Hemophilia A (HA), a rare recessive X-linked bleeding disorder, is caused by either deficiency or dysfunction of coagulation factor VIII (FVIII) resulting from deleterious mutations in the F8 gene encoding FVIII. Over the last 4 decades, the methods aimed at determining the HA carrier status in female relatives of HA patients have evolved from phenotypic studies based on coagulation tests providing merely probabilistic results, via genetic linkage studies based on polymorphic markers providing more accurate results, to next generation sequencing studies enabling highly precise identification of the causative F8 mutation. In parallel, the options for prenatal diagnosis of HA have progressed from examination of FVIII levels in fetal blood samples at weeks 20–22 of pregnancy to genetic analysis of fetal DNA extracted from chorionic villus tissue at weeks 11–14 of pregnancy. In some countries, in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) has gradually become the procedure of choice for HA carriers who wish to prevent further transmission of HA without the need to undergo termination of pregnancies diagnosed with affected fetuses. In rare cases, genetic analysis of a HA carrier might be complicated by skewed X chromosome inactivation (XCI) of her non-hemophilic X chromosome, thus leading to the phenotypic manifestation of moderate to severe HA. Such skewed XCI may be associated with deleterious mutations in X-linked genes located on the non-hemophilic X chromosome, which should be considered in the process of genetic counseling and PGD planning for the symptomatic HA carrier. Therefore, whole exome sequencing, combined with X-chromosome targeted bioinformatic analysis, is highly recommended for symptomatic HA carriers diagnosed with skewed XCI in order to identify additional deleterious mutations potentially involved in XCI skewing. Identification of such mutations, which may profoundly impact the reproductive choices of HA carriers with skewed XCI, is extremely important. Hemophilia A (HA) is a rare bleeding disorder resulting from either deficiency or dysfunction of coagulation factor VIII (FVIII), caused by deleterious mutations in the F8 gene encoding FVIII (NM_000132.3). The F8 gene is located at the end of the long arm of the X chromosome in Xq28. Therefore, HA is a recessive X-linked disorder manifested predominantly by hemizygous male patients and transmitted by heterozygous female carriers.F8 mutation in most cases. In parallel, the options for prenatal diagnosis of HA have progressed from fetal blood sampling providing diagnostic results at weeks 20–22 of pregnancy , as well as deep intronic mutations potentially creating alternative splicing sites [Significant advances in molecular biology techniques have greatly facilitated the identification of causative F8 gene mutations in HA patients. Early mutation analyses based on PCR amplification of F8 promoter, coding regions, and splice junctions revealed causative mutations in almost all patients with mild and moderate HA, but in only half of patients with severe HA . Furtherng sites ,25. IdenThe aim of prenatal diagnosis is either to prevent the birth of a child with HA or to determine specific precautions required for the prevention of potential obstetrical complications during pregnancy and delivery of a child diagnosed with HA.In the early period of phenotypic diagnosis of HA, prenatal diagnosis offered to established HA carriers was based on fetal sexing by amniocentesis at week 16 of pregnancy, followed by measurement of FVIIIC levels in fetal blood samples obtained from male fetuses at week 20 . HoweverThe introduction of the chorionic villus sampling (CVS) technique in the early 1990s has enabled early prenatal diagnosis of HA during weeks 11–12 of pregnancy ,27. ExamThe development of highly sensitive PCR methods assisting in the identification of fetal DNA in maternal blood has enabled early determination of fetal sex by examination of Y chromosome sequences at weeks 5–10 of pregnancy ,30. EarlThe attitude towards prenatal diagnosis of hemophilia significantly varies between different world regions. In view of the continuous advances and increasing availability of diverse therapeutic options in developed countries, hemophilia patients are now capable of leading a normal life, with life expectancy equivalent to that observed in the general population. Therefore, acceptance of prenatal diagnosis has been gradually decreasing in these countries. However, prevention of severe hemophilia is crucial in developing countries, where patients rarely survive beyond childhood in view of inadequate therapy due to the extremely limited availability of therapeutic products. Beyond national public health policies and the availability of hemophilia prevention options in various world regions, personal attitudes to living with hemophilia have a significant impact on the carriers’ choices regarding giving birth to an affected child. Personal beliefs, as well as religious and cultural constraints, greatly contribute to these attitudes, which must always be carefully considered in the setting of genetic counseling.In the Israeli National Hemophilia Center, the approach of early fetal sexing at week 7–8 of pregnancy, followed by CVS for prenatal diagnosis of HA in pregnancies with male fetuses, has evolved into the standard of care successfully implemented in the National Hemophilia Prevention Program over the last 2 decades. In Poland, prenatal testing for hemophilia is less widespread and hemophilia is not considered an indication for pregnancy termination in view of the rapidly improving management of the disease. A study comparing the attitudes towards prenatal diagnosis of hemophilia and termination of pregnancy in Iran and Italy demonstrated a much higher rate of acceptance of abortion among Iranian participants (58.2%) as compared to Italian participants (16.7%). The significantly greater rate of acceptability of abortion in the Iranian population may be attributed to differences in the quality of patient care in the two countries .Preimplantation genetic diagnosis (PGD) involves in vitro fertilization (IVF) in conjunction with genetic diagnosis of HA in the embryo, followed by transfer of an embryo free of HA to the HA carrier. Single cells biopsied from cultured embryos at the stage of 6 to 8 cells are examined for the hemophilic haplotype using multi-allelic STR markers within and in close proximity to the F8 gene. Predetermined informative STR markers are co-amplified with AMELX/Y indel polymorphism for sex determination in a single-tube PCR reaction ,33,34,35Heterozygous female HA carriers are typically asymptomatic because their FVIII levels correspond to approximately half of the concentration found in healthy individuals, which is generally sufficient for normal hemostasis . HoweverThe genetic mechanisms underlying the manifestation of HA phenotype in HA carriers include homozygosity or compound heterozygosity for F8 gene mutations ,39, TurnStudies investigating the genetic causes of HA phenotype in HA carriers indicate that skewed XCI accounts for the majority of cases . XCI is Although extremely skewed XCI can be accidental, such a rare phenomenon may be mediated by a mechanism of selection against a deleterious mutation in a gene located on the silenced X chromosome bearing the intact F8 gene copy.Pavlova et al.  , or a mosaic carrying the mutation in some of her tissues, which may include her germline cells . In such a scenario, the apparently non-carrier mother may be at substantial risk of giving birth to additional HA patients.Previous studies suggest that the occurrence of mosaicism may be associated with the type of mutation causing HA. A large international study by Antonarakis et al.  conducteIn a study of a large Chinese cohort that included 257 families with sporadic HA cases, Lu et al. demonstrated mosaicism in 22.5% of mothers determined to be non-carriers by analysis of their white blood cell DNA . A similIn view of the relatively high incidence of mosaicism among mothers of sporadic HA patients, comprehensive genetic analysis aimed at detecting mosaicism, using highly sensitive methods such as quantitative PCR or NGS in DNA samples extracted from various tissues, such as buccal cells, hair follicles, and urothelial cells must be conducted in order to enable accurate assessment of the risk of HA recurrence in the future offspring. In some cases, analysis of ovarian cells for the causative F8 mutation may be necessary .The conventional approach currently used for molecular diagnosis of HA is based on screening for the common intron 22 and intron 1 inversions, sequencing of all the 26 F8 exons, splice junction regions, promoter and 3′-untranslated region, and analysis of copy number variation (CNV). Despite such a comprehensive analysis, the causative F8 mutation remains unidentified in 1–5% of HA patients. In these cases, the causative F8 mutation might be a deep intronic variant creating a de novo acceptor splice site or donor splice site. Several groups have used whole F8 gene sequencing by next-generation sequencing (NGS) in order to identify the pathogenic mutation in HA patients who remained undiagnosed following application of the conventional diagnostic approach. Using whole F8 gene sequencing combined with splicing functional analyses, Dericquebourg et al.  have recThe aforementioned studies emphasize the importance of whole F8 gene sequencing combined with splicing functional analyses for elucidation of the disease-causing mutation in genetically undiagnosed HA patients. However, such comprehensive studies are not yet broadly available in routine clinical practice, especially in developing countries. Therefore, in hemophilia centers with no access to NGS technology, carrier detection in female relatives of a HA patient with no identified F8 mutation should be based on comprehensive genetic linkage studies combined with analysis of FVIIIC and VWF Ag levels.Currently, fetal genotyping involves invasive procedures such as chorionic villus sampling, amniocentesis, or fetal blood sampling. All these procedures are associated with a substantial risk of fetal loss.The discovery of cell-free fetal DNA in maternal plasma  has led The presence of additional deleterious X-linked mutations in symptomatic HA carriers may influence their reproductive choices , and, thHighly accurate molecular biology techniques enabling precise diagnosis of the HA-causing mutation are not yet widely available in routine clinical practice. Expanding access to mutation analysis is especially relevant for developing countries, where carrier detection and prenatal diagnosis of HA are crucially important in view of the low availability of therapeutic products; thus, worldwide collaboration projects such as My Life, Our Future initiative , successF8 mutation in DNA extracted from their blood cells), based on analysis of multiple tissues  using highly sensitive technologies, is relatively expensive and time-consuming. However, the identification of mosaicism is highly informative for both the suspected carrier and genetic counselors. Detection of the causative F8 mutation in any of the tissues examined may suggest that the mutation is present in the suspected carrier’s ovaries as well, and therefore, the risk of HA recurrence should be discussed and managed accordingly.Detection of mosaicism in mothers of sporadic HA patients who appear to be non-carriers (i.e., not exhibiting the causative Finally, symptomatic hemophilia carriers require extensive genetic analysis beyond their HA carrier status. The ultra-rare phenomenon of manifestation of hemophilia in a female, as discussed above, may indicate carriership of severe X-linked disease on the non-hemophilic X-chromosome. Therefore, we recommend whole exome sequencing with X chromosome-targeted bioinformatic analysis for all symptomatic hemophilia carriers with skewed XCI. A hemophilia carrier diagnosed with an additional X-linked deleterious mutation should receive comprehensive genetic counseling with the aim of appropriately informing and guiding her reproductive decisions.


