with regards to CRC, it should be pointed that diet quality is important as KDs with large quantities of artificial foods may have a negative influence on the gut microbiome and offset the otherwise beneficial effects of ketosis . This waTo achieve therapeutic ketosis, strict dietary compliance is required and this is often difficult specifically in terminally ill patients such as metastatic CRC patients. One review reported that the studies implementing the KD had low adherence to KD but the drop-out rates varied greatly between the different studies. The major reason behind low adherence were related to limitations in monitoring and delivery, unpalatability of meals and difficulty integrating the dietary pattern into family life . Weight Based on this, the use of exogenous ketone supplementation such as ketone esters (liquid form), ketone salts (powder form), and medium chain triglycerides (MCTs) which induce ketosis in a similar way that a KD might is justified. The making of β-HB from exogenous ketone supplementation is not influenced by carbohydrate intake, and thus administration of these supplements would reach therapeutic ketosis by following a normal healthy diet that does not involve carbohydrate restriction. MCTs are quickly absorbed, energy-dense, dissolve in water and have no taste. Supplementation with exogenous MCTs as a source of dietary fat was not well tolerated as high consumption of MCTs results in gastrointestinal side effects including diarrhea, dyspepsia, and flatulence. Oral administration of ketone esters fully metabolizes to βHB and acetoacetate. The two most prominent forms of ketone esters include (R)-3-hydroxybutyl (R)-3-hydroxybutyrate ketone monoester (KME) and R,S-1,3-butanediol acetoacetate ketone diester (KDE). Ketone esters are safe, tolerable, and effective ketogenic mediators ,104,105.Beside the greater feasibility of using exogenous ketone supplementation than following a strict KD diet , severalColorectal cancer is one type of cancer which is influenced by dietary patterns and body mass index. The majority of CRC cells exhibit an altered metabolism that is expressed by increased glycolysis and decreased oxidative phosphorylation. The KD, which is characterized by very low carbohydrate and high fat intake, has the potential to provide energy in the form of ketone bodies to all normal cells and deprive CRC cells of glucose which is needed for proliferation and metastasis. Moreover, the KD and specifically the ketone body βHB were found to play major role in several pathways such as IGF, PI3K, mTOR, and TNFα. Recently, the ketone body βHB exhibited the ability to suppress epithelial cell proliferation and inhibit tumor growth. The ketone body acts on cancer cells through regulation of Hopx, a known regulator of CRC. Furthermore, βHB requires a surface receptor Hcar to induce Hopx expression and suppress proliferation of intestinal epithelial cells . Current evidence on the role of KD in CRC comes mainly from in vivo or in vitro studies and few human trials. In these studies, KD improved quality of life and attenuated many of the side effects of cancer treatment. Although there is still limited evidence to support the use of KD as an adjunct therapy in mCRC patients, the recent discoveries of the potential role of KD specifically βHB in CRC, are encouraging and many trials investigating the efficacy of this dietary therapy are underway. Several registered clinical trials are currently ongoing to investigate the case for a KD as a supportive therapeutic option in oncology mainly in breast, brain, prostate, and renal cancers. It is necessary that following the finding of the important role of βHB in CRC that future trials study its effect in complementation with standard treatment for CRC. Implementing the KD is not without disadvantages. Problems with palatability and strict dietary restrictions increase the dropout rate. Additionally, some reported side effects including gastrointestinal side effects such as constipation and weight loss prevent patients from adhering to the diet. To circumvent the downfalls of KD, exogenous ketone supplementation, providing adequate amounts of the ketone body βHB, is starting to be put into use. It is likely that future implementation of KD will be in the form of exogenous ketones where they will be administered alongside standard therapy in mCRC as they might pose as a more viable alternative to the KD. Additionally, another advantage of ketone supplementation is their tolerability and their ability to be formulated and titrated in a way that minimizes their gastrointestinal side effects, all of which pave the way for an acceptable and tolerable option for mCRC patients. Recently, a beneficial association between KD and epigenetic modulation has surfaced. Implementation of KD resulted in changes in DNA methylation, histone modifications and miRNA levels . Data on


