Substantial advances have been made in our understanding of the molecular biology of cancer, leading to profound progression in the fields of diagnosis and treatment of non-small cell lung cancer (NSCLC). More recently, many uncommon mutations are gaining attention in NSCLC, including epidermal growth factor receptor exon 20 insertion (EGFR 20ins), anaplastic lymphoma kinase (ALK), human epidermal growth factor receptor 2 (HER2), rearranged during transfection (RET), v-Raf murine sarcoma viral oncogene homolog B (BRAF), neurotrophic tropomyosin receptor kinase (NTRK), and others . PharmacXu et al. reported that two patient-derived xenografts in zebrafish embryos from two patients harboring EGFR 20ins received precision treatment. Zebrafish were inoculated with tumor cells and cultured in osimertinib-containing medium to predict a clinical response. Their study demonstrated the applicability of zebrafish models for testing targeted drugs. Yang et al. identified five metastatic NSCLC patients with EGFR p.L747P mutation and found that afatinib achieved numerically longer progression-free survival (PFS). Dynamic simulations and in vivo experiments demonstrated that afatinib had the best binding affinity and significantly inhibited p.L747P-mutant tumor growth. Feng et al. reported three locally advanced NSCLC patients with EGFR sensitive mutations switching to aumolertinib, a novel third generation EGFR-TKI, as neoadjuvant therapy after 1-2 cycles of preoperative chemotherapy neoadjuvant therapy. Excellent tumor remission and downstaging were achieved to allow surgical treatment, and no tumor recurrence was observed until the latest follow-up. This may indicate that aumolertinib was clinically applicable and could be a viable option in the neoadjuvant phase of therapy.In this special issue, we compiled a series of papers including original research, reviews and case reports that focus on recent advances and challenges in the treatment of lung cancer patients with uncommon driver mutations. For rare EGFR mutations, Peng et al. reviewed recent studies and summarized the efficacies and safety profiles of ALK-TKIs and other therapies with data from preclinical and clinical trials. They also proposed several key points regarding treatment sequencing strategies, resistance mechanisms of ALK-TKIs and toxicity problems when giving these drugs.Nowadays, ALK-altered NSCLC can be treated with a variety of effective ALK inhibitors and a number of next-generation ALK-TKIs have already been developed. Yu et al. reviewed available data and described the biological function of HER2 and its dysregulation in NSCLC, as well as clinical characteristics of patients. Further, they provided a comprehensive overview of traditional and emerging therapies including chemotherapy and monoantibody, non-selective TKIs, new-generation TKIs and antibody-drug conjugates (ADCs). Of note, ADC-based therapy seems to provide the best clinical outcomes among all treatment regimens, which sheds new light on the management of HER2-altered NSCLC.HER2 aberrations are comprised of three distinct formations: mutation, amplification and overexpression. They were originally discovered in breast and gastric cancer, but their role in lung cancer is gaining increasing attention. Meng et al. retrospectively analyzed the characteristics and clinical outcomes of patients with RET-fusion-positive NSCLC receiving RET-TKI, multi-kinase inhibitor (MKI), chemotherapy and immunotherapy-based regimens from three centers. The results showed that RET-TKI remained the best choice for a better response rate and PFS. Chemotherapy, especially with angiogenesis inhibitors, was still a good choice, while the other two regimens should not be recommended for this patient group. Zhou et al. reported a case involving a stage IIIA lung adenocarcinoma patient harboring RET rearrangement who was treated with pralsetinib as neoadjuvant target therapy. Pralsetinib exhibited a significant response and transformed the unresectable tumor into a resectable one. Additional clinical trials are warranted to verify the effect of pralsetinib for locally advanced NSCLC.There are two real-world studies concerning treatment options for RET-fusion positive NSCLC patients. Yan et al. and Sun et al. discussed the diagnostic challenges of BRAF mutations, therapeutic strategies and post-therapeutic evolutionary pathways of BRAF, and also the mechanisms of resistance to BRAF-TKIs. NTRK fusion has become increasingly studied in lung cancer. Liu et al. provided a panoramic view of the function of NTRK genes, the diagnostic techniques for NTRK fusions, the clinical data on TRK inhibitors and their resistance mechanisms. These reviews provide us with a comprehensive view of the current landscape of BRAF and NTRK alterations in NSCLC, but there are still many unsolved issues to be addressed.BRAF activation consists of several distinct forms including V600 and non-V600 mutations, rearrangements, fusions, in-frame deletions and insertions. Personalized medicine has revolutionized the therapeutic landscape of lung cancer with molecular alterations in the past two decades. Since most uncommon mutations are associated with poor to moderate efficacy of immunotherapy, their identification and the development of new-generation drugs are pivotal for designing improved treatment strategies. Attempts to treat other rare mutations are being investigated in addition to those discussed above, such as MET, ROS1, FGFR, STK11, etc. The growing use of next-generation sequencing (NGS) platforms has expanded the panel of genes that can be detected and targeted, and noninvasive liquid biopsy techniques will provide more information regarding efficacy monitoring and resistance mechanisms in real-time. With novel therapies springing up, further investigation should continue to evolve and lead to improve patient outcomes.Finally, we thank all the authors for their inspiring contributions to this Research Topic and hope that these papers will provide our readers with a deep understanding of the current status and future directions in the treatment of NSCLC patients with uncommon mutations.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This study was supported by the National Natural Science Foundation of China , the Science and Technology Commission of Shanghai Municipality (grant number: 19411971100) and the Shanghai Shenkang Hospital Development Center (grant number: SHDC12020110).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Respiratory diseases are a major cause of illness in veterinary medicine, leading to extensive economic losses, especially in animal production settings, with detrimental consequences to animal health and welfare –5. InfecFrucchi et al., report that BCoV, as well as Pasteurella multocida and Mannheimia haemolytica, were frequently detected in nasal swabs samples from heifer calves in high-production dairy herds in Brazil, suggesting a role of BCoV in Bovine respiratory disease complex (BRDC). Molecular characterization of BCoV N and S1 genes in swab samples revealed that the detected strains are ancestrally different from strains previously reported in the region. The high frequency of BCoV (56%) detection and the identification of a different variant circulating in Brazil suggest that BCoV may be a major BRDC pathogen among heifer calves. Corroborating with this finding, Soules et al. experimentally infected calves intranasally with virulent BCoV. The detection of BCoV in bronchoalveolar lavage, nasal turbinate, and trachea along with histopathologic lesions in the upper and lower respiratory tract further implicates BCoV plays a role in respiratory disease in cattle.Bovine Coronavirus (BCoV) has long been identified as a causative agent of cattle diarrhea. However, its role as a respiratory pathogen is less clear. Nelsen et al. at SDSU screened swine lung clinical samples submitted to the diagnostic laboratory for the presence of porcine rotavirus A (RVA) genome by quantitative reverse transcription PCR (qRT-PCR). RV was detected in 30.8% (22 samples) of lung samples obtained from conventionally reared pigs with respiratory signs. In situ hybridization (ISH) showed that RV genome localization in positive lung samples was restricted to alveolar and interstitial macrophages and bronchiolar epithelial cells. Analysis of 120 archival formalin-fixed and paraffin-embedded lung samples by ISH revealed another 10 RV-positive cases with similar RV genome localization patterns.Rotavirus (RV) is also known to cause acute gastroenteritis worldwide, affecting various species, including pigs, cattle, poultry, and humans. While epidemiological studies report respiratory symptoms concurrently with fecal and nasal shedding, RV respiratory infections have scarcely been investigated. Mycoplasma hyopneumoniae is another relevant swine respiratory pathogen. This bacterium is the primary agent in