Gene editing tools such as CRISPR/Cas9 are often thought of as a means to prevent gene expression; however, a more subtle and yet powerful approach is the enhancement of gene expression by precise deletion of repressor sites within a gene of interest. Recent reports demonstrate that editing sequences required for post-transcriptional regulation can result in transcript that is more stable or more translated, with consequent trait improvement in crop plants. Within this approach, gene editing of miRNA target sites is remarkably promising because of the typical conservation of miRNAs and their targets, and thus the likely ease with which the technique might be transferred from model to crop species. Here, we argue that gene editing of miRNA target sites is preferable over deletion of the miRNA genes themselves, given that it avoids the pitfalls of unspecific derepression of miRNA target(s) beyond the gene of interest.Agricultural biotechnology is undergoing a revolution with the development of CRISPR/Cas gene-editing tools  complex that can specifically recognize and cleave a target site within the genome. Such an RNP complex is formed with an engineered non-coding RNA  and a Cas protein  has been done in tomato ; however, the advantages of using more subtle approaches are becoming apparent. Gene knockout using CRISPR/Cas9 has enabled crop trait improvement such as increased yield, improved quality and stress resistance, and acceleration of breeding (hybrid breeding) (LsGGP2 uORF using CRISPR/Cas9 in lettuce (Lactuca sativa) (LsGGP2). Sweeter strawberries (Fragaria vesca) have also been produced by enhancement of translation via uORF gene editing  and proteins  (Oryza sativa) improvement (GROWTH REGULATING FACTOR 4 (OsGRF4) and OsGRF8 coding sequences leads to enhanced transcript stability with consequent trait improvement (SQUAMOSA promoter-binding protein-like 13 (TaSPL13) in wheat has been edited using CRISPR/Cas9, producing different indels and resulting in a 2-fold increase in gene expression (SPL family, because the target sequence for miR156 is found in the coding sequence in all transcripts except for that of TaSPL13. As the CRISPR/Cas technology matures, gene editing will become more precise and predictable, for example with tools such as APOBEC-Cas9 (In plants, the stability and translation of several transcripts are tightly regulated by specific miRNAs that display sequence complementarity to binding sites within target transcripts. miRNA binding sites and the miRNAs themselves are usually conserved, in contrast to other inhibitory elements such as uORFs. miRNAs have also been targets of gene editing, and several reports have shown that CRISPR/Cas can be used to knockout specific miRNAs (recently reviewed by rovement , with larovement . Indeed,rovement . In caseIt is increasingly apparent that the path from gene to protein to function is far from orthogonal, given the myriad of regulatory mechanisms that modulates the abundance and activity of virtually all molecules found in a living organism. During transcription, modulations of processes that define the mature transcript sequence, such as transcription start-site usage, the polyadenylation signal, and alternative splicing, are commonly associated with the presence of regulatory elements, without necessarily altering the encoded amino acid sequence. For instance, differential transcript start-site usage modulates the presence of uORFs in response to light in the model plant Arabidopsis by producing transcripts with a shorter 5ʹ-UTR lacking inhibitory uORFs, thus exhibiting enhanced translation (Box 1.Most plant miRNAs are specific to a limited number of target transcripts, usually defined by perfect base pairing between the miRNA seed region and the target transcript . HoweverPlant miRNAs guide transcript cleavage between their nucleotides 10 and 11. Therefore, a mismatch in the cleavage site abolishes transcript cleavage, which is usually readily quantified by RNA sequencing and quantitative PCR. However, a cleavage-impaired miRNA–argonaute complex might still drive inhibition of translation , and bec


Epithelial-mesenchymal transition (EMT) is a cellular reprogramming process that converts epithelial cells into mesenchymal-like cells with migratory and invasive capabilities. The initiation and regulation of EMT is closely linked to a range of transcription factors, cell adhesion molecules and signaling pathways, which play a key role in cancer metastasis and drug resistance. The regulation of ferroptosis is intricately linked to various cell death pathways, intracellular iron homeostasis, and the protein network governing iron supply and storage. The ability of ferroptosis to disrupt cancer cells and overcome drug resistance lies in its control of intracellular iron ion levels. EMT process can promote the accumulation of iron ions, providing conditions for ferroptosis. Conversely, ferroptosis may impact the regulatory network of EMT by modulating transcription factors, signaling pathways, and cell adhesion molecules. Thus, ferroptosis related genes and signaling pathways and oxidative homeostasis play important roles in the regulation of EMT. In this paper, we review the role of ferroptosis related genes and their signaling pathways in regulating cancer EMT to better understand the crosstalk mechanism between ferroptosis and EMT, aiming to provide better therapeutic strategies for eradicating cancer cells and overcoming drug resistance. EMT (EMT) is a major cause of cancer metastasis and characterized by proliferation, division, invasion and migration. Therefore, there is a need for an effective EMT treatment strategy, which can be broadly classified into surgical and nonsurgical approaches . Most coIn 2012, a novel programmed cell death form distinct from necrosis and apoptosis, called ferroptosis, was discovered . FerroptIn the course of EMT, epithelial cells lose their apical-basal polarity and cell-cell adhesion, and acquire spindle-shaped mesenchymal shapes to gain the ability to migrate and invade, which are closely related to the invasion, migration and metastasis of cancer . During Ferroptosis is closely related to EMT, which are important biological processes associated with the growth of cancer. The latest research indicates that drug-resistant cancer cells that have Patients undergoing EMT are more susceptible to ferroptosis, indicating that they are more prone to being killed by ferroptosis inducers compared to non-drug-resistant cancer cells . It alsoUnderstanding the interaction of EMT with ferroptosis is critical to overcoming the challenges of cancer therapy . It is i22.1Recently, it has been demonstrated that high mesenchymal status is closely related to GPX signaling. GPX4 is involved in the induction of ferroptosis in cancer cells. The EMT-mediated transcription factor ZEB1 regulates and induces EMT via TGF-β/Smad, Furthermore, it can promote ferroptosis through direct inhibition of the activity of GPX4 and activation of the PPARγ (PPARγ) pathway . TGF-β cIt has been shown that targeting GPX4 or inducing ferroptosis may be an effective way to overcome drug resistance. Targeted regulation of GPX4 is promising as a novel approach to cancer therapy. The further study of the role of GPX4 in the development and therapy of cancer will contribute to a better understanding and application of this strategy.The CD44-dependent increase in iron endocytosis promotes iron-dependent demethylase activity, which promotes the expression of genes associated with EMT signaling, thereby sensitising cancer cells to iron transfer . Data fr2.2Nrf2 is a transcription factor with a basic leucine zipper domain that partially supports tumor cell proliferation by activating the oxidative stress response . rf2 regNrf2 binds directly to the anti-oxidation reaction element region of the system Xc− Subunit promoter, and promotes the expression of the system Xc− . InhibitNrf2 plays a key role in the regulation of EMT. Studying Nrf2, as well as its downstream targets, will help to better understand the mechanisms involved and guide the development of new therapeutic strategies. Deep study of Nrf2’s function and regulation network will be helpful to enhance the efficacy of cancer therapy, particularly in overcoming therapeutic resistance and inhibiting metastasis.2.3BACH1 is a hemoglobin-binding transcription factor, which plays an important role in the regulation of oxidative stress, hemoglobin, and iron metabolism . BACH1 rBACH1 can also inhibit Nrf2 signaling pathway by binding to Nrf2. Deletion of BACH1 increased expression of Nrf2-regulated genes, especially HO-1 . BACH1 pBACH1 plays an important role in the regulation of EMT. Further research on the function and regulation of BACH1 will help us better understand the interplay between EMT and ferroptosis and provide guidance for the development of new therapeutic strategies. At the same time, the BACH1/Nrf2 regulatory networks are being studied, and their potential applications in cancer therapy are discussed.3ROS are highly reactive molecules that are important regulators of signaling pathways. They are tumorigenic in nature and act as secondary messengers in the cellular signaling cascade to induce and maintain the oncogenic phenotype of cells as well as sustain cancer development by inducing DNA damage and promoting the proliferation, survival, and migration of tumor cells . ROS actROS plays an important role in the development of cancer and may affect EMT and ferroptosis. As iron chelators, dithiocarbamate derivatives, such as DpdtbA, can interfere with the generation of ROS induced by ferritinophagy and may inhibit EMT. Understanding this mechanism will be helpful in the development of cancer metastasis and drug resistance, and in the development of therapeutic strategies. The exploration of ROS, EMT, and ferroptosis signaling pathways will provide a better understanding and strategy for cancer therapy.3.1P53 is a tumor suppressor protein and a key and complex ROS regulatory factor in cancer development. It has both antioxidant and pro-oxidant functions . It has P53 plays an important role as a regulator of ROS in the development of cancer. It influences the proliferation, survival and EMT of cancer cells through induction of ROS and regulation of PI3K/AKT/mTOR. Deep Research on the mechanism of P53 in regulating ROS, ferroptosis, and EMT will help to better understand the molecular mechanisms underlying cancer progression and provide new insights into the development of therapeutic strategies that target these pathways.3.2Nrf2 is an important factor in the regulation of ROS. When ROS levels increase, the degradation of Nrf2 is disrupted by Keap1, allowing Nrf2 to stabilize and enter the cell nucleus to activate a protective antioxidant response . By induDeep research on the role of Nrf2 in the regulation of ROS, ferroptosis, and EMT may help to understand the complexity of cancer development and therapy. As one of the regulatory mechanisms produced by ROS, iron sequestration plays an important role not only in the induction of ferroptosis, but also in the regulation of EMT and activation of Keap1/Nrf2/HO-1 pathway through the influence of ROS. Therefore, further research on the mechanism of Keap1/Nrf2/HO-1 signaling pathway will contribute to the development of new therapeutic strategies, especially in the treatment of cancer.3.3The Wnt/β-catenin signaling pathway plays an important role in the development and progression of cancer by regulating cell proliferation and apoptosis. When Wnt ligands (such as Wnt3a and Wnt1) are secreted, they bind to Frizzled receptors and low-density lipoprotein receptors, thereby blocking the pathway of β-catenin degradation and activating downstream gene transcription Cyclin D1, and WISP . These g3.4Autophagy is a cellular self-digestive process that maintains cellular homeostasis and function by degrading and recycling harmful or ageing substances within the cell. Recent studies have found that autophagy is closely related not only to cell survival and death, but also to some important biological processes such as ferroptosis and EMT .ROS-induced autophagy is a process that gives expression to key regulators of ferritin degradation and transferrin receptor 1 during ferroptosis, which has been suggested by recent studies to be a process of autophagic cell death that can be termed autophagy-dependent ferroptosis , 60. FirSecondly, autophagy can also influence the onset of ferroptosis by regulating the clearance of ROS. It was found that autophagy can remove intracellular ROS and reduce oxidative stress in cells . AutophaIn addition, autophagy is also closely related to the occurrence of EMT Figure 1Taken together, ROS is involved in various response pathways affecting tumorigenesis, and a comprehensive understanding of the regulation of different cellular responses can facilitate the clinical application of ROS or ROS-regulated pathways to formulate and improve the shortcomings of current cancer therapies.Therefore, the above ferroptosis related genes and their signaling pathways can be regulated to modulate EMT occurrence in cancer cells Figure 24Significant progress has been made in the field of tumor treatment in recent years, including the increasing use of targeted drugs and immunotherapy. However, the problem of tumor drug resistance remains challenging. Therefore, reversing or inhibiting EMT has become a potential cancer treatment strategy to suppress the migration and metastasis of tumor cells to distant sites. Resistant cancer cells, upon transition to a mesenchymal state, become resistant to apoptosis induced by conventional therapies but are highly sensitive to ferroptosis inducers due to changes in their metabolic state. By inhibiting or activating ferroptosis related genes and their signaling pathways, the regulation of EMT by ferroptosis can be achieved, thereby improving the prognosis and treatment outcomes of cancer and providing new strategies to address clinically challenging cancers.Further research on the interaction mechanism between ferroptosis and EMT not only helps us gain a deeper understanding of the mechanisms of tumor metastasis and recurrence but also provides new strategies for tumor treatment. Exploring and confirming the deeper relationship between these two processes can help us better grasp the diversity and complexity of tumor development, thus guiding clinical practice and the development of treatment strategies. Ferroptosis and EMT have significant implications in cancer treatment. Reversing or inhibiting EMT and modulating genes and signaling pathways related to ferroptosis can bring about breakthroughs in tumor treatment. Further research will help uncover the interplay mechanisms between these two processes and provide a theoretical basis for the development of more effective cancer treatment strategies.WM: Writing – original draft. ZZ: Funding acquisition, Resources, Writing – review & editing. LS: Writing – review & editing. QW: Writing – review & editing. WF: Writing – review & editing. YT: Writing – review & editing. YH: Writing – review & editing. YW: Writing – review & editing.