Hypophosphatemic rickets is a rare disease that results in bone deformities. However, little is known about bone turnover and bone mass disorders in this disease. This retrospective study included 12 children aged 1–16 years diagnosed with hypophosphatemic rickets. Parameters of calcium-phosphate metabolism and bone turnover markers were analysed. Bone mineral density was assessed with the use of dual-energy X-ray absorptiometry, and indices of quantitative ultrasound examination of tibiae and radial bones were analysed. In the majority of patients, hypophosphatemia and hyperphosphaturia were present. The assessed bone turnover markers showed increased bone formation. Increased pyridinoline levels were found in 5 out of 12 patients. Bone mineral density was decreased only in one patient. Decreased values of quantitative ultrasound examination were observed in all the analysed patients. Conclusions: (1) Bone metabolism disturbances, reflected in the increased values of bone turnover markers and worse bone quality, were found in the group of patients with hypophosphatemic rickets. (2) It is crucial to determine bone turnover markers, dual-energy X-ray absorptiometry findings and indices of quantitative ultrasound examination in order to monitor progress of the disease, as well as treatment effects. Among all cases of rare genetically conditioned rickets, hypophosphatemic rickets is diagnosed most commonly; its incidence is estimated at 1 in 20,000 births ,4. The sIrrespectively of inheritance pattern, all congenital forms of hypophosphatemic rickets present similar phenotypes including short height along with age and typical changes in the skeleton. Characteristic bone symptoms of hypophosphatemic rickets include varus deformity of the lower extremities, frequently severe, which appears after the child starts walking—usually in the 2nd year of life—that also progresses with age and may lead to gait disturbances and require surgical correction ,7 Figur. Other symptoms present also in different types of rickets include the following: short stature (non-treated adults reach body height of 130–165 cm), weakening of muscle strength and exercise performance, expansion of long bone metaphyses, rachitic bracelets and rachitic rosary. Skull bone deformities may include craniosynostosis, protruding forehead and hypoplastic central part of the face, as well as peridental lesions and elongation of teeth crowns. Adults in whom the disease was not diagnosed and no treatment was implemented develop multiarticular pains, chronic fatigue, depression, symptoms suggestive of ankylosing spondylitis and entezopathies  ,8,9,10. Laboratory tests show significant hypophosphatemia with hyperphosphaturia that is caused by decreased phosphate reabsorption in kidney tubules. Increased phosphaturia in hypophosphatemic rickets is accompanied by a decrease in the tubular reabsorption of phosphate (TRP) below 85%.Calcium levels remain in normal ranges or are slightly decreased. Alkaline phosphatase (ALP) activity is elevated in hypophosphatemic rickets, and a decrease in its activity is a good marker of treatment efficacy. The bone formation marker osteocalcin and the bone resorption marker pyridinoline may be elevated, which reflects accelerated bone remodelling ,7,11. The levels of the hepatic vitamin D metabolite (25OHD) remain normal as long as cutaneous synthesis is maintained or the patient receives relevant vitamin D supplementation in preventive doses, the same as in the healthy population. The levels of the renal metabolite may be decreased in FGF-23-dependent rickets or increased in FGF-23-independent rickets ,11. Treatment depends on the form of hypophosphatemic rickets and on clinical disease manifestation. In the FGF-23-dependent form, in children, treatment starts with the administration of active vitamin D analogues such as alfacalcidol (at a dose of 1–3 ug once daily) or calcitriol. Additionally, if there is vitamin D deficiency, cholecalciferol supplementation should be started in accordance with typical guidelines for a given population. This should be the sequence of treatment: vitamin D products before the treatment with phosphates prevents occurrence or exacerbation of preexisting secondary hyperparathyroidism, hypercalciuria and consequent nephrocalcinosis. After one week of treatment with vitamin D, phosphates are started at a dose of 40–70 mg/kg/day in 3 to 8 doses, optimally without a night-time break. The effect of conventional treatment in growth has been related to the age at diagnosis and onset of treatment, and some patients do not respond to this treatment. In recent years, burosumab, a recombinant human IgG1 monoclonal antibody, has been used for treatment. Among patients treated with the recombinant human IgG1 monoclonal antibody, significant improvements in serum phosphate levels and a reduction in pain alongside clinical improvement of patients have been demonstrated.As low stature is one of the leading symptoms of hypophosphatemic rickets, it is growth hormone (GH) that has so far most often been considered an alternative treatment option for hypophosphatemic rickets. GH treatment can increase short-term linear growth in these patients before puberty, but clinical observations have shown that it has been ineffective in some patients and has caused worsening in limb deformity, radiographic deterioration and biochemical abnormalities. When pharmacological treatment is ineffective in patients with significant bone deformities, surgical