the swine enzootic pneumonia infectious and plays a role in the swine respiratory disease complex (SRDC) with significant economic losses to the swine industry worldwide. Despite its impact, control and prevention are challenging due to the limited characterization of the antigenic repertoire, virulence profile, and resistance to antimicrobials. Additional complicating factors regarding M. hyopneumoniae epidemiology are distinct circulating strains and the intrinsic resistance against β-lactam antibiotics, sulfonamides, and trimethoprim. A few reports have shown acquired antimicrobial resistance against some antibiotics and associated resistance mechanisms. However, a clear picture of the virulence and pathogenicity of M. hyopneumoniae is still missing, and the potential impact of strain variability on disease severity is likewise not always well-defined. Therefore, the identification and characterization of M. hyopneumoniae strains circulating within a population or geographical region are essential. In the current topic, Zong et al. evaluated the growth and morphology, pathogenesis, and antimicrobial sensitivity characteristics of an M. hyopneumoniae strain isolated from Chinese native Enshi black pig lungs (M. hyopneumoniae strain ES-2). The study also identified 2 genes only present in pathogenic M. hyopneumoniae strains, with great potential as a virulence marker and a tool in clinical diagnosis.Bordetella bronchiseptica is responsible for severe respiratory disease in dogs, swine, and rabbits and is a significant contributor to canine infectious tracheobronchitis . While upper respiratory infections and their usual clinical signs can have obvious implication for the ability of these dogs to carry out their scent detection duties, the potential impact of the intranasal anti-Bordetella vaccine administration on the olfactory capabilities of dogs have not been evaluated. The delivery to the nasal mucosa epithelium elicits a local immune response, and this localized inflammation could cause hyposmia/anosmia, hampering the dog's ability to conduct detection work safely and effectively. Collins et al. evaluated different anti-Bordetella vaccine regiments, including oral and intra-nasal vaccine delivery. Luckily, odor thresholds were not influenced by any vaccine strategy. Single intranasal or oral Bordetella vaccine did not impact detection by dogs, while prime with an oral vaccine followed by intranasal boost led to a slight increase in time to detection but not the ability to detect the target odor.Although usually considered pets, dogs are frequently raised as working animals. Dogs serve, for example, as guides for vision-impaired owners, livestock guardian dogs, and as scent detectors. Canine olfactory detection has a broad range of purposes, including detection of drugs and explosives, medical detection, or search and rescue –12. No dStudies included in this Research Topic portrayed the ever-evolving nature of respiratory infectious diseases, especially under intensive production settings that favor transmission and spread. This Research Topic aimed to highlight and promote discussion regarding respiratory diseases in veterinary science. The complied research portraited the investigation of new pathogens, but mostly ways to re-think old pathogens and their role in newly identified problems. It is noteworthy that detection, treatment, and prevention of respiratory diseases in animals is not only limited to protecting animal health and welfare but also supports food security and has a profound positive impact on human health.MM, FB, and AJ-K wrote the editorial. All authors contributed to the article and reviewed and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The gut commensal microbes modulate human immunity and metabolism through the production of a large number of metabolites, which act as signaling molecules and substrates of metabolic reactions in a diverse range of biological processes. There is a growing appreciation for the importance of immunometabolic mechanisms of the host-gut microbiota interactions in various malignant tumors. Emerging studies have suggested intestinal microbiota contributes to the progression of multiple myeloma. In this review, we summarized the current understanding of the gut microbiome in MM progression and treatment, and the influence of alterations in gut microbiota on treatment response and treatment-related toxicity and complications in MM patients undergoing hematopoietic stem cell transplantation (HSCT). Furthermore, we discussed the impact of gut microbiota-immune system interactions in tumor immunotherapy, focusing on tumor vaccine immunotherapy, which may be an effective approach to improve anti-myeloma efficacy. Multiple myeloma (MM) is the second most common hematological malignancy with significant heterogeneity, characterized by the accumulation of malignant plasma cells in the bone marrow that in the majority of cases secrete large amounts of monoclonal immunoglobulin paraproteins , 2. DespThe intestinal microbiota plays a key role in many aspects of human physiological processes including nutrient metabolites uptake, immune regulation and response, reaction to infection –10, hemaHere, we discuss the role of gut microbiota in the host immune response, summarize the gastrointestinal microbiota profile of MM patients and discuss the potential mechanisms of gut microbial alterations in myeloma progression. In addition, we summarized the correlation between gut microbiota and the outcome of HSCT in MM patients. Finally, we explored the value of clinical applications of gut microbes in MM, mainly the predictive biomarker role and the potential roles in immunotherapy with vaccination.The immune system is accountable for recognizing, adapting, and responding to a myriad of foreign and self-molecules and is thereby vital in both pathological and physiological conditions . NumerouIt is well known that intestinal commensals can maintain the epithelial barrier and intestinal integrity by synthesizing and releasing a myriad of metabolites. These metabolites can enter the circulation and can act as metabolic substrates or induce the production of inflammatory cytokines to exert immunomodulation and influence the immune response . For exaMM oncogenesis is a complex process, impacted by genetic and environmental factors with various pathological mechanisms. Although gene expression programs seem to be driven by primary genetic events, there is a sequence of molecular events that are caused by these abnormalities, including secondary genetic abnormalities, as well as genetic events of monoclonal gammopathy of undetermined significance (MGUS) and MM progression such as aberrant nuclear factor-κB (NF-κB) signaling . In addiFirmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria , showing a promising therapeutic strategy. In conclusion, these results suggest that specific bacteria may directly influence the migration of immune cells and the secretion of inflammatory cytotoxic factors and play an important role in tumor progression.The gastrointestinal tract provides an important site for crosstalk between the microorganisms and the host immune system . IntestiAnaerostipes hadrus, Clostridium butyricum, and Clostridium saccharobutylicum, which are the main SCFAs-producing microorganisms, compared to healthy controls , compared to the healthy population  represents another metabolite related to gut microbiota. Glutamine is a nutrient that can be used as a carbon or nitrogen source to support intracellular energy production and biomass synthesis, and it can also serve as a nitrogen donor to meet the increased demand for nucleotide biosynthesis in cancer cells to support cancer growth , 60. To NH4+) . MM cellepletion , 62. Fecpulation . Researcynthesis . These rSeveral observational studies in patients with hematologic malignancies have established that different HSCT outcomes such as risk of death, recurrence/progression of disease after transplantation and transplantation-related adverse events are largely associated with the integrity of gut microbes or the abundance of certain bacteria –65. GeneNewly diagnosed MM patients are highly sensitive to first-line therapy and high-dose chemotherapy with autologous HSCT (auto-HSCT) is an important treatment regimen for MM patients . HoweverBacteroidetes was correlated with milder gastrointestinal symptoms, whereas enrichment in Blautia and Ruminococcus was correlated with a high incidence of diarrhea and post-transplant nausea and vomiting. The researchers also found that a high abundance of beneficial bacteria such as butyrate-producing Eubacterium hallii and Faecalibacterium prausnitzii were implicated in minimal residual disease (MRD) negativity  dominance has been shown to be associated with subsequent bacteremia during allo-HSCT, and Enterococcal dominance led to a 9-fold increased risk of