Ester et al report the findings from a 2-arm cluster randomized controlled trial nested within a hybrid effectiveness-implementation study, which involved a 12-week exercise and behavior change program for rural and remote Canadians (Exercise for Cancer to Enhance Living Well [EXCEL]). The addition of 23 weeks of app-based physical activity monitoring to the EXCEL program did not result in significant between-group differences in physical activity at 6 months. While several behavior change techniques were included in the initial 12-week intervention, additional techniques were embedded within the mobile app. However, there is currently a lack of evidence regarding how many and which behavior change techniques are the most effective for people with cancer and if these differ based on individual characteristics. Potentially, the use of the mobile app was not required in addition to the behavior change support delivered to both groups as part of the EXCEL program. Further research should involve participants who may be in most need of behavioral support, for example, those with lower levels of self-efficacy. Suggestions for future research to tailor behavior change support for people with cancer are discussed. Ester et al  recentlyIn the initial 12-week component, both groups received the EXCEL “exercise and educate” program, which involved 5 educational topics delivered on the web and targeted the following behavior change techniques (BCTs): instruction on how to perform the behavior, goal setting, action planning, information about health consequences, and social support , and as Over three-quarters (32/42) of participants who withdrew from the EXCEL trial within the first 12 weeks were in the intervention group; however, no withdrawals occurred between 12 and 24 weeks. This raises the question of whether the participants’ burden of using the mobile app along with participating in the EXCEL program was too high or whether participants simply felt it was not required. Findings from participant interviews will provide valuable insights into their app usage experiences and preferences. The app included several features that facilitate mobile health app use in the posttreatment setting, including having a cointervention alongside the app , easy navigation, being a single app housing all required information, visual graphs, and information on energy levels. Barriers to uptake associated with technical problems were addressed by troubleshooting support provided by EXCEL staff. Additional factors that may have improved uptake include app integration with PA trackers and the inclusion of relevant educational videos . Time siIn their conclusion, Ester et al  highligh


Extensive research has been conducted to gain a deeper understanding of the deregulated metabolic pathways in the development of trisomy 21 (T21) or Down syndrome. This research has shed light on the hypothesis that oxidative stress plays a significant role in the manifestation of the T21 phenotype. Although in vivo studies have shown promising results in mitigating the detrimental effects of oxidative stress, there is currently a lack of introduced antioxidant treatment options targeting cognitive impairments associated with T21. To address this gap, a comprehensive literature review was conducted to provide an updated overview of the involvement of oxidative stress in T21. The review aimed to summarize the insights into the pathogenesis of the Down syndrome phenotype and present the findings of recent innovative research that focuses on improving cognitive function in T21 through various antioxidant interventions. By examining the existing literature, this research seeks to provide a holistic understanding of the role oxidative stress plays in the development of T21 and to explore novel approaches that target multiple aspects of antioxidant intervention to improve cognitive function in individuals with Down syndrome.The guides -base systematic review process (Hutton et al.  Trisomy 21 (T21), also known as Down syndrome (DS), is the most prevalent genetic disorder affecting fetuses  and the Enhancing the QUAlity and Transparency Of health Research (EQUATOR) network  encompass various dietary phytochemicals such as polyphenols, quinones, flavonoids, catechins, coumarins, and terpenoids, which are considered crucial exogenous AOX.Previous research has already demonstrated the ability of AOX administration to impede the advancement of neurodegenerative diseases. However, the precise mechanism of action of AOX has yet to be fully elucidated. Ongoing investigations are aimed at further understanding the specific ways in which AOX exert their protective effects in DS and other related conditions , levels of total and oxidized glutathione (GSH), concentrations of heat shock protein (HSP) and thioredoxin (TRX), in amniotic fluid samples. The investigation utilized techniques such as slot-blot analysis, enzymatic assays, and Western blot. The study findings demonstrated that oxidative stress occurs early in pregnancies affected by trisomy 21 (T21), as evidenced by increased protein oxidation, lipid peroxidation, reduced levels of GSH and TRX, and the induction of the HSP response. These processes have been implicated in the DS phenotype, providing insights into the role of oxidative stress in T21 pathogenesis . The study revealed that the group of children with T21, consisting of 20 participants, exhibited higher levels of protein carbonyls in comparison to the healthy control group, which consisted of 18 individuals . Conversely, the study found that the antioxidant capacity was comparable between the two groups. The primary focus of this investigation was to assess the hypothesis that individuals with T21 exhibit an elevated oxidative stress status . Likewise, Kamatsu et al. conducted an assessment of the concentration of 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker, in saliva samples obtained from individuals with T21. The findings of the study confirmed the presence of oxidative stress in T21 patients based on the elevated levels of the oxidative stress marker  , lipid metabolism , and inflammation  . Pharmacological investigations have been conducted utilizing the Ts65Dn mouse model of DS. One study involved administering α-tocopherol to pregnant Ts65Dn female mice starting from the day of conception throughout pregnancy, which resulted in a reduction in cognitive impairment. Furthermore, young mice were supplemented with α-tocopherol from birth until the completion of the behavioral testing period. The findings of this study indicated that α-tocopherol supplementation led to a mitigated cognitive impairment, accompanied by decreased lipid peroxidation levels when compared to the control group  during maternal inheritance and cell division. By delving into these aspects, researchers can shed light on the underlying mechanisms contributing to mitochondrial dysfunction in T21 and potentially identify targets for therapeutic interventions  gene resulted in a reduction in congenital impairments. The research aimed to inhibit the ISR-inducing double-stranded RNA-activated protein kinase by enhancing the function of the eukaryotic translation initiation factor 2-eukaryotic translation initiation factor 2B complex (eIF2-eIF2B). This intervention facilitated proper translation and synaptic transmission, leading to increased synaptic activity. By silencing the ISR, significant improvements were observed in the formation of long-term memories in mice with DS  therapy was evaluated using rat middle cerebral artery occlusion. The results demonstrated neuroprotective effects and improvements in neuronal function  concentrations. This suggests that α-tocopherol supplementation in the diets of children with DS may alleviate oxidative stress at the DNA level  were enrolled and supplemented with EGCG  or placebo over a 3-month period. The intervention with EGCG showed improvements in visual recognition memory, working memory performance, psychomotor speed, and social functioning in individuals with T21(De la Torre et al. DYRK1A expression have been not proposed or recommended (Fayçal Guedj, Bianchi, and Delabar 2014; Faycal Guedj et al. DYRK1A expression. Therefore, EGCG acts as a mimic of multiple kinase inhibitors simultaneously (Bain et al. Despite the promising results obtained during in vivo studies, further DS treatment protocols aiming to inhibit the Neuropharmaceutics represents the most promising avenue for addressing cognitive impairments among DS patients, making it the largest potential growth sector in the pharmaceutical industry (Pardridge Studies investigating the impact of oxidative stress on the occurrence of congenital fetal impairments in T21 patients have revealed a complex interplay of multiple metabolic pathways. Despite extensive research, it has not been possible to determine the potential of antioxidant supplementation in reducing cognitive impairment in T21 individuals. However, the integration of various oxidative stress-related pathways holds promise for the discovery of novel therapies in the future. In the coming decades, the challenges in the field of T21 will involve not only identifying effective therapies to mitigate cognitive defects but also understanding the molecular and cellular mechanisms underlying the characteristic phenotype resulting from genetic triplication. Exploring multitargeted antioxidant interventions in DS individuals will provide insights into the molecular and cellular mechanisms governing their nervous system function, leading to the discovery of potential therapies to overcome cognitive impairment. Among the clinical trials conducted to date, the inhibition of DYRK1A combined with cognitive training has shown significant improvements in visual recognition memory, inhibitory control, and adaptive behavior in adults with T21. Moreover, novel techniques such as intranasal delivery have been evaluated to improve cognitive impairments in DS individuals, with promising results. However, further studies in this field are still required.