procedures such as corrective osteotomy, epiphysiodesis or epiphyseal cartilage block of the knee and ankle joints using Blount staplers are used. Corrective osteotomies are rarely performed in children under 6 years of age, as pharmacological treatment very often improves lower limb deformities in younger children. This procedure is usually postponed until the growth process is complete. Severe deformities may require earlier treatment, and then a newer, less invasive method is used, epiphysiodesis, which induces corrective, differential growth of the growth plate ,6,7,12.The aim of this study has been to analyse bone turnover markers and bone mineral density in children with hypophosphatemic rickets. So far, there are only a few reports in the literature on the assessment of bone mineralisation and bone turnover markers in children with hypophosphatemic rickets.From a clinical point of view, in patients with metabolic bone disease, and therefore also hypophosphatemic rickets, it is important to assess bone quality in terms of potential fracture risk. Therefore, the authors analysed the results of the gold standard for assessing bone mineral density, DXA, and the less commonly performed non-invasive ultrasound QUS examination. The analysis will select appropriate diagnostic methods to assess bone quality and thus the potential risk of fractures.Biochemical parameters of bone turnover, such as alkaline phosphatase, osteocalcin and pyridinoline, were assessed in all the patients. Calcium and phosphate levels were measured in serum, in 24-h urine collection and/or in the first morning urine portion. Serum parathormone activity (PTH) as well as both renal and hepatic vitamin D metabolite levels were measured. Furthermore, TRP was calculated according the following formula: TRP% = 100 − [1 − (P in urine × creatinine in serum)/(P in serum × creatinine in urine)]). Serum concentrations of calcium, phosphate levels and ALP activity were determined using routine analytical methods. Serum values of 25-OHD and PTH were determined using electrochemiluminescence detection , as was osteocalcin . Pyridinoline was assayed with chemiluminescence . Bone mineral density was assessed with dual-beam X-ray absorptiometry (DXA) using the same scanner (GE Lunar Prodigy) in total body and spine mode. Z-score was calculated for all the patients. Quantitative ultrasound examination (QUS) was assessed with a Sunlight Omnisense 7000P apparatus. The speeds of the ultrasound waves  along the tibiae and radial bones were measured. X-ray examination of lower extremities was also performed in all patients. The influence of hypophosphatemic rickets on skeletal mass is not good, especially for children, although studies report variable results. As reported in literature, most untreated adults have normal indices of bone mass. Studies using dual-energy X-ray absorptiometry (DXA) in these patients also suggest that bone mineral density (BMD) is increased at the spine but not at cortical sites. Quantitative ultrasound (QUS) is a non-invasive and radiation-free technology that is used for bone mass and quality assessment. Studies in adults confirm that the information provided by QUS measurements is independent of BMD because it only relates to cortical bone .This assessment included 12 patients aged 1–16 years (6 girls and 6 boys) treated in the Department of Paediatric Propedeutics and Bone Metabolic Diseases and in the Outpatient Clinic of Osteporosis and Other Bone Metabolism Diseases. The patients were diagnosed with hypophosphatemic rickets based on clinical symptoms and biochemical disease markers; in eight children, the diagnosis was confirmed by genetic testing. The study was retrospective.Medical history and clinical analysis of the examined patients showed that various deformities of the lower limbs were the main symptoms in all the patients and that it was the reason for the patients’ referrals for laboratory tests. Additionally, 4 out of 12 children had gait disturbances, the same number of patients suffered from bone and joint pain, and 1 out of these 12 children had experienced a finger fracture. Family history was positive in 5 children. All patients were treated with phosphates, and three of them underwent corrective surgery .The analysis of calcium–phosphate metabolism results showed that hypophosphatemia was present in the majority of patients (10 of 12) and documented in consecutive measurements; serum calcium levels were normal in all patients. Hyperphosphaturia occurred in the majority of children from the analysed group (10 of 12). Increased urine phosphate excretion was recorded both in the 24-h urine collection and in the calculated phosphate elimination ratio in the first morning urine portion. Both hypophosphatemia and hyperphosphaturia are typical findings for the disease in question. In our study the aforementioned biochemical changes were the first stimulus for the further diagnosis of hypophosphatemic rickets. Calculated phosphate reabsorption in the renal tubules was decreased in 8 out of 12 patients. The levels of the renal vitamin D metabolite were normal in all 12 children, whereas those of the hepatic metabolite were decreased in as many