Vancomycin-resistant Enterococcus bacteremia, while Proteobacteria domination led to 5-fold increased risk of gram-negative rod bacteremia  infection  often destroy the gastrointestinal mucosal barrier and the stability of intestinal flora structure, usually characterized by lower microbial diversity and a decrease in beneficial intestinal commensal bacteria or an increase in opportunistic pathogens. In patients with low intestinal microbial diversity, the intestinal flora is typically dominated by a single genus, including herichia , 71. Thecteremia . The inccteremia . Additioic fever , suggestnfection . Excitinnfection , a commonfection . The aboHelicobacter Pylori and gastric cancer) or an identifiable characteristic structure, preferably with characteristic alterations that can be observed in antecedent disease. It is well established that MM develops through gradual evolution, usually from an asymptomatic precursor entity, such as MGUS or smoldering multiple myeloma (SMM), to transform and develop into symptomatic MM  in stool and serum by targeted metabolomics may improve the ability to predict clinical outcomes. However, more studies are needed to explore whether biomarkers from the microbiome will have clinical relevance in MM.Immunotherapy, which relies mainly on immune cells inside or outside the tumor microenvironment to specifically recognize and attack tumor cells, is an effective treatment for many cancers. It is well known that the interaction of antigenic components of the gut microbiota and the products of their metabolic activity with host cells affects the immune response and can promote immunomodulatory effects and a variety of tumor-suppressive effects . In addiCancer vaccines show great potential for the immunotherapy of malignant tumors due to their low toxicity and sustained immune intervention. Cancer vaccines mainly use specialized antigen-presenting cells  to recognize antigenic substances such as tumor cells or tumor-associated antigens (TAAs) or peptides and activate naïve antigen-specific T cells to induce a specific and persistent immune response to tumor antigens in the host, thereby specifically clearing minimal residual tumor lesions or significantly inhibiting tumor proliferation and preventing cancer recurrence , 84. HowFirmicutes and Bacteroidetes phyla in linear sequence as well as in structure and conformation, which together account for 90% of the intestinal microbiome, including melanoma antigen-C1/cancer testis antigen 7 (MAGE-C1/CT7), MAGE-A3, and MAGE-A10  and Alphaproteobacteria (gen. Bosea) associated with amino acid metabolism. This suggests that treatment-induced ecological dysregulation plays an important role in the prevention of vaccine-induced immune responses. Similarly, as mentioned previously, the gut microbial composition of MM patients receiving HSCT is disrupted. Therefore, this favorable environment for vaccine action, in addition to the release of tumor antigens due to cell death , such as therapeutic blockade of PD-1 and PD-L1, have been shown to be an effective treatment for a variety of solid tumors or hematologic malignancies. Plasma cells in MM patients highly express PD-L1 and single-agent PD-1 blockers have a favorable safety profile, however, there is no single-agent activity for MM patients . Moreove T cells  and impr T cells . It is p T cells , 106 to + T cell immunity, thereby overcoming immunosuppression and enhancing anti-myeloma immunity  and prolonged survival after CAR-T treatment, in addition, Bifidobacterium and Leuconostoc were found to be abundant in MM patients with severe CRS while Butyricicoccus were abundant in patients with mild CRS  T-cell therapy, as one of the most important therapeutic strategies for hematological malignancies, has attracted extensive attention in recent years. However, due to individual patient characteristics, the efficiency and associated immune-related adverse effects of CAR-T therapy vary considerably among patients. The gut microbial composition exhibits great individual specificity , which mmild CRS . Bifidoboduction . Thus, BCAR-T therapy involves isolating T cells from a patient’s blood, genetically modifying them to express CAR receptors on their surface, and reinjecting them into the patient so that they can increase the immune response . IntestiIn conclusion, the gut commensal microbiota is critical in regulating the function of the immune system, and several studies have shown a significant role for the gut microbiota in MM pathogenesis and progression by influencing inflammatory pathways and host metabolism. Gut microbial metabolites can influence host immunity, and in particular, the most studied SCFAs may significantly influence the outcome for patients with MM. After auto-HSCT, higher gut microbiota diversity linked with better outcome, which may be a rationale for avoiding injury to the microbiota through judicious use of broad-spectrum antibiotics. Recent studies suggest that gut microbes may influence vaccine efficacy, including the ability of antigens to elicit protective immune responses and the ability of the immune system to respond properly to antigenic stimulation, by acting as immunomodulators and natural vaccine adjuvants. Thus, it seems highly probable that there is a role of the gut microbiota in combination with vaccine immunotherapy for MRD in MM after receiving HSCT. Despite the important role of the microbiota in modulating the vaccine immune response, further investigations are needed to definitively demonstrate this possibility.LZ wrote and reviewed the manuscript. YX, YL, and JZ reviewed and proofread the manuscript. All four authors provided substantial contributions to this review, drafted and critically revised the manuscript, designed and created figures. All authors contributed to the article and approved the submitted version.


Migraine is a disabling neurological disease characterized by moderate or severe headaches and accompanied by sensory abnormalities, e.g., photophobia, allodynia, and vertigo. It affects approximately 15% of people worldwide. Despite advancements in current migraine therapeutics, mechanisms underlying migraine remain elusive. Within the central nervous system, studies have hinted that the cerebellum may play an important sensory integrative role in migraine. More specifically, the cerebellum has been proposed to modulate pain processing, and imaging studies have revealed cerebellar alterations in migraine patients. This review aims to summarize the clinical and preclinical studies that link the cerebellum to migraine. We will first discuss cerebellar roles in pain modulation, including cerebellar neuronal connections with pain-related brain regions. Next, we will review cerebellar symptoms and cerebellar imaging data in migraine patients. Lastly, we will highlight the possible roles of the neuropeptide calcitonin gene-related peptide (CGRP) in migraine symptoms, including preclinical cerebellar studies in animal models of migraine. Migraine is a debilitating primary headache disorder commonly characterized by moderate or severe headaches that can be aggravated by routine activity ICHD-3, . MigrainThe cerebellum is widely known for integrating non-motor sensory signals and controlling motor function , ditans (5-HT1F receptor agonists), gepants [calcitonin gene-related peptide (CGRP) receptor antagonists] as abortive drugs, and CGRP and CGRP receptor monoclonal antibodies as preventative treatments  and antiemetics for abortion, and beta blockers and anticonvulsants for prevention  works independently of CGRP or 5-HTThe cerebellum has three main divisions: the cerebrocerebellum, the vestibulocerebellum, and the spinocerebellum  is a region known to modulate multiple aversive behaviors , adjacent to the thalamus  or DL-homocysteic acid stimulations of the posterior cerebellar vermal lobule VI modulated the neuronal activity of the lumbosacral spinal cord in response to non-noxious and noxious visceral  or somatic  stimuli: an increase in spinal cord neuronal activity in response to visceral stimuli was observed after cerebellar stimulation, while varied responses to somatic stimuli were seen in rats  and non-motor dysfunction in migraine patients.As mentioned above, vestibular nuclei and the cerebellum are closely connected and work in concert to influence posture, equilibrium, and vestibuloocular eye movements . FHM is an autosomal dominant subtype of migraine with aura characterized by fully reversible motor weakness ICHD-3, , and disNon-motor function in migraine patients may contribute to attack-related disability and interfere with work performance and personal life. However, it is often neglected by clinicians and little is known regarding cognitive dysfunction in migraine patients  reveals that the comparison to healthy controls, migraine patients have decreased axonal integrity in vermal lobule VI extending to the bilateral