Results: Neonatal rats with CP exhibited sensorimotor impairment and decreased spatial exploration. CP rats spent significantly less time rearing  and engaged in multi-dimensional nose sweeping and sniffing  than sham controls. Conclusions: These pilot findings of harmonized translational and precision biobehavioral assays provide an opportunity for increased expediency of clinical trials at the earliest stages of brain development.Background: Cutting-edge neonatal programs diagnose cerebral palsy (CP) or “high risk of CP” using validated neurobehavioral exams in combination with risk history and neuroimaging. In rat models, digital gait analyses are the gold standard adult assessment, but tools in infant rats are limited. Refinement of infant rat neurobehavioral correlates of CP will establish translational behavioral biomarkers to delineate early mechanisms of CP in both humans and rodent models of CP. Objective: To facilitate precision medicine approaches of neurodevelopmental health and integrate basic and clinical research approaches for CP, we developed and piloted a new assay of neonatal rat neurobehavior to mimic human neonate exams. Methods: Our established rat model of CP secondary to chorioamnionitis (CHORIO) that induces bilateral motor impairment reminiscent of spastic CP was used. On postnatal day 10 (P10), 5 min videos were recorded of 26 (6 sham and 20 CHORIO) animals moving freely in a cage were reviewed by an evaluator trained in the human General Movements Assessment (GMA). Non-blinded observation revealed two behaviors that differed between rat pups in each group . Each video was re-coded for these criteria by an evaluator blind to group status. Differences between sham and CP groups were analyzed using a Mann–Whitney U-test or Student’s  Cutting-edge neonatal neurodevelopmental follow-up programs utilize empirically validated tools to support early detection of cerebral palsy (CP) ,2,3,4,5.Human infant writhing movements occur from 10 weeks of fetal life to 10 weeks post-term age equivalent ,8,9. NorHuman infant fidgety movements occur between ages 3 and 5 months (corrected degree for prematurity if applicable) . These mThe combination of cramped synchronized motor movements in the writhing stage and then later absent fidgety movements is highly predictive of a diagnosis of cerebral palsy in human infants . This isPresently, the gold standard approach to identifying motor deficits akin to CP in adult rats  utilizes computer digital gait analysis ,12,13,14Lack of harmonization between human clinical methods and animal methods limits the translation necessary for clinical trials of promising pharmacological interventions for neurologic injury and CP, and precise translational biobehavioral assays capturing healthy and abnormal state-trait domains are urgently needed. Therefore, the goal of the current study was to facilitate development of a translational methodology that could be readily utilized to detect neurologic injury leading to CP versus neurological health in rats during ages that are equivalent to humans in the neonatal and late infancy period. Specifically, we sought to develop and pilot a new biobehavioral assay for a rat model that mirrored the human infant General Movement Assessment.All experiments were performed in strict accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Johns Hopkins University School of Medicine . Protocols were developed and performed consistent with National Research Council and ARRIVE guidelines . SpragueTo induce a CP-like injury in rats, pregnant Sprague Dawley rats underwent abdominal laparotomy on embryonic day 18 (E18) to induce CHORIO as previously reported ,23,24,25In preparation for developing a behavioral coding scheme of rat motor behaviors, literature was reviewed about typical motor and sensorimotor development in rats . On postt-test (p < 0.05 level of significance).Differences between 6 sham  and 20 CHORIO/CP  were analyzed using a Mann–Whitney U-test or Student’s U = 5.500, p = 0.03  compared to the sham controls (5.5 ± 0.96 episodes), shown in .t (10) = 8.104, p = 0.007 , rats with CP also exhibited sensorimotor impairment and decreased spatial exploration. Specifically, there was a statistically significant difference in the amount of time CP rats spent rearing (17.85 ± 1.60 s) compared to sham controls (34.8 ± 2.89 s), shown in .Prechtl’s General Movement Assessment in human infants examines motor movements that are spontaneously generated motor movements involving the whole body that present in a variable sequence and evolve over the first 5 months of the human infant’s life . These mThe present pilot study provides some data that there are rat age specific movements of neurodevelopmental health  that correlate to equivalent human infant motor movements assessed on the General Movements Assessment over the first 5 months of life. Decreased frequency or absence of specific sensorimotor behaviors in rats appear to predict the risk of manifesting CP-like symptoms in rats similar to how specific sensorimotor behaviors in human infants predict the risk of CP. Specifically, the frequency of multi-dimensional nose sweeping and sniffing in neonatal rats are predictive of neurologic injury and CP-like symptoms by adulthood in rats, similar to the writhing movements observed in human neonates and characterized by the General Movement Assessment. Additionally, much like the absence of normal fidgety movements on General Movement Assessment are further predictive of CP risk in human infants within the first 3–5 months of life, reduced amounts of engagement in early rearing behaviors in rats at P17 is also predictive of CP-like symptoms.The rat model of CP utilized in this study has previously provided a great deal of information about the mechanisms of injury underlying the behavioral manifestations of CP by adulthood, including motor and cognitive deficits ,15,16,19Presently, clinical practice guidelines for diagnosis of CP have shifted, placing an important emphasis on the early detection of CP in humans . The Beyond the development of translational motor assessments, the use of analogous measures of cognition is also critical, as similar cognitive-behavioral deficits have also been appreciated in humans, with brain injuries leading to CP in humans and CP-like motor impairments in rats. These cognitive-behavioral deficits include fewer social interactions compared to shams and deficits in visual discrimination and reversal learning on touchscreen operant chamber testing. Such cognitive-behavioral findings are in line with deficits in selective visual attention, and executive dysfunction, including problems with behavioral initiation and inhibition, as well as cognitive flexibility that are frequently observed among humans diagnosed with CP ,19. As sIn the future, additional examination of inter-rater reliability and external validity is warranted. It is critical that following training, there is a high level of consistency between evaluators for the coded behaviors. Further examination of these methods across multiple samples is also necessary to assure the results are generalizable across future studies and clinical trials.Beyond the development of translational motor assessments, the use of analogous measures of cognition is also critical, as similar cognitive-behavioral deficits have also been appreciated in humans with brain injuries leading to CP in humans and CP-like motor impairments in rats. These cognitive-behavioral deficits include fewer social interactions compared to shams and deficits in visual discrimination and reversal learning on touchscreen operant chamber testing. Such cognitive-behavioral findings are in line with deficits in selective visual attention, and executive dysfunction, including problems with behavioral initiation and inhibition, as well as cognitive flexibility that are frequently observed among humans diagnosed with CP ,19. As sLimitations to the current study included the small sample size of our control group, and the reliability and validity of these methods were not fully explored. Concerning reliability and validity, the specific animal behaviors were only coded by one blinded evaluator, limiting the information regarding inter-rater reliability. Additionally, these methods were not examined across multiple samples, which currently limits the external validity.Harmonization of clinical and preclinical methodologies is critical for facilitation of translational work, particularly in the area of perinatal brain injury as the field moves towards implementation of clinical trials. The gold standard measurement of CP-similar motor behaviors in adult rats has been successful using Digigait; however, with the push for early diagnosis of CP, diagnosis routinely occurs before 1 year of age and often as early as infancy. The lack of early diagnostic tools in rats that mirror the tools used in clinical practice and the incongruity in animal and human outcome models makes it much more challenging to develop translational studies and clinical trials that specifically target and evaluate therapeutic interventions within the neonatal period. In a search to identify early models for better translational work, we identified two behavioral biomarkers that differentiated brain injured rats from shams at early ages. Nose sweeping and sniffing at P10 (early infancy) and time spent rearing at P17 (late infancy) both identified rats who were at a high risk of CP in adulthood, similar to Prechtl’s General Movement\Assessment in terms of infants and at a 3-month corrected age. Our pilot findings of harmonized translational and precision biobehavioral assays provides an opportunity for increased expediency of clinical trials at the earliest stages of brain development, when there is an opportunity to mitigate injury that may result in a later diagnosis of CP.