as 7 of 12 of the subjects.The assessed bone turnover markers showed, in all patients, increased bone formation markers. Accordingly, values of ALP and osteocalcin were found elevated in 10 of 12 and 7 of 12 patients, respectively. The levels of pyridinoline, a bone resorption marker, were higher than the upper limit of reference values in 5 of 12 patients. Only in two patients (P1 and P10) did both bone formation and bone resorption markers remain normal, whereas in 5 of 12 children, increased values of at least one bone formation marker and one resorption marker were observed. The results are presented in In 9 of 12 patients, the bone mineral density was assessed by densitometry. In the total body projection, bone density was decreased only in one girl with the most pronounced short stature, in whom the correct diagnosis and treatment had been delayed. On the other hand, we observed an elevated Z-score in the spine Z-score programme (>2.0) in one (1) child. In all analysed patients, decreased Z-scores were present on quantitative ultrasound bone examination at at least one measurement site within the tibiae and/or radial bones .Hypophosphatemic rickets is a rare disease with a diverse genetic background. Diagnosis is based on clinical symptoms in combination with imaging and laboratory results. In addition to measurements of basic calcium–phosphate metabolism parameters, where significant hyperphosphaturia accompanied by hypophosphatemia predominate, the assessment of bone turnover markers (both bone formation markers and bone resorption markers), e.g., ALP, osteocalcin and pyridinoline, is indicated. Increased activity of these markers reflects increased bone metabolic activity that even in the absence of fractures may cause exacerbation of bone deformities, e.g., varus deformity of the lower extremities. So far, there are only a few studies that analyse bone turnover and bone mineralisation in children with hypophosphatemic rickets ,15,16.In our study, the analysis of the calcium-phosphate balance results showed that the majority of patients had hypophosphatemia, but serum calcium levels were normal in all patients. Hyperphosphaturia was present in the majority of children in the analysed group. Calculated renal tubular phosphate reabsorption was reduced in 8 of 12 patients. The bone turnover markers assessed showed mainly increased activity of bone formation markers. Bone resorption marker levels were higher than the upper limit of reference values in about half of the patients. In only two did both bone formation and bone resorption markers remain normal. The assessment of bone mineral density using densitometry, in 9 of 12 patients, showed that bone mineral density was normal. In the total body projection, bone density was reduced in only one girl in whom the correct diagnosis and treatment had been delayed. All analysed patients had reduced Z-scores on the quantitative bone ultrasound examination.Bone turnover markers in the assessment of hypophosphatemic rickets were analysed by Nagata et al. . The autShanbhogue et al.  conducteDue to the clinical manifestation of the disease that hypophosphatemic rickets predominantly affects the motor system, the assessment of bone mineral density seems important, and the BMD may be decreased or increased in densitometric measurements.Colares et al.  reportedCheung et al.  assessedThe above reports show that a normal DXA result does not guarantee that the patient has normal BMD and is appropriately treated. Quantitative CT is a better method for this assessment.The QUS technique that we have used for the examination of our patients is a simple and non-invasive method which does not expose the patient to radiation. It was found that the measurement of sound velocity (SOS) along a long bone gave information not only about bone density but also about its’ microarchitecture, cortical thickness and elasticity ,19,20. DTaking all into account, in children with hypophosphatemic rickets, typical biochemical disturbances such as chronic hyperphosphaturia and hypophosphatemia may be accompanied by accelerated bone turnover; therefore, monitoring of bone formation and bone resorption markers is also recommended in this disease. Disturbances of mineral metabolism in hypophosphatemic rickets may manifest in the form of either increased or decreased BMD. The decreases in the indices of QUS in all examined patients suggest poorer bone cortex quality in this disease.In the evaluated group of children with hypophosphatemic rickets, bone metabolism abnormalities were found manifested in elevated bone turnover markers and poorer bone quality. These parameters need to be monitored during the treatment process.In children with hypophosphatemic rickets, the assessment of bone status cannot be based solely on the result of a densitometric examination because a normal result does not indicate correct bone quality. An important complementary role is played by bone ultrasound examination, which is non-invasive but rarely performed and which, by assessing cortical bone, gives better information on bone quality. Abnormal bone quality can predispose to fractures, just as in patients with normal bone mineral density.