lobules V and VI. These analyses also revealed myelin damage in the right inferior cerebellum peduncle which is composed of cerebellar inputs and outputs  and functional magnetic resonance imaging (fMRI) scans. During the interictal phase, regions in the bilateral posterior lobe in the cerebellum showed spontaneous activity deficiencies in migraine patients compared to healthy controls  and fMRI techniques which may contribute to hypersensitivity to various sensory modalities like visual, auditory, olfactory, and somatosensory stimuli in patients who experience migraine , which was reported to induce migraine-like headaches in migraine patients  calcium channel. Cav2.1 channel is vital for neurotransmitter release , calcitonin receptor-like receptor (CLR), and receptor component protein (RCP). Recently, a second CGRP-responsive receptor, amylin subtype 1 receptor (AMY1), which is comprised of RAMP1 and calcitonin receptor (CTR), has also been identified  of the cerebellum play a causal or modulatory role, or is it the consequence of migraine attacks? In this respect, preclinical studies are necessary to resolve these queries. There are many migraine animal models  findings that cerebellar connections with pain/migraine-related regions and manipulations alter pain; (2) cerebellar symptoms in migraine patients and; (3) changes in structure, activity, functional connectivity, and metabolism in migraine patients or migraine animal models, all suggest that the cerebellum plays a role in migraine. Based on reports that cerebellar manipulations affect neuronal activities in pain-related brain regions, we propose that the cerebellum might play a modulatory role in migraine. This could happen by cerebellar modulation of the descending pain pathway From clinical and preclinical studies described above, the cerebellar regions that most frequently show abnormalities or have been investigated in pain and migraine are Crus I, Crus II, the vermal lobules VI and VIII, lobules IV–VIII, the MN, and the lateral cerebellar nucleus. Future studies dissecting the specific functions of different cerebellar subregions and their circuits will help reveal cerebellar contributions to migraine pathophysiology. Towards this goal, CGRP and its receptors in the cerebellum might be a possible contributor to migraine. Pharmaceutical and optogenetic approaches to modulate CGRP and its receptors may provide new avenues to reveal cerebellar mechanisms and treat migraine pathophysiology.The contents do not represent the views of Veterans Administration or the United States Government.MW, JT, and ND drafted the manuscript. KP, AR, and LS critically revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Institutes of Health (R01 NS075599); VA-ORD (RR&D) MERIT (1 I01 RX003523-0); Career Development Award (IK2 RX002010); and Center for Prevention and Treatment of Visual Loss (VA C6810-C).


Climate change continues to threaten global crop output by reducing annual productivity. As a result, global food security is now considered as one of the most important challenges facing humanity. To address this challenge, modern crop breeding approaches are required to create plants that can cope with increased abiotic/biotic stress. Metabolomics is rapidly gaining traction in plant breeding by predicting the metabolic marker for plant performance under a stressful environment and has emerged as a powerful tool for guiding crop improvement. The advent of more sensitive, automated, and high-throughput analytical tools combined with advanced bioinformatics and other omics techniques has laid the foundation to broadly characterize the genetic traits for crop improvement. Progress in metabolomics allows scientists to rapidly map specific metabolites to the genes that encode their metabolic pathways and offer plant scientists an excellent opportunity to fully explore and rationally harness the wealth of metabolites that plants biosynthesize. Here, we outline the current application of advanced metabolomics tools integrated with other OMICS techniques that can be used to: dissect the details of plant genotype–metabolite–phenotype interactions facilitating metabolomics-assisted plant breeding for probing the stress-responsive metabolic markers, explore the hidden metabolic networks associated with abiotic/biotic stress resistance, facilitate screening and selection of climate-smart crops at the metabolite level, and enable accurate risk-assessment and characterization of gene edited/transgenic plants to assist the regulatory process. The basic concept behind metabolic editing is to identify specific genes that govern the crucial metabolic pathways followed by the editing of one or more genes associated with those pathways. Thus, metabolomics provides a superb platform for not only rapid assessment and commercialization of future genome-edited crops, but also for accelerated metabolomics-assisted plant breeding. Furthermore, metabolomics can be a useful tool to expedite the crop research if integrated with speed breeding in future. In this era of global warming and unchecked population growth, food security is becoming a much more pressing concern. The dream of a world without hunger is only possible if agricultural productivity is significantly enhanced to fulfill the growing food requirements around the globe due to population growth and other factors ,2. It isMetabolomics is emerging as a powerful tool for crop improvement. Metabolomics involves the comprehensive characterization of the metabolome using advanced analytical chemistry technologies. The metabolome is formally defined as the complete set of low molecular weight (MW < 1500 Da) primary and secondary metabolites found in an organism . The metFrom the perspective of plants and plant breeding, metabolomics can be readily used as a diagnostic tool to assess the plant performance, to probe vital metabolic markers linked to biotic/abiotic stresses tolerance, perform mutant characterization, and conduct robust ecotype detection . MetabolIn this review, we will discuss the applications of advanced metabolomics for crop improvement  studies and genome wide association studies (GWAS) have been the traditional approaches to identify broad, macroscale phenotype-to-genotype relationships. However, the macroscale phenotypes used in these studies  are often “ambiguous” or not clearly defined, as they represent the downstream effects of hundreds of genes, proteins, and metabolites. These unclear effects are compounded by difficult-to-measure environmental and epigenetic effects. As a result, the apparent strength or influence of these genetic effects on macroscale plant phenotypes is greatly diminished or difficult to detect. By focusing on molecular-scale phenotypes, such as metabolite concentrations that can be quantitatively measured, one can identify more compelling and higher performing gene-phenotype relationships. As a result, metabolic qualitative trait loci (mQTLs) studies and metabolic genome-wide association studies (mGWAS) have emerged as powerful tools  to disseFor example, mGWAS analysis has proved to be crucial for dissecting the genetic and metabolic architecture of rice by identifying the genes associated with the natural variation seen in the rice metabolism. GWAS was used to identify 6.4 million SNPs from 529 different rice strains and identified 36 candidate genes that modulate metabolite levels of at least 34 known primary and secondary metabolites. The results showed that this technique can be crucial to perform molecular phenotypic trait mapping for the goal of rice improvement . Yadav eA comparative analysis using a combination of phenotypic genome-wide association studies (pGWAS) and mGWAS was carried out on a diverse collection of rice accessions. LC-MS/MS-based metabolomic analysis allowed the identification of more than 830 metabolic features having high heritabilities for flavonoids and amino acids. These results indicated a relationship among phenotype-metabolites that can be beneficial for complex phenotype trait mapping . Matros Metabolomics-based mGWAS analysis for individual metabolite content rations subjected to climatic stresses will give deep insight and knowledge about metabolic diversity and discover key metabolic regulators in response to different stresses. For instance, LC-MS/MS-based metabolomics was used to perform untargeted metabolic profiling of 282 inbred lines (ILs) of maize. This led to the identification of 3991 mass features that were then compared to >29 million SNP markers. Numerous hotspots for QTLs were identified that control a citrate synthase-like gene and benzoxazinoid biosynthesis genes that may play key roles in the synthesis of pathogen defensive metabolites. The genetic loci identified would be useful for marker-assisted breeding to develop resistant maize lines . LC-MS bLiu and coworkers analyzed 286 soybean varieties with a SNP