Breast cancer is the most common cancer affecting women worldwide. Investigating metabolism in breast cancer may accelerate the exploitation of new therapeutic options for immunotherapies. Metabolic reprogramming can confer breast cancer cells (BCCs) with a survival advantage in the tumor microenvironment (TME) and metabolic alterations in breast cancer, and the corresponding metabolic byproducts can affect the function of tumor-associated macrophages (TAMs). Additionally, TAMs undergo metabolic reprogramming in response to signals present in the TME, which can affect their function and breast cancer progression. Here, we review the metabolic crosstalk between BCCs and TAMs in terms of glucose, lipids, amino acids, iron, and adenosine metabolism. Summaries of inhibitors that target metabolism-related processes in BCCs or TAMs within breast cancer have also served as valuable inspiration for novel therapeutic approaches in the fight against this disease. This review provides new perspectives on targeted anticancer therapies for breast cancer that combine immunity with metabolism. According to the latest statistics, breast cancer remains the most frequently diagnosed cancer worldwide despite advances in treatment, and women aged 20-59 years with advanced stages of the disease still face a significant risk of mortality. Approximately 287,850 new cases of invasive breast cancer and 51,400 new cases of ductal carcinoma in situ among U.S. women were reported in 2022, and the incidence of female breast cancer continues to increase at a rate of approximately 0.5% per year Breast cancer is highly heterogeneous, and the tumor microenvironment (TME) actively contributes to this heterogeneity. Moreover, the TME serves as a key player in the multi-process steps of breast cancer malignant progression; therefore, the introduction of immunotherapy to target the TME has a very high potential to personalize the treatment of breast cancer patients and improve prognosisCancer metabolic reprogramming serves as a bridge between intracellular stress and cancer behavior, which is a typical sign of the malignant progression of cancer. It confers a growth advantage to tumor cells and influences immune cell differentiation and function, thereby promoting cancer progressionThis paper reviews the importance of the metabolic dialogue between BCCs and TAMs in the biological progression of breast cancer. The first part focuses on describing the dialogue between breast cancer cells undergoing metabolic reprogramming and TAMs, emphasizing that breast cancer cells undergoing metabolic reprogramming affect the polarization of TAMs and, thus, cancer progression during dialogue with TAMs. We then summarized the dialogue between TAMs undergoing metabolic reprogramming and BCCs. The second section provides an overview of the current metabolic drugs that target BCCs and TAMs. A summary of these drugs is presented in Table Tumor cells appropriately regulate their metabolism to maintain a high proliferation rate and adapt to survive in unfavorable microenvironments. In this regard, the crosstalk between BCCs undergoing metabolic reprogramming and TAMs is closely related to breast cancer progression.Cancer cells drive carcinoma progression via distinct metabolic pathways. Aerobic glycolysis is closely associated with breast cancer progression. The state of "aerobic glycolysis" or the Warburg effect is typical of glucose metabolic reprogramming in breast cancerThe immunosuppressive TME blocks immunotherapy for cancer, and metabolic modulation of the TME is a promising strategy to improve immunotherapyGenerally, cancer cells have higher levels of glycolytic enzymes and glycolytic transporter proteins than normal cells do, which correspondingly produce high lactate levels and promote malignant progression The metabolic reprogramming of tumor cells can also be modulated by immune cells in the TME. An increasing number of studies have indicated that TAMs in the TME can affect the aerobic glycolysis of tumor cells in different ways, thereby affecting the occurrence and development of breast cancer. Recent studies have shown that myeloid lncRNA HISLA is encapsulated in extracellular vesicles (EVs) secreted by TAMs, preventing the interaction between PHD2 and HIF-1 α and inhibiting the hydroxylation and degradation of HIF-1 α, thereby enhancing the aerobic glycolysis of BCCs. Moreover, lactate secreted by tumor cell glycolysis has an upregulatory effect on HISLA in macrophages, which can form a positive feedback loop between tumor cells and TAMs. Therefore, glycolysis and chemotherapy resistance in breast cancer can be effectively inhibited by blocking HISIA expression in TAMsLipids are a group of substances closely related to metabolic diseases. These molecules include phospholipids, triglycerides, cholesterol, cholesteryl esters, and sphingolipidsEnzymes or related products of tumor cell lipid metabolism can polarize macrophages toward the M2 type and promote the development of breast cancer. Analysis of these data suggests that fatty acid (FA) biosynthesis is the most important process in the early stages of breast cancer is the process of fatty acid (FA) biosynthesis metabolismProstaglandin E2(PEG2) is a metabolite of arachidonic acid produced by COX2, and numerous studies have shown that PGE2 derived from tumor cells can polarize TAMs toward the M2 type Increasing evidence suggests that amino acid metabolism is active in cancer cell growth, signaling, oxidation, and immunosuppression in the TME. Among these, 15 amino acids are significantly elevated in breast cancer samples compared to normal samples and can be used as markers for the early diagnosis of breast cancerGlutamine is the most abundant amino acid in the blood, and cancer cells preferentially take up glutamine compared to immune cells within the TMEIron metabolism disorders are closely associated with cancer, and macrophages play a crucial role in iron metabolismTAMs are essential components of TME. To survive in harsh tumor environments, TAMs must also undergo metabolic adaptations. These metabolic adaptations cause changes in one's functional phenotype, affecting both the "promoting" and "inhibiting" ends of the breast cancer spectrum. In addition to TAMs undergoing metabolic adaptation, some products produced by BCCs can affect TAMs' metabolism and cancer progression.TAMs can influence cancer progression by reprogramming lipid metabolism, which also involves FAs, arachidonic acid (AA), and cholesterol (CHOL) pathways. In contrast to tumor cells, the lipid metabolism of TAMs can lead to both pro- and anti-tumor effects. Lipid metabolic reprogramming of macrophages by stimuli, products, or secreted factors is an important feature of TAMs that can affect the regulation of TAMs in the TME and thus influence cancer progressionStudies have shown that lipid accumulation and the pro-tumor function of TAMs are closely linked. It has been reported that caspase-1 can cleave peroxisome proliferators activate receptor γ (PPARγ) at aspartate 64. PPAR γ can translocate to the mitochondria and interact with medium-chain acyl-CoA dehydrogenase (MCAD). This attenuates MCAD activity and inhibits fatty acid oxidation, leading to lipid droplet accumulation in TAMs and promoting their differentiation into a pro-tumor phenotypeInterestingly, there is evidence that BCCs in TME have a "contradiction" with TAMs in lipid metabolism, and they have opposite lipid metabolism reprogramming during tumor progression. Leukotrienes are a group of pro-inflammatory lipid mediators derived from AA, and 5-lipoxygenase (5-LO) is a key enzyme in leukotriene production. In TAMs, 5-LO is downregulated through MerTK (a receptor tyrosine kinase) after the recognition of apoptotic cancer cells, and activation of its transcriptional repression through c-Myb.5-LO expression deficiency leads to a reduced ability of TAMs to recruit T cells and exert anti-tumor effects. Therefore, the inhibition of MerTK in the TME may enhance the antitumor immune response in treating breast cancer. However, in BCCs, enhanced expression of 5-LO and its products promotes cell proliferation and inhibits apoptosis in cancer cellsCombined with previous studies, we found that disrupting the lipid-mediated crosstalk between mesenchymal and tumor cells or TAMs by targeting enzymes, receptors, or bioactive lipids inhibits the pro-tumor function of TAMs in lipid metabolism, induces tumor regression, and inhibits cancer metastasis is a very promising strategy. The implementation of lipid metabolism interventions in different cells can result in different effects. Therefore, there is an urgent need to explore new approaches to more precisely target the metabolic pathways between TAMs and tumor cells or other immune cells in the microenvironment. The purposeful development of relevant targeted inhibitors and drugs will lead to a better understanding of the role of metabolic reprogramming in tumor therapy.Studies have shown that altered glucose metabolism patterns in TAMs can lead to immunosuppressive functions and ultimately promote tumor growth and metastasis Figure a79. The M2-type macrophages have a higher level of CARKL expression and decreased PPP, subsequently restricting the glycolytic processTAMs can also adjust their intracellular metabolism to adapt to appropriate polarization according to the availability of oxygen and different parts of the malignant tissues. Studies have shown that TAMs in tumor-anoxic areas can produce phenotypes that promote angiogenesis and invasionIn addition, tumor-derived products or related signals can affect glycolysis in macrophages, thus promoting cancer. Previous studies have shown that tumor cells can release cytokines, such as CSF1, IL-34, and VEGFA, which can downregulate the glycolysis level of TAMs and induce polarization to M2In summary, TAMs can dynamically adjust their metabolic patterns according to different signals or interactions and maintain the corresponding polarization phenotype according to different metabolic patterns. Metabolic flux and metabolic intervention in TAMs may further improve tumor immunotherapy; however, selective targeting of TAMs metabolism in vivo remains a continuous challenge.The INOS/ARG axis is a regulatory center of macrophage immunometabolism. Arginine metabolism is important for crosstalk between TAMs and BCCs. Tumor cells prefer to shift from the NO synthesis pathway to the polyamine synthesis pathway during arginine metabolism to satisfy their growth and proliferation requirements Glutamine is an important energy source for macrophages and is essential for their physiological functionsTumor cells must express high levels of ferritin to meet their iron requirements. Ferritin is generally expressed in the tumor stroma, and macrophages are a major component of the tumor stroma. Different macrophage phenotypes play different functional roles in iron release. M1-type macrophages isolate iron under inflammatory conditions, whereas M2-type macrophages prefer iron release. Tumor phenotypes that interfere with iron sequestration can inhibit breast cancer growth and metastasis to some extentThe release of iron from TAMs into the TME is an influential aspect of tumorigenesis. Hepcidin  was found to be a major regulator of iron metabolism, and its expression was associated with IL-6 signal transduction and transcriptional activator STAT3 signaling pathways, suggesting that hepcidin could be directly targeted using a neutralizing antibody approach to inhibit tumor growth and metastasis Adenosine is an important extracellular signaling molecule that accumulates in the TME. The adenosine pathway regulates tumor cell proliferation and apoptosis. Adenosine can activate apoptosis in tumor cells through different adenosine receptors in a caspase-dependent or caspase-independent manner. Stimulation of the A2B receptor (one of the subtypes of adenosine receptors) in tumor cells can cause a consequent change in the phenotype of immune cells via the adenosine-adenosine receptor systemIn addition to breast cancer, the general mechanism of tumor cell/TAM crosstalk via metabolic reprogramming has been frequently mentioned in other cancers. Metabolic crosstalk between tumor cells and TAMs largely determines tumor heterogeneity and the conditions that regulate antitumor immunityTherefore, targeting TAMs and cancer cell metabolism may have therapeutic implications. There is substantial evidence that changes in the TME can promote the resistance of some antitumor agents to conventional therapies Although many other potential mechanisms of metabolic remodeling in BCCs and TAMs in the microenvironment remain unclear, there is growing evidence that inhibition of relevant metabolic enzymes may be a reasonable approach to breast cancer treatment. It has been previously demonstrated