The epidemics of obesity and type 2 diabetes mellitus (T2DM) pose enormous threats to human health. Weight loss of at least 5%–10% attenuates the severity of these disorders and their complications. Diet and lifestyle modification are necessary but not sufficient for sustained weight loss in most people. Historically, antiobesity medications have been modestly effective, with numerous adverse side effects. Bariatric surgery offers sustained weight loss and potential remission of T2DM, but this treatment is impractical for large populations and may be associated with serious surgical and other complications, such as malabsorption and micronutrient deficiencies. The development of analogs of the gut peptide glucagon-like peptide 1 (GLP1) suggested considerable potential for gut hormones for treatment of T2DM and obesity. This culminated with regulatory approval of the GLP1 receptor (GLP1R) agonist semaglutide, which leads to clinically significant weight loss and improves T2DM control in most patients. The discovery of polyagonist drugs that activate multiple gut-brain pathways promises to further transform the management of obesity and T2DM. Recently, the gastric inhibitory polypeptide receptor/GLP1R (GIPR/GLP1R) co-agonist, tirzepatide, became the first polyagonist approved for the treatment of T2DM; it induces a staggering 22% average weight loss in patients with obesity  promote weight gain. The development of safe and effective antiobesity therapies has lagged significantly. Moreover, the FDA standard of 5% weight loss was barely attained by many approved drugs. This began to change when investigators first sought to harness the therapeutic potential of gut hormones, which mediate communication between peripheral organs and the brain. The incretin hormone GLP1 was discovered as a cleavage product of preproglucagon produced by L cells in the small intestine to enhance postprandial insulin secretion and reduce blood glucose . Native While GLP1 analogs were the first to make it to market, owing largely to the serendipitous discovery of exendin-4, GLP1 is one of many hormones that modulates glucose metabolism and regulates appetite. Although glucagon has largely been portrayed as the canonical counterregulatory glucose control hormone, which acts via the glucagon receptor to raise blood glucose, it also exerts lipolytic, thermogenic, and anorexigenic actions . To asseToday, numerous other gut hormone polyagonists are in the drug development pipeline for obesity and T2DM. Glucagon receptor/GLP1R co-agonists were the first gut peptide polyagonists to be developed by engineering glucagon to render it a less specific ligand for the glucagon receptor, while also targeting GLP1R . In rodeThe discovery of and regulatory approval process for gut hormone polyagonists have outpaced our understanding of their underlying mechanisms of action. How do receptors expressed in peripheral organs and the brain mediate the complex metabolic and behavioral effects of these drugs? Sophisticated mouse genetic, molecular, and neuroscience approaches have begun to unravel how these molecules may exert synergistic effects on metabolism via their activity at different receptors. Determining the exact mechanisms by which GIPR agonism potentiates the effects of GLP1R agonism is a particularly active area investigation, given the remarkable clinical success of tirzepatide . GIP potAs noted above, long-acting GIP analogs induce dose-dependent weight loss, an effect that is abrogated in GIPR-knockout mice . Thus, aThe role of glucagon agonism in the treatment of metabolic disease. The mechanisms underlying the anorectic effects of glucagon are poorly understood, but they are abrogated by vagotomy and may involve hepatic futile cycling and/or secretion of FGF21 . Others The dual incretin agonist tirzepatide is the first pharmacological agent that rivals the efficacy of bariatric surgery. However, several important issues must be addressed. To date, nearly all GLP1 agonists and tirzepatide are administered as daily or weekly subcutaneous injections, with prolonged dose-escalation paradigms to minimize gastrointestinal side effects. This limits their use in some patients. Although an oral formulation of semaglutide coupled to an absorption enhancer is available, its bioavailability is low and it lacks the full glucose and body weight lowering effects compared with injected semaglutide. Therefore, the development of potent, small-molecule, orally bioavailable incretin receptor (poly-)agonists and allosteric modulators is an active area of drug development , 28. As