chip that identified 54,294 SNPs. This SNP analysis was combined with a targeted metabolomic study that identified 52 metabolites for lipid metabolism and six oil-related traits. Additionally, multi-omics analysis identified 284 genes associated with oil-related traits. This work also allowed the authors to construct 133 genetic networks related to soybean metabolism, of which only 24 were previously known . Target Advances in genomics and metabolomics technologies have allowed mQTL mapping for diagnostic markers identification to study the plant performance and enabled to develop high density maps of candidate genes controlling the various metabolic pathways . MetabolShi et al.  examinedThe mGWAS and mQTL studies outlined earlier are now giving plant scientists a much clearer idea about the metabolic genes and metabolic gene networks involved in a variety of metabolic activities for many important crop plants. This set of improved functional genome annotations is opening the door to performing much more focused metabolic profiling. In particular, metabolomics is now being extensively used to study plant responses to biotic and abiotic stress. This is helping to elucidate the unique metabolic pathways and metabolic biomarkers linked to stress tolerance and disease resistance in globally important crop plants. In many cases, the pathways and genes associated with these metabolic biomarkers to stress are now well known.Rhizoctonia solani. This metabolomic analysis revealed an increased production of ADP, mucic acid, jasmonic acid, and glyceric acid in resistant cultivars (32R), while sensitive cultivars (29S) increased their production of inosine monophosphate. These data suggest that different metabolic strategies are adopted by different rice cultivars to fight R. solani infection and that metabolite-based selection and breeding for increased levels of these pathogen-fighting metabolites may improve rice resistance to this widespread fungus. The ascomycete fungal pathogen known as Fusarium graminearum, is a common crop pathogen that causes fusarium head blight (FBH). FBH is a devastating disease specific to wheat and barley and is responsible for billions of dollars in economic losses worldwide each year. With climate change and global warming, FBH in wheat is of increasing concern. FBH infection causes shifts in the amino acid composition of wheat, resulting in shriveled kernels and contaminating the remaining grain with mycotoxins, mainly deoxynivalenol, which inhibits protein biosynthesis, and zearalenone, an estrogenic mycotoxin. These toxins cause vomiting, liver damage, and reproductive defects in livestock. They are also harmful to humans when they appear in contaminated food [Suharti et al.  used twoted food .QTL-Fhb2 allele. Untargeted metabolomics showed increased accumulations of fatty acids, flavonoids, glycerophospholipids, lignins, phenylpropanoids, and terpenoids in FHB resistant RILs. In addition, transcriptome profiling detected numerous mycotoxin detoxification genes, transcription factors, receptor kinases, and disease resistance genes. By combining these datasets, several candidate resistance genes within the QTL-Fhb2 allele, including cinnamyl alcohol dehydrogenase (CAD), 4-coumarate—CoA ligase (4CL) and ABC transporter-4 (ABC4)—were identified.In a study described by Dhokane et al. , recombiFusarium graminearum. These metabolites can potentially be used as disease resistant biomarkers, or they may be used in metabolite-based breeding studies to select for increased levels of these pathogen-fighting metabolites. Indeed, the genes and metabolic pathways in wheat are already known for GABA, spermine, lactic acid, and putrescine production [In a related work, Cuperlovic-Culf et al.  conducteoduction .TaTIR1 gene. This result and the multi-omics data produced by this study provide a much more detailed mechanistic understanding of the wheat response to F. graminearum. Kim et al. [Rp1-D gene against P. sorghi The results showed that these defensive genes involved in secondary metabolism including terpenoid, flavonoids and phenylpropanoid pathways. Furthermore, many transcriptional factors such as MYB100, BZIP84, and WRKY53 were detected as targeted metabolites for specific stress response.While many resistance genes against wheat FHB have been identified, we still lack a good understanding about the stress-regulation mechanisms associated with these genes. Recently, Su and colleagues  employedm et al.  performeHvMPK3) and chitin elicitor receptor kinase (HvCERK1). Furthermore, knockouts of HvCERK1 gene in the tolerant barley genotype showed how important its role was in the FHB resistance mechanism and in the biosynthesis of disease-resistant metabolites.Karre et al.  performeTilletia indica). KB is a floret-infecting disease that damages wheat kernels and causes them to emit a pungent fishy odor arising from the fungal teliospores. This obviously reduces grain quality. In a multi-omics study, MALDI-TOF/TOF mass spectrometry was employed to perform a proteomic analysis of highly virulent and weakly virulent isolates of T. indica. This led to the detection of 21 proteins that were differentially expressed in the highly virulent isolates. Protein and DNA sequencing data from the identified T. indica proteins led to the determination that certain candidate pathogenicity proteins were expected to produce oxalic acid. This illustrates how integrated tools that combine proteomics with metabolomics offer an excellent platform to detect virulency-associated compounds in plant pathogens [Kernal bunt (KB) is another economically important disease of wheat caused by smut fungus  in the tolerant genotype. These findings highlight the benefits of integrating metabolomics and transcriptomics in next-generation breeding programs to create climate-resilient varieties [The hyper-production of primary metabolites including specific amino acids such as branched-chain amino acids (BCAAs) also appear to provide a defense mechanism against severe environmental stresses. For example, in a study, integrated metabolomics and transcriptomics were used to study the BCAT genes and their corresponding metabolites in two durum wheat genotypes. Molecular characterization and expression analysis of the BCAT genes revealed the crucial role of the arieties .The malting quality of barley can be degraded by post-anthesis drought stress. This can significantly enhance the β-amylase and grain protein and decrease β-glucan and grain weight. To decipher the molecular mechanisms associated with malting and drought stress, Hong et al.  employedSalinity is another major abiotic stress that negatively influences crop growth and crop yield. Xu and co-workers  exploredIn another study conducted by Pan and colleagues , the salA number of other examples of metabolomics studies have also been published exploring metabolic and transcriptomic changes arising from heat tolerance , cold toAs highlighted here, there have been a large number of studies using integrated metabolomics to understand responses to biotic/abiotic stress and to help with metabolomics-assisted selection and breeding for crop improvement. The integration of metabolomics with other omics approaches will, no doubt, significantly enhance the efficiency and accuracy of future breeding programs, particularly for the screening of novel metabolic traits associated with biotic and abiotic stress resistance.Genetic engineering of plants for improved yields, stability, and herbicide resistance dates from the early 1990s. Some of the first genetically modified (GM) crops include the FLAVR SAVR tomatoes, introduced in 1992 , and RouIn contrast to transgenic plant engineering, plant metabolic engineering or metabolic editing is a promising strategy that does not require the introduction of foreign or non-plant genes into the host plant genome. Instead, metabolic engineering or metabolic editing is a method that accelerates the selective breeding process by modifying existing genes in the host plant’s specific metabolic pathways. These changes simply direct the biochemical reactions to produce more of the desired products . The purMetabolic editing has many significant applications in crop improvement such as increased production of secondary metabolites to confer abiotic stress tolerance in crop plants . Recent The advent of gene editing has led to the view that metabolically edited plants can and should be considered as non-GMO . This is4′OMT2, which regulates benzylisoquinoline alkaloid (BIA) metabolism, was efficiently disrupted by CRISPR/Cas9 cassette mutagenesis to reduce the production of thebaine and morphine [Generally, more than one gene is involved in a metabolic pathway, and therefore, editing only one gene at a time would not be sufficient for a useful metabolic editing result. Multi-gene or multiplexed gene editing via CRISPR/Cas9 appears to be the most