that when patients with breast cancer are resistant to paclitaxel and trastuzumab, treatment with lactate dehydrogenase inhibition can significantly improve Previous studies have shown that some old drugs can achieve potent therapeutic effects by targeting breast cancer metabolism. Some targeted drugs do not act directly on BCCs, but rather by activating cancer-suppressing macrophages, thereby preventing the growth of cancer cells. Lipid homeostasis is closely linked to cancer. One of the mechanisms regulating cholesterol homeostasis in macrophages is the liver X-receptor (LXR)/adenosine triphosphate-binding cassette transporter A1 (ABCA1) axis. When the ATP-binding cassette transporter G1 (ABCG1) is deficient, macrophage cholesterol accumulation activates NF-kB, leading to M1-type polarization of macrophages and production of TNFα and NO for antitumor immune effectsWhile it is well known that M1-type macrophages can withstand a wide range of ROS levels. M2-type macrophages are more susceptible to the cellular redox status; therefore, targeting the redox sensitivity of macrophages to develop relevant strategies and drugs is a promising strategy for treating breast cancer. For example, Griess et al. found that the macrophage ROS regulator MnTE-2-PyP5+ inhibits the IL4-stimulated polarization of M2 macrophages by reducing STAT3 activation, thereby reducing angiogenesis and metastasis in breast cancerStudies have shown that epigenetic regulation by inhibiting class IIa histone deacetylases (HDACs) is a promising approach for exploiting the antitumor potential of macrophages. TMP195, a class IIa HDAC inhibitor that alters the transcriptional profile of macrophages, reduces macrophage-mediated tumor growth in preclinical breast cancer modelsThe metabolic reprogramming of tumor and immune cells in the TME is increasingly being recognized as a key pathway contributing to the complex dialogue between these cells. It has been found that the treatment of liver cancerMacrophages are highly plastic and heterogeneous. In breast cancer, metabolites produced by TAMs metabolism affect BCCs directly or indirectly by affecting their polarization, thereby impacting cancer progression. In turn, the metabolism can also affect the function and polarization of TAMs. It is fascinating that the metabolic reprogramming of these two cell types can sometimes form a feedback pathway. However, there are limitations and unanswered questions regarding precisely targeting tumor-associated macrophages and tumor cells to specific metabolic targets. First, BCCs and TAMs are metabolically heterogeneous and may show the same or opposite effects when treated with drugs specifically targeting lipid metabolism. Secondly, altered amino acid metabolism significantly affects both BCCs and TAMs. However, there are very few reports on amino acid metabolism in breast cancer; therefore, further research is urgently needed on the mutual dialogue between tumor cells and TAMs and the mechanism of action. Third, we found that when targeted metabolic drugs were used, some metabolic pathways were shared by normal cells. Ensuring the specificity of targeted TAMs or tumor cell metabolism is also worth considering when developing targeted metabolic drugs. Therefore, further understanding of the metabolic mechanisms between targeted immune cells and tumor cells may help us understand the hyper-progression of immunometabolic therapy for breast cancer.In addition, recent novel techniques have provided new ideas regarding the immunometabolism of TAMs for future cancer therapies. For instance, tumor-derived exosomes represent a potential mode of metabolic crosstalk between cancer cells and TAMs. The extent of the exosomal influence on TAMs polarization in cancer patients and the potential pathways for targeted therapy using exosomal transport prompted us to investigate blocking drugs corresponding to breast cancer


Zeolitic Imidazolate Frameworks (ZIF) find applicationin storageand dissipation of mechanical energy. Their distinctive propertieslinked to their (sub)nanometer size and hydrophobicity allow for waterintrusion only under high hydrostatic pressure. Here we focus on thepopular ZIF-8 material investigating the intrusion mechanism in itsnanoscale cages, which is the key to its rational exploitation intarget applications. In this work, we used a joint experimental/theoreticalapproach combining in operando synchrotron experiments during high-pressureintrusion experiments, molecular dynamics simulations, and stochasticmodels to reveal that water intrusion into ZIF-8 occurs by a cascadefilling of connected cages rather than a condensation process as previouslyassumed. The reported results allowed us to establish structure/functionrelations in this prototypical microporous material, representingan important step to devise design rules to synthesize porous media. Among the vast number of MOFs, only ahandful have been studied for this application,16 owing to their good chemical,thermal, and mechanical stability, and the relatively inexpensivechemicals and synthesis protocol for their production; they predominantlybelong to the hydrophobic microporous family of zeolitic imidazolateframeworks (ZIFs).17 Arguably, the mostextensively studied microporous material to date is ZIF-8, which hasbecome a reference for microporous materials in the area of waterintrusion–extrusion and related applications.20 Traditionally,research in the field focused on the empirical evaluation of energyabsorption performance of ZIF-8 and other microporous media. However,attention has recently turned toward the investigation of the microscopicmechanism of hydrophobic ZIF wetting.21 Deepening the understanding of hydrophobic wetting has been themain goal of several groups in the scientific community in recentyears, exploring the effects of confining water in hydrophobic nanopores,the interactions of water with different hydrophobic surfaces, andthe liquid and vapor interfaces in nanoconfinement under hydrostaticpressure.27 In a recent work, Sun et al.,28 proposedthe intrusion of ZIF-8 to be governed by the condensation of vaporpresent in the nanoscale cages, postulating that the kinetics of theintrusion process is determined by the intrinsic length (single nanometer)and time scales (nanoseconds) necessary for critical water clustersto nucleate inside individual cages.In this context, metal–organic frameworks (MOFs) have arisenas a promising class of materials to achieve breakthrough performancein energy absorption during mechanical impact.28 pavedthe way to such investigation. Indeed, the hysteresis, i.e., the differenceof thermodynamic conditions between intrusion and extrusion of a nanoporousmaterial, determines how much energy per intrusion-extrusion cycleis dissipated. Hysteresis, in turn, depends on how quickly the systemreaches the equilibrium state corresponding to the given thermodynamicconditions, whose microscopic determinants are investigated here.Understanding the key rate-limitingstep of intrusion is of paramountimportance to design improved materials for energy dissipation andthe key contribution of Sun et al.2O. We reveal that theintrusion of water in ZIF-8 proceeds by the cascade penetration ofwater in connected cages, forming coherent domains of ZIF-8 cageswith alike structural characteristics. This mechanism is fundamentallydifferent from the previously proposed condensation-driven intrusionin individual ZIF-8 cages. We show that the proposed cascade intrusionmechanism crucially depends on the hydrogen bonds which form acrossneighboring cages, which would instead be irrelevant in the condensationscenario. On a larger scale, water intrusion progresses by the formationand growth of coherent domains of wet cages, which progressively andcooperatively advance through the MOF crystallite. Our conclusionssupport new design criteria for MOFs for energy applications, hingingon pore connectivity rather than on individual cage properties.In this work, combining intrusion experiments, in operando synchrotronradiation, atomistic simulations, classical continuum theories, andstochastic models, we report some important insights into the multiscalemechanism of ZIF-8 MOF wetting by H29 during water intrusion at 30 °Cby in operando synchrotron radiation and intrusion porosimetry. a during the experiment,namely the expansion of a during H2O intrusion. We have already reported thenegative compressibility of ZIF-8 upon intrusion in a recent article.21 The analysis of the shape of the diffractionpattern peaks allowed us to identify the strain (Supporting Information). Additionally,we were able to identify the coherent crystalline domain size, i.e.,the largest domain of crystallites with the same structural characteristics.In fact, wet cavities are larger than dry ones . Considering that wet ZIF-8 cagesundergo an expansion relative to dry ones,21 intrusion introduces some strain in partly filled ZIF-8 crystallitesdue to the size mismatch between wet and dry cages becomes the smallest coherent domain in the ZIF-8 crystallite; fromthis point on, the largest coherent domain becomes the wet one, whichsteadily grows 1b while Summarizing, synchrotron datasuggest that ZIF-8 is intruded bywater following a cascade mechanism by progressive filling of connectedcages, which account for the observed shrinkage and growth of coherentdomains.z-oriented ZIF-8 slab is immersed in bulkwater, 30 Time scales accessible by atomistic simulations, tens of nanoseconds,are too short to observe the wetting of the slab; thus, the processwas accelerated using the restrained molecular dynamics (RMD). RMDforces the system to visit the microscopic states corresponding toan overall level of wetting of the ZIF-8 computational sample as measuredby the number of water molecules within the MOF slab. It is worthremarking that these microscopic states are sampled with a statisticaldistribution consistent with the constant number of particles, pressure,and temperature ensemble and in quasi-static conditions, i.e., inconditions consistent with the experimental ones . RMD also allows us to compute the free energyprofiles characterizing the wetting process.21 At a variance with previous simulations focusing on a three-periodicZIF-8 bulk sample, with water condensation investigated by Grand CanonicalMonte Carlo simulations,28 in which newwater atoms are inserted at random locations within the material,our approach allows ZIF-8 wetting to take place according to mostprobable mechanism without excluding a priori neither condensationnor cascade penetration.To clarify the molecular mechanism of wetting of complexmicroporoussystems, we performed molecular dynamics atomistic calculations inwhich a To identify the actual wetting mechanism, we computedthe numberof molecules in each of the 28 cages comprising the ZIF-8 slab 2a. If in2O/cage is zero,some water molecules inside the surrounding wet cages are presentnear the connecting 6MR apertures. When wetting starts, i.e., when#H2O/cage grows, these molecules penetrate the cage andthe free energy grows. This can be explained considering that in thisearly stage the penetrating water molecules are undercoordinated;i.e., they form fewer hydrogen bonds than in the initial state.31 This energetically unfavorable process is drivenby the external pressure. The free energy ceases to increase at thetransition state (free energy maximum) which occurs when water moleculespenetrating from neighboring wet cages can form hydrogen bonds betweenthem . This brings us to propose that the key structural parameter controllinghysteresis is the pore connectivity, including the number and distanceof connecting apertures through which water molecules can propagatecapillary penetration. To some extent, these key characteristics arerelated to the cage size of the MOF, but the latter characteristicis not as important for determining hysteresis via limiting the sizeof critical liquid-like water nuclei, as previously proposed.28To confirm that the wetting mechanism is due tocascade penetrationrather than condensation, we performed additional free energy calculationsfor the wetting of a single cage but allowing water to enter fromonly one of the surrounding wetting cages .The results of the single cage wettingsimulation clearly showthat the microscopic ZIF-8 wetting mechanism is not condensation butthat the transition state is determined by the formation of a hydrogenbond bridge between water molecules penetrating from neighboring wetcages, rather than by the competition of free energy gain correspondingto larger liquid-like water clusters and the free energy penalty,which characterize condensation processes (see n fully wet cages (∼40 H2O per cage) and focus on two cases, (i) one in which the wet cagesare randomly distributed in the ZIF-8 crystallite and (ii) the otherin which they form a