promising metabolic editing system, as it allows multiple genes to be removed or inserted as shown in morphine .LCY-E, LCY-B1, LCY-B2, Blc, and SGR1 were selected for metabolic editing in the carotenoid biosynthesis pathway via multiplexed CRISPR/Cas9. The resulting metabolically edited tomato plant demonstrated a five-fold increase in the accumulation of lycopene. Similarly, in a study described by Li et al. [SSADH, CAT9, TP1, TP2, and TP3 which are involved in the GABA shunt metabolic pathway. This multiplexed metabolic editing led to a 19-fold enhancement in the biosynthesis of GABA in the engineered tomato plants. GABA is produced in large amounts during fruit development in tomatoes and is considered a health-promoting functional compound. It is also involved in various regulatory pathways, and GABA homeostasis directly influences the growth and development of many plants [Metabolic editing has not only been applied to alter the production of alkaloids, but it has also been used to change the levels of other secondary metabolites, such as lycopene. Lycopene is considered as a crucial product of the carotenoid biosynthesis pathway and is known to improve the quality and color of ripe tomato fruit. In a study described by Li et al. , five toi et al. , the pYLy plants .GmFNSII-1, GmF3H1, and GmF3H2 involved in isoflavonoid synthesis. Metabolic profiling of the metabolically edited plants revealed an enhanced production isoflavone content. Additionally, the higher levels of these isoflavones appeared to reduce the integrity of the protein coat of the soybean mosaic virus, which led to an increased resistance of the engineered soybean plant to the soybean mosaic virus.Leguminous plants, such as soybeans, have a diverse variety of isoflavonoid metabolites, which are known to have a positive effect on human health. They also play a significant role in regulating plant-environment or plant-pathogen interactions. A recent study by Zhang et al.  showed hMetabolic processes in plants can be regulated at several points including transcriptional and post-transcriptional (exon splicing) check points, as well as via translational, post-translational modifications, or protein-protein interactions. To date, most plant-based gene editing or metabolic editing efforts have focused on transcriptional modifications. However, metabolic editing may soon begin to focus on translational changes or protein engineering edits (such as amino acid changes to reduce or enhance protein stability or activity) and post-translational modifications to change protein–protein interactions . The useAs noted earlier, GM or transgenic crops are currently subject to complex regulatory rules and severe market controls due to the lack of social acceptance and widespread public health concerns . OutsideOne of the best routes to assess the safety, phenotype, and nutritional value of transgenic crops is through taking a snapshot of their metabolomes, because metabolomics and proteomics-based analysis are more closely related to endpoint phenotypes as compared to genomics and transcriptomics. Indeed, all the manipulations performed at the genomic level for plant GMOs should ultimately be “readable” or detectable at their metabolic and nutritional level . As a reAspergillus niger phyA2). The phyA2 gene encodes phytase and can catalyze the hydrolysis of phytic acid to release inorganic phosphorus. The increased phosphorus and decreased phytic acid in the transgenic maize could eliminate the need for phosphorus supplementation when maize is used as animal feed. This metabolomic study demonstrated that the concentration of nine metabolites were altered in transgenic maize compared to conventional (non-transgenic) maize lines. The altered metabolites included tyrosine, glucosaminate, myo-inositol hexakisphosphate (IP6), raffinose, citrate, phosphate, indole-3-acetyl-aspartate, 2-hydroxyglutarate, and gamma-glutamyltyrosine. Only the concentration of IP6 was downregulated while the concentration of all other eight metabolites were found upregulated in transgenic maize. All nine of these metabolites are commonly found in all plant crops and their concentrations in maize fell well within the ranges found for other crop plants or other food products commonly consumed around the world. In other words, this study confirmed that the PhyA transgenic modification introduced no safety risk to these GMOs [Metabolomics tools have also been used to distinguish between GMO and non-GMO maize and to probe the possible risks due to changed metabolic concentrations arising from the transgenic modifications. In a study conducted by Bernillon et al. , two traese GMOs .In 2013, Clarke and coworkers demonstrated a safety assessment analysis for transgenic soybeans (with engineered resistance to the herbicide Mesotrione) via a non-targeted metabolomics approach using UHPLC/MS/MS and GC/MS methods. This approach allowed the detection and quantification of allantoin, delta-tocopherol, myo-inositol-hexakisphosphate, ectoine, citrulline, asparagine, ribitol, phytate galactinol, gulano-1,4-lactone, genistin, and glycitin. Their results indicated that the metabolite composition of transgenic soybeans was not statistically different from that seen within the natural variation of non GMO soybean varieties . MetabolThe risk assessment of metabolically edited or gene-edited crops is likely to become a new area where metabolomics can play a role as elaborated in Speed breeding is an emerging technology in which the generation cycle of a crop is reduced to allow the more rapid introduction of new traits into plants. This innovation was inspired by NASA, which was attempting to grow wheat plants in space and by scientists from the University of Queensland who proposed the term “speed breeding” . Speed bTypically, a 22 h light period and 2 h dark period is applied under controlled temperatures to enhance the plants’ photosynthetic activity. Under normal conditions, only 1–2 generations of a given crop can be produced per year, but with speed breeding, up to six generations of a given crops can be produced in a single year . Speed bSpeed breeding can potentially be integrated with metabolomics approaches to fast-track crop improvement as illustrated in As yet, there is no published example of metabolomics-assisted speed breeding, however we believe it is only a matter of time before examples start appearing in the literature. We expect that metabolomics-assisted speed breeding will help in the rapid phenotyping of plants, in accelerating the detection of new metabolic pathways, in rapid gene identification, and the mapping of new genes controlling different stress-responsive mechanisms in crop plants.Metabolomics-assisted speed breeding will also provide an excellent platform to learn about the stress memory mechanism in plants and the effects of epigenetic modifications in crop plants at a metabolite level. We believe that metabolomics-assisted speed breeding will open many new avenues for crop improvement.Climate change, reduced land quality, shrinking arable land area, and population growth are all major threats to global food security. While many of these issues require social or political solutions, there are a number of scientific options that may help address some of these challenges. As we have attempted to show in this review, a convergence of innovations in genomics, bioinformatics, and metabolomics have created a powerful version of metabolomics. Metabolomics combines advances in metabolomics technologies, genomics technologies, and bioinformatics methods to allow a much more detailed exploration of gene–metabolite–phenotype relationships. As we have shown, metabolomics has emerged as an excellent tool for crop improvement and plant biology research. In particular, metabolomics is allowing the deciphering of novel metabolic pathways, the detection and functional annotation of new metabolic genes or gene networks, the deconvolution of different stress-responsive metabolic pathways, the detection of novel genes responsible for stress tolerance, and the elucidation of the relationship between genes, metabolites, and phenotypes in a wide variety of crop plants. With advances in gene editing and metabolite editing, along with advances in speed breeding and improved GMO risk assessment, the discoveries made by metabolomics could soon lead to healthier, safer, more nutritious, more resilient, faster growing crops. Metabolomics will also play a crucial role in the characterization of gene-edited and speed-bred crops. This integration of metabolomics into the regulatory process will also prove to be very beneficial for GMO risk-assessment and commercialization of gene edited-crops. The metabolite-based safety-assessment of gene-edited crops will improve the current methods of risk-assessment of biotech crops and address public concerns related to their social acceptance.


Antimicrobial resistance (AMR) is considered a major global health problem and location-specific policy are warranted with urgent priority. A recent systematic analysis has estimated 4.95 million deaths worldwide associated with bacterial AMR in 2019 [AMR has especially highlighted important infection control implications in the Intensive Care Unit (ICU) settings where multidrug resistant (MDR) organisms are being isolated with growing frequency .Staphylococcusaureus (MRSA), vancomycin-resistant enterococci (VRE), Acinetobacterbaumannii, Enterobacteriaceae that produce extended-spectrum beta-lactamases and/or carbapenemases , and carbapenem-resistant Pseudomonasaeruginosa, along with emerging MDR fungal pathogens, with Candidaauris currently menacing ICUs worldwide [The most threatening MDR bacteria are methicillin-resistant orldwide .European Antimicrobial Resistance Surveillance Network (EARS-Net), coordinated by European Centre for Disease Prevention and Control, found that the overall number of reported isolates at EU/EEA level increased in 2020 compared to 2019 for almost all bacterial species, with Italy having worrisome and statistically significant increasing trends in Acinetobacter spp., VRE and CRE isolation .The National Healthcare Safety Network (NHSN), United States’ largest electronic surveillance system for tracking healthcare-associated infections managed by the Centers for Disease Control and Prevention (CDC), reported in their latest summary statistically significant differences in the percentages of pathogens with nonsusceptibility to selected antimicrobials between hospital wards and ICUs, with ICUs tending to have a higher resistance rate for bloodstream infections .Prevention and control of MDR organisms require an extensive financial and human resource commitment, along with specific scientific knowledge and administrative leadership, in order to be successful.Although some encouraging new antimicrobials have been approved, the number of novel compounds with a new mechanism of action remains small . While nImprovement in prevention strategies might especially prove helpful in low-resource health systems, where access to antibiotics is limited and nonetheless the highest burden due to AMR was found .Strategies to prevent the emergence and spread of MDR pathogens in ICUs can be divided into two major categories: antibiotic stewardship (AS) (to reduce selective pressure)  and infeIPC was defined by the World Health Organization as an evidence-based approach which attempts to prevent transmission of avoidable infections, including those caused by MDR, acquired during the provision of health care services . Such inAlthough appropriate antibiotic therapy administration has contributed to reduce selective pressure , by limiMany risk factors have been linked to the likelihood of MDR carriage and/or infection, the common ones being prior exposure to antibiotics, prolonged hospital and ICU length of stay, indwelling devices, comorbidities and local epidemiology , 17–19. Empiric isolation is meant for symptomatic cases in which a transmissible infectious disease is suspected but tests have not yet confirmed it. Such strategy is largely adopted in common infectious diseases where symptoms clearly ring the bell of possible diffusion, like contact precautions in diarrhoea or droplet isolation in influenza . By contIn the present paper, we describe a single-centre strategy with the aim of raising some interesting cause for reflection on ICU patient management as far as AMR is concerned. The following strategy refers to a polyvalent Bufalini Hospital ICU , consisting of 23 beds organized into three multibed rooms, one double-bed room and three single-bed rooms. The ICU admits an average of 1,000 patients per year, 280 of which are trauma patients and about 500 are surgical (both emergency and elective). Besides accurate antimicrobial stewardship program .Although such a precautionary policy is well known through hospitals, in literature only a few authors described in detail their protocols. Among these, Abella Álvarez et al.  found thIn this paper we describe our current strategy of placing patients under pre-emptive contact precautions whenever specific risk factors identifies possible MDR colonization, followed by constant monitoring for events that might change the assessment.Since universal isolation seems unrealistic, ineffective and in certain cases detrimental, scrupulous patient selection is warranted , 27. TheOn the other hand, patients not needing unnecessary isolation and contact precautions are spared, thus facilitating nurses’ assistance and medical care. For instance, during SARS-CoV-19 outbreak, the augmented use of personal protective equipment was supposed to cause higher levels of blood culture contaminations, which in turn spoils correct diagnosis and treatment . Other rWe regard monitoring as another key point in IPC. Prevention strategies must be followed by adequate and constant monitoring in order to critically judge their benefits and their disadvantages. Clinic, radiology, procalcitonin trend, surveillance cultures and periodic score calculations are ready-to-use tools but have some predictive limitations. Monitoring local epidemiology through periodic surveillance and systematic screening of new admissions, as well as monitoring afferent wards’ epidemiology, has the double advantage of guiding antimicrobial stewardship and of proving IPC programs’ effectiveness. Mathematical models like transmission coefficient  were designed for the purpose , 34. AutPseudomonasaeruginosa biofilm was found to be significantly less susceptible to disinfection by two oxidative biocides [Despite lack of evidence, we also advocate that IPC interventions should be customized in respect of certain pathogens once they are confirmed. It is well known, in fact, that some MDR organisms such as Candida spp. and Acinetobacter have the ability to adhere to surfaces by means of biofilm formations , 37. Notbiocides . Yu et abiocides . We coulbiocides , but untTesting the effectiveness of different cross-colonization control strategies requires studies with large population size, rigorous screeening measures implementation and a large amount of data on individual patient characteristics and risk factors. This paper is intended to be a call for research on the topic, starting of course with the results of our center.The purpose of this brief report is to illustrate the logic behind a pre-emptive and risk factors-based approach to potential MDR carriers. Aside from sharing knowledge and practices, we also intend to underline how these strategies need to be individualized according to local epidemiology, resources and patient’s characteristics. The importance of developing risk stratification tools has the purpose of keeping CPs as proportionate as possible, in opposition to a blind policy of massive and non-selective screening and isolation. Advantages of a reasoned and customized attitude include costs containment, decreased unnecessary stress to patients and reduced workload for personnel. The final effect would be a beneficial virtuous cycle where effective contact isolation leads to less need for isolation by means of keeping MDR rates low, while healthcare workers’ satisfaction and understanding would lead to even more efficacy by encouraging them to accurately observe protocols.