single, droplet-like, coherent domain. Accordingto our recent finding that water molecules can form hydrogen bondsacross secondary apertures of porous systems,32 the droplet-like state is energetically favored. MD simulationsreveal that hydrogen bonding across 6MR apertures stabilizes the systemby 6.5 kBT (see the Supporting Information) per pair of adjacentwet cages connected by the aperture. Thus, the droplet-like configurationis energetically favored because it minimizes the number of wet cagesin contact with dry ones. Indeed, the number of “interface”wet–dry 6MR apertures is proportional to the surface of thecoherent domain of wet cages which tends to be minimized for energeticreasons. It is worth remarking that the argument developed here isanalogous to the one at the basis of the capillarity, which predictsthat isolated droplets are spherical to minimize surface energy; similarlyto classical capillarity,33 intrusion inZIF-8 is controlled by a competition between bulk-like volume termsfavoring intrusion—mainly the liquid pressure—and thesurface cost related to the interface of wet–dry cages whichintroduces a (free) energy penalty that disfavors the formation ofmany small wet domains.The simulated microscopic penetrationmechanism suggests that thereis a correlation between the wetting state of neighboring cages, whichcan favor the formation of coherent domains of wet or dry cages, causingin turn different structural and mechanical characteristics (strain).It is helpful to explain the formation of these coherent domains interms of the macroscopic theory of capillarity. Let us consider thecase of kBT reduction of the wettingbarrier per surrounding wet cage (see Supporting Information for the details). The simulated crystallite hasthe same type of connectivity of ZIF-8. Mimicking experiments, inour model, we linearly increase the pressure in a ramp, although forcomputational reasons we use compression rate orders of magnitudefaster than the typical experimental ones (106 MPa/s vs3.16 × 10–6 MPa/s). The results of our model,shown in Supporting Information for a full movie of the process) while the random filling of cagesby condensation is negligible. Because of the high rate at which thepressure is changed, the system forms straight “liquid fronts”connected by curved edges which bear memory of the square geometryand of the initial conditions. In an infinitely long calculation,one would reach the surface with minimal energy—a single sphericalempty domain in the center. Additionally, we remark that the advancingflat front of Atomistic simulations, with their (relatively)small computationalsample preclude a direct empirical confirmation of the wetting mechanismon the crystallite scale. Thus, we developed a stochastic model inwhich cages undergo wetting or drying with a probability dependingon the number of wet neighbors, coherently with the results in In summary, via a combination of experimental andtheoretical approacheswe have clarified the intrusion mechanism of ZIF-8 under hydrostaticpressure, which proceeds by cascade filling of neighboring cages.In operando synchrotron radiation tracked the evolution of domainsof wet/dry cages during intrusion, indicating the formation coherentdomains of wet cages which grow during the process. RMD simulationsgave microscopic evidence of this process, bringing to the conclusionthat coherent domains are formed due to penetration of water in connectedZIF-8 cages, rather than water condensation in individual ones, aspreviously proposed. The detailed wetting mechanism of a single cagehas been analyzed, showing that the kinetic bottleneck of the processis the formation of hydrogen bonds bridging water molecules acrossneighboring apertures. This suggests that tuning the intrusion orextrusion characteristics of hydrophobic MOFs can be obtained by optimizingthe pore connectivity; in particular, the geometrical and chemicalcharacteristics of the apertures are crucial to favor or disfavorthe formation of hydrogen bonds, which, in turn, drive the cascadefilling of neighboring cavities. Finally, on the crystallite scale,the shape of the domains of wet cages seems compatible with the classicalcapillarity concept of surface energy because they tend to minimizethe surface area. Indeed, a stochastic model informed by MD simulationsconfirms the prevalence of cascade penetration in coherent domainsas opposed to a condensation scenario.Figure S5). OtherZIFs, e.g., ZIF-12, with different morphologies and cage and aperturesizes, might show some differences with respect to the mechanism discussedabove, which, however, we expect to be quantitative rather than qualitative.We plan to investigate this in the future.The critical characteristicsresponsible for the water intrusionmechanism if ZIF-8 is the presence of interconnected cages. Thus,we expect a similar mechanism to hold also for other ZIF MOFs. Forexample, for ZIF-67, which is an isomorph to ZIF-8, simulations showcage-by-cage intrusion (


Hepatic encephalopathy, which is a serious complication, and sarcopenia are undesirable consequences in cirrhosis. Transjugular intrahepatic portosystemic shunt increases the risk of hepatic encephalopathy. We investigated the effect of sarcopenia on the incidence of post-transjugular intrahepatic portosystemic shunt hepatic encephalopathy.2)/ height (m2).Clinical data of patients who underwent transjugular intrahepatic portosystemic shunt were extracted retrospectively. Computed tomography images at L3 level of scans performed prior to transjugular intrahepatic portosystemic shunt were analyzed to assess skeletal muscle index—expressed as skeletal muscle area : 2.8 (1.08-7.4), P  = .02] were higher, with sarcopenics having 3 times higher risk of hepatic encephalopathy and 8 times higher risk of multiple episode of hepatic encephalopathy . In multivariate analysis, age , serum albumin , and skeletal muscle index  were independently associated with post-transjugular intrahepatic portosystemic shunt hepatic encephalopathy.Of 210 patients who underwent transjugular intrahepatic portosystemic shunt, complete information was available in 79 . Post-transjugular intrahepatic portosystemic shunt hepatic encephalopathy developed in 29 (37%) patients. In patients who developed hepatic encephalopathy, both serum ammonia [177.6 ± 82.5 vs. 115.5 ± 40.5 µg/dL, Sarcopenia is present in nearly half of the cirrhotic patients undergoing transjugular intrahepatic portosystemic shunt, which increases the risk of a single episode of hepatic encephalopathy by 3-fold and that of multiple episodes of hepatic encephalopathy by 8-fold after transjugular intrahepatic portosystemic shunt procedure. Increased skeletal muscle index is associated with decreased risk of hepatic encephalopathy. This retrospective study highlights a high prevalence (50%) of sarcopenia in patients with cirrhosis at the time of undergoing the transjugular intrahepatic portosystemic shunt (TIPS) procedure.This pre-TIPS sarcopenia significantly increases the risk of developing both single and multiple episodes of post-TIPS hepatic encephalopathy (HE).Low muscle mass does predict post-TIPS HE, thus highlighting a careful screening of patients from a nutritional angle before performing the TIPS procedure.1Patients with cirrhosis having the complications of recurrent variceal hemorrhage and ascites that are refractory to standard treatment often require the procedure of transjugular intrahepatic portosystemic shunt (TIPS). Transjugular intrahepatic portosystemic shunt is an interventional radiology technique consisting of an artificial conduit from the portal to the systemic venous circulation created to resolve portal hypertension in order to manage these 2 complications, thus acting as a bridging therapy to liver transplantation.2Hepatic encephalopathy (HE), a syndrome of altered neurological function due to metabolic deregulations, is yet another commonly seen complication in patients with cirrhosis. Though the pathogenesis of HE is very complex, but the key factor is increased concentration of gut-derived ammonia which bypasses the liver due to portosystemic shunts. The shunt connection between the splanchnic and systemic circulation formed in TIPS reduces the complications of variceal bleed and ascites but ironically shows increased association with the complication of HE. With 20%-31% of patients demonstrating either new or worsening HE after TIPS, it is considered a major drawback of this procedure as the ammonia-rich blood bypasses the liver detoxification process making its way straight to the systemic circulation and manifesting as HE.3 which adversely affects disease outcome in terms of increased risk of infections, development of hepatocellular carcinoma, poor quality of life, poor outcome of liver transplantation, and also increased hospital costs.5 In a failing liver, the muscle acts as an alternative site for ammonia detoxification.6 Reduction in muscle mass and even quality may lead to an increase in the circulating ammonia levels.7 Hyperammonemia per se reduces muscle protein synthesis and enhances muscle protein degradation.8 Hence, a reduced muscle mass may further augment the manifestation of post-TIPS HE in a cirrhotic patient.Sarcopenia, defined as loss of skeletal muscle mass, is a major cause of malnutrition seen in patients with cirrhosis. Almost 65%-90% of patients with cirrhosis have sarcopeniaThough a few studies have examined the relationship between sarcopenia and HE after TIPS, there are no Indian studies to suggest the same association. Hence, the present study was planned to investigate the prevalence of sarcopenia in patients who underwent TIPS and to study the effect of sarcopenia on the incidence of post-TIPS HE.This study conforms to the provisions of the Declaration of Helsinki. Ethics Committee approval and informed consent were not taken due to the retrospective nature of the study as the data were collected from the hospital records.In this retrospective observational study, the clinical records of 210 cirrhotics who underwent TIPS at our institute from October 2010 to January 2018 were reviewed . PatientData related to clinical characteristics, laboratory tests, and follow-up records were extracted from the hospital information system. Medical records were accessed using the hospital accession number from the database for demographic (age and gender), clinical  score, Model for End Stage Liver Disease score, and presence of HE), and radiological (CT scan) information at baseline and follow-up. Information on the day of TIPS was considered as a baseline and that on hospital re-admission was considered as a follow-up.The patient’s records were reviewed to look for episodes of HE after the TIPS procedure. Hepatic ncephalopathy was considered when there was a record of at least 1 hospital admission due to HE as defined by the West Haven criteria. Hepatic encephalopathy episodes after TIPS were categorized as HE absent (−), HE once (HE+), and more than once as multiple episodes of HE (HE++).2) was defined as the sum of the area of paraspinal, psoas, transverse abdominis, interior/exterior oblique, and rectus abdominis muscle at the level of the L3 region. Skeletal muscle was identified based on predefined CT density of −29 to +150 Hounsfield unit (HU).9 Muscle was measured using the paint tool of the software, with red color coding (2)/height (m2). Sarcopenia was defined as SMI <50 cm/m2 in males and <39 cm/m2 in females.10All CT scans were performed on Discovery 750 HD 64-row spectral CT scanner . Single-slice CT images at the third lumbar vertebra of the scans performed within 3 months prior to the TIPS procedure were analyzed using image analysis software Slice-O-MaticV4.3 . The skeletal muscle area  was assessed by the mean radiological muscle attenuation of the psoas muscle at the L3 level. Bilateral psoas muscle attenuation expressed in the Hounsfield unit was measured using the hospital standard Picture archiving and communication system (PACS) imaging software. An oval region of interest of 1.5 cme muscle .11 Measut-test and categorical variables by Chi-square test. Wilcoxon rank-sum test was used for non-normal data. Besides this, univariate and multivariate logistic regression was also applied. P < .05 was considered statistically significant. All analyses were carried out using Statistical Package for the Social Sciences software version 22 .Data are presented as number (%), mean ± standard deviation, or median (range) as appropriate. Continuous variables were analyzed using Student’s Of the 210 patients who underwent TIPS, complete information was available for 79 patients. The demographic and clinical characteristics of patients included in the study are given in Of the 79 patients who underwent TIPS, half (53%) of the patients had sarcopenia at baseline. The comparison of clinical, biochemical, and muscle parameters between sarcopenics and non-sarcopenics is shown in Hepatic encephalopathy developed in 29 (37%) patients within a period of 1-9 months after the TIPS procedure. P  = .02; OR (95% CI): 2.8 (1.08-7.4), P  = .02], with sarcopenics having almost 3 times higher risk of developing HE.Also, the prevalence of sarcopenia was significantly higher in patients who developed post-TIPS HE as compared to those who did not develop HE , thus taking the risk of multiple episodes of HE among sarcopenics to be 8 times higher than non-sarcopenics.Further, on multivariate analysis , age, se1 At our liver disease super specialty center, we conducted this retrospective study on patients who underwent TIPS for management of either refractory ascites or variceal bleed. Our assessment of sarcopenia in this group of patients revealed an alarmingly high prevalence of sarcopenia in almost half (53%) of these patients.Transjugular intrahepatic portosystemic shunt procedure, a therapeutic modality for treating cirrhotics with complications related to portal hypertension, is a double-edged sword with the qualms of development of HE after the procedure. A good number of patients who undergo TIPS procedure demonstrate new or worsening HE thereafter. Hepatic encephalopathy may be pure sequelae of liver failure or a result of a combination of both liver failure and portosystemic shunting.16 These wide variations could be due to the different diagnostic methods used for the assessment of sarcopenia like anthropometry,15 bioelectrical impedance analysis,16 and CT.13 In the present study, we used the gold standard technique of CT for muscle mass quantification to define sarcopenia. Notwithstanding the use of an advanced method of muscle quantification, our study falls short of the availability of complete information in all 210 patients who underwent TIPS; hence, the prevalence of sarcopenia could further be an underestimation.There exists a wide variation in the reported percentage of sarcopenia among cirrhotics.17 Likewise, the serum ammonia levels in the present study were commensurate with the incidence of post-TIPS HE. Not only was the prevalence of sarcopenia higher but also serum ammonia levels were significantly elevated in patients who developed HE after the TIPS procedure. Similar to our findings, a recent study done by Hanai et al16 also reported a higher prevalence of minimal HE (MHE) in patients with sarcopenia as compared to non-sarcopenics. Two more studies have also reported significantly higher ammonia levels in patients with muscle depletion or sarcopenia.15Over the last decade, sarcopenia, the principal cause of malnutrition in cirrhosis, has been recognized as one of the additional factors responsible for the development of HE, thus further compounding the issue of post-TIPS HE. Patients with liver cirrhosis are prone to the development of skeletal muscle abnormalities because of reduced dietary intake, reduced hormone levels, inactivity, increased inflammation, and hyperammonemia. Hyperammonemia has a cumulative effect in accruing HE in a cirrhotic patient. The reduced capacity of the liver to detoxify ammonia elevates its concentrations which contributes to sarcopenia by interfering with the muscle protein synthesis pathways, that is, upregulating the myostatin secretion and stimulating muscle autophagy. Muscle, which potentially can play a compensatory role in ammonia detoxification, in the setting of sarcopenia, fails to detoxify ammonia, which in turn augments sarcopenia and further causes hyperammonemia; thus forming a vicious cycle.Almost a 3 times higher risk of HE and 8 times higher risk of developing multiple episodes of post-TIPS HE in a cirrhotic patient, with pre-existing sarcopenia, poses an important query on pre-TIPS sarcopenia assessment and risk stratification prior to TIPS procedure. The artificial channel in TIPS created between the portal vein and the hepatic vein exposes the brain to the gut-derived ammonia and other toxic substances, thereby enhancing the probability of HE in a cirrhotic patient with an already failing liver.18 studied the role of myosteatosis in the development of HE and found that myosteatosis and sarcopenia were both more frequent in patients who developed MHE. Merli et al15 also showed that the prevalence of both overt and MHE was significantly higher in patients with muscle depletion and those with decreased muscle strength as compared to those without these alterations. Muscle strength and myosteatosis are both considered markers of muscle quality, and hence, these studies prove that not only the reduced muscle mass but also muscle quality is the driving force for the development of HE in patients with liver cirrhosis.Apart from the muscle mass, our study also highlights the association of muscle quality with the increased incidence of HE. A recent study by Bhanji et al13 reported significant improvement in SMI and muscle attenuation after a mean follow-up of almost 9 months following the TIPS procedure, with Psychometric Hepatic Encephalopathy Score and ammonia levels significantly improving in patients who showed >10% improvement in SMI. In a very recent study, TIPS creation was found to be associated with a significant increase in cross-sectional area and attenuation of truncal musculature after a median follow-up of 13.5 months.19 In yet another similar study, a significant increase in the total psoas and paraspinal muscle area and reduction in visceral fat volume have been observed in cirrhotic patients after the TIPS procedure.20Contrastingly, few observational studies have associated TIPS creation with reversal of muscle mass and improvement in body composition as well. Gioia et al21 and also improvement in the circulating levels of insulin-like growth factor 1 or even decrease in the muscle myostatin levels.22 Nevertheless, the improvement in muscle mass after TIPS procedure in these studies is quiet paradoxical to the previously reported literature with observations of increased incidence of post-TIPS HE associated with sarcopenia.Though the literature does not elucidate very clearly about the molecular mechanisms behind the improvement in SMA after TIPS procedure, yet there are a few indications toward some plausible reasons like improved dietary intake after resolution of ascitesThe present study did have its own limitations, like a relatively small sample size due to lack of complete information from the hospital records, missing information on patients having MHE or those with HE being admitted to some other hospital, and also the information related to precipitating factors such as electrolyte imbalance, constipation, etc., all these which are intrinsic to a retrospective study. Nonetheless, the present study clearly indicates that the risk of post-TIPS HE is manyfold higher in patients who have sarcopenia, and each unit increase in the skeletal muscle index decreases the risk of post-TIPS HE by 7.5%. With nearly half of the cirrhotics undergoing TIPS having sarcopenia, which reduces ammonia detoxification capacity and blatantly increases the risk of post-TIPS HE, a baseline sarcopenia risk assessment, appropriate stratification, and a prudent patient referral to the TIPS procedure are the need of the hour. Early and effective nutritional habilitation and physical training programs are very promising to get better outcomes of TIPS in terms of reduced incidence of HE and overall better quality of life.


Here, we report a case of subacute combined degeneration (SCD) of the spinal cord in the setting of nitrous oxide poisoning seen at the Medical Ward, Queen Elizabeth Hospital Birmingham Our patient was a 28-year-old lady who presented with impaired sensations in the lower limbs and difficulty walking for approximately one and a half months. Her clinical symptoms did not match common neurological conditions. Upon detailed history, she revealed that she had been frequently using nitrous oxide recreationally for several years. Although her baseline investigations were normal, her magnetic resonance imaging (MRI) of the spine showed bilateral symmetrical T2 hyperintense signal changes in the dorsal columns extending from C2 to C6 spinal segment. Based on history, clinical findings, and MRI of the cervical spine, the diagnosis of SCD of the spinal cord was made, and her symptoms fully resolved with treatment. Nitrous oxide is commonly used as an anesthetic in dental procedures. However, its increasing recreational use can cause vitamin B12 deficiency which can cause peripheral neuropathies and myelopathies leading to severe neurological consequences.Here, we report a case of subacute combined degeneration (SCD) of the spinal cord in the setting of nitrous oxide poisoning which presented to the Medical Ward, Queen Elizabeth Hospital Birmingham.A 28-year-old lady was admitted to our medical unit with complaints of tingling and numbness in both of her hands and feet for a month. She admitted to a history of recreational use of nitrous oxide inhalation, smoking cannabis/marijuana, and consuming amphetamine. She had a power of 5/5 in all limbs with diminished reflexes and impaired pain sensation in glove and stocking distribution. There was an absent joint position and vibration sense. The routine blood examination including full blood counts and liver and kidney functions were normal. Vitamin B12 level was low (188 pg/mL) with normal homocysteine and methylmalonic acid (MMA) levels. The patient was seen by the neurology team and was advised to undergo magnetic resonance imaging (MRI) of the brain and whole spine with contrast. The brain MRI was normal. However, MRI of the spine showed bilateral symmetrical T2 hyperintense signal changes in the dorsal column extending from C2 to C6 level which is typical of SCD of the cord, as described in sagittal Figure  and axiaShe was managed as a case of SCD of the spinal cord likely due to functional vitamin B12 deficiency secondary to nitrous oxide inhalation. She was started on intramuscular vitamin B12 treatment as per British National Formulary (BNF) guidelines and her symptoms started to resolve. At the one-month office follow-up, the patient reported overall symptomatic improvement. She was discharged on treatment with the advice to stop nitrous oxide use and complete vitamin B12 injections as per the BNF protocol. Her neurophysiological studies showed reduced peroneal motor responses with prolonged distal latencies which supported a predominantly demyelinating neuropathy. Also seen was a slowing of some motor and sensory conduction velocities, mainly in the lower limbs, and borderline f-wave responses from tibial nerves. There was mildly reduced recruitment and no signs of active denervation, which is consistent with the characteristics of demyelinating neuropathy. The diagnosis of SCD of the spinal cord was established based on the neurophysiological and radiological findings.SCD is characterized by demyelination of the spinal cord arising due to a deficiency of vitamin B12, which can cause symptoms such as spastic paraparesis and impaired vibration and proprioception sense, leading to sensory ataxia and difficulty in walking. The symptoms occur due to the development of lesions in the cord’s lateral corticospinal tracts and dorsal columns, respectively .Nitrous oxide has various conventional medical applications, such as its use as an anesthetic agent during dental and surgical procedures. However, due to its easy availability over the counter, it can sometimes be abused. Various studies have reported an association between chronic recreational uses of nitrous oxide and subsequent gait disorder by the deactivation of vitamin B12 and its downstream effect on methionine synthesis . It has In cases of nitrous oxide neurotoxicity, similar to classic SCD, patients typically report symptoms such as limb numbness, limb weakness, and gait disturbance. However, unlike other causes of vitamin B12 deficiency, nitrous oxide-induced vitamin B12 deficiency often presents with a normal hematological pattern . Age andIn treating patients with SCD, usually, abstinence from nitrous oxide with parenteral injections of hydroxocobalamin (vitamin B12 ) lead to greater symptomatic improvement . PreviouThe course of symptom resolution is not associated with sex, hemoglobin level, serum vitamin B12, or MRI manifestations at the time of admission and at follow-up visits . HoweverNitrous oxide recreational abuse can lead to permanent neurological manifestations and early diagnosis and treatment can be dramatic in long-term prognosis. Clinical staff must be aware of the various types of presentations of neurotoxicity related to nitrous oxide abuse, and chronic nitrous oxide abuse should be the part of differential of patients who present with atypical neurological symptoms.