BNCT is a biologically targeted, densely ionizing form of radiation therapy that allows for increased tumor cell kill, while reducing toxicity to surrounding normal tissues. Although BNCT has been investigated in the treatment of head and neck cancers and recurrent brain tumors, its applicability to breast cancer has not been previoulsy investigated. In this review we discuss the physical and biological properties of various boronated compounds, and advantages and challenges associated with the potential use of BNCT in the treatment of breast cancer. BNCT is a high LET radiation therapy modality that allows for biologically targeted radiation delivery to tumors while reducing normal tissue impacts. Although the clinical use of BNCT has largely been limited to phase I/II trials and has primarily focused on difficult-to-treat malignancies such as recurrent head and neck cancer and recurrent gliomas, recently there has been a renewed interest in expanding the use of BNCT to other disease sites, including breast cancer. Given its high LET characteristics, its biologically targeted and tumor specific nature, as well as its potential for use in complex treatment settings including reirradiation and widespread metastatic disease, BNCT offers several unique advantages over traditional external beam radiation therapy. The two main boron compounds investigated to date in BNCT clinical trials are BSH and BPA. Of these, BPA in particular shows promise in breast cancer given that is taken up by the LAT-1 amino acid transporter that is highly overexpressed in breast cancer cells. As the efficacy of BNCT is directly dependent on the extent of boron accumulation in tumors, extensive preclinical efforts to develop novel boron delivery agents have been undertaken in recent years. Preclinical studies have shown promise in antibody linked boron compounds targeting ER/HER2 receptors, boron encapsulating liposomes, and nanoparticle-based boron delivery systems. This review aims to summarize the physical and biological basis of BNCT, the preclinical and limited clinical data available to date, and discuss its potential to be utilized for the successful treatment of various breast cancer disease states. Given the densely ionizing nature of these high-LET particles, the biological impact of BNCT on tumor cells, compared to a reference radiation type such as X-rays, is expected to be higher. As the high LET particles deposit their energy within a radius of <10 μm, the resulting DNA damage only occurs within the diameter of a single tumor cell, largely sparing adjacent normal tissues. The dense ionization tracks produced along the DNA by high-LET radiation has been shown to produce greater DNA double strand breaks in comparison to low-LET counterparts. Additionally, studies indicate that the clustered DNA damage produced following high-LET radiation is more difficult to repair and is more likely to lead to genomic instability and cell death [Boron neutron capture (BNCT) is an emerging radiation treatment modality that is aimed at improving tumor control while limiting damage to normal tissues. BNCT falls under the category of high linear energy transfer (high-LET) radiation, which is a form of densely ionizing radiation that causes clustered, irreparable direct DNA damage in a less oxygen dependent manner, in comparison to traditional low-LET X-ray-based therapies. Treatment with BNCT involves the targeted delivery of boronated compounds to tumor cells, followed by the irradiation of tumors with epithermal neutrons. The interaction of the nonradioactive boron-10 atoms , poor tumor penetration by the thermal neutrons, and the accumulation of boronated compounds in normal tissues. Despite the discontinuation of US clinical trials, BNCT continued to be investigated in Asia and Europe with the use of tumor selective boronated compounds and better penetrating epithermal neutrons beams, leading to multiple successful clinical trials. In one such trial involving malignant gliomas, the intracranial tumor bed was directly injected with a boronated compound and treated with neutron irradiation. These patients demonstrated an impressive 5-year survival rate of 58%. Similarly, Mishima and colleagues successfully treated 22 patients with cutaneous melanoma with BNCT between the years of 1987 and 2002. This study reported a complete response rate of 68.2% and a partial response rate of 23% ,5.To date, clinical use of BNCT has largely been limited to small Phase I/II trials in difficult to treat tumors or reirradiation situations. BNCT clinical trials have been carried out in a variety of disease sites, including recurrent glioblastoma, primary and recurrent head and neck cancer, sarcoma, meningioma, melanoma, hepatocellular carcinoma, and malignant mesothelioma ,7. A briPotential clinical advantages of BNCT over traditional external beam radiation therapy include its biologically targeted, tumor selective nature, its potentially greater clinical impact given the ability to create more irreparable double strand breaks in tumor cells in a non-oxygen dependent manner, and its capability to largely spare adjacent normal tissues while adequately treating tumor volumes. Despite these potential therapeutic advantages, challenges associated with the widespread adoption of BNCT include the limited availability of suitable neutron beams, difficulty ensuring adequate and selective boron accumulation in tumors, and our limited knowledge regarding BNCT radiation dosimetry and treatment planning aspects . Due to Although breast cancer is the most common malignancy in females worldwide, there has been no prospective clinical investigations regarding the feasibility of BNCT in breast cancer. To our knowledge, the only published data regarding the use of BNCT in breast cancer are case reports. In one such case report, Fujimoto et al. described the treatment of a 65-year-old woman diagnosed with a locoregional recurrence of her previously treated breast cancer within the left axilla. This disease was deemed unresectable due to the involvement of the axillary nerve and was first treated with chemotherapy and conventional radiation therapy without a durable response. BNCT was therefore administered. For the delivery of BNCT, the patient was placed in the supine position and IV boron was infused. The left axilla was then treated with neutron beams directed anteriorly and posteriorly. MRI of the axilla obtained 2 months following completion of treatment demonstrated a significant decrease in tumor mass and the reduction of pain . Despite910B atoms per cell or 20 μg per each gram of tissue [An ideal boron compound for BNCT is one that preferentially accumulates in tumor, has low systemic toxicity, and is rapidly cleared from blood and normal tissues following delivery. The minimum concentration of boron-10 needed to yield lethal cellular damage following BNCT is approximately 1 × 10f tissue . Since tf tissue ,11. Secof tissue .10B (12 times more) than BPA [Once administered intravenously, BSH enters tumor cells via passive diffusion through the plasma membrane . Studiesthan BPA . Hatanakthan BPA . Kageji than BPA . BSH hasthan BPA . Investithan BPA . Anotherthan BPA . While t®, was also approved by the Ministry of Health in Japan for clinical use. This measure came following promising phase II clinical trial data which showed Steboronine to be highly tumor selective and improve the objective response rate in recurrent and locally advanced head and neck SCC compared to historical controls [BPA primarily enters cells through an amino acid transporter known as the L-amino acid transporter (LAT-1) . BPA hascontrols .In addition to these traditional boron delivery agents, there has been increased interest in developing targeted boronated therapies to increase selective tumor uptake and limit the normal tissue toxicity of BNCT. These third-generation boron delivery compounds may be divided into low molecular weight and high molecular weight agents. For instance, Boron-containing amino acids and polyhedral borane clusters are examples of low-molecular weight boron delivery agents. These compounds are of interest due to their high boron content, chemical stability, ease of incorporation into organic molecules, and selectivity for tumors . Other lHigh molecular weight compounds such as monoclonal antibodies and other receptor-targeting moieties have also shown promising results. Nakase et al. (2020) described an antibody-based receptor targeting method using an Fc-Binding Peptide Conjugate. known as Z33. This conjugate was demonstrated to recognize and interact with the Fc domain of human IgG, making it an ideal linker to attach to receptor targeting antibodies. To deliver boron to cell, the Z33 peptide was then linked to the heavily boronated compound, dodecarbonate, and bound to the Fc component of Cetuximab. Human epidermoid carcinoma cell line A431 was treated with EGF and incubated with the Z33- DB- cetuximab compound, which led to its internalization through macropinocytosis and the intracellular release of large quantities of boron. Although this concept is still under investigation, similarly formulated antibody-conjugated boron compounds targeting HER2 or ER receptors may offer novel methods of selectively targeting breast tumors with BNCT in clinical settings [Boron compounds could also be conjugated to lipids to create boron-loaded liposomes. The linkage of boron containing liposomes to monoclonal antibodies such as Cetuximab has allowed for in-vitro targeting of epidermal growth factor receptor (EGFR) expressing rat glioma cells . HER2-taAnother interesting area of research with regard to BNCT involves the development of boron cluster-modified therapeutic nucleic acids that may be utilized in gene therapy and BNCT. In one study by Kaniowski et al. (2017), antisense oligonucleotides targeting the EGFR receptor were synthesized by conjugation of DNA-oligonucleotides with a boron cluster alkyl azide component. In vitro experiments using Hela cells transfected with these constructs showed promising gene silencing and prooxidative properties leading to reactive oxygen species production. Transfected cells showed reduced cell proliferation. The authors noted that this type of dual-action compound could have binary activities of targeting EGFR overexpression in cancer and serving as a novel, targeted boron carrier for the purposes of BNCT .Polymeric nanoparticles have also been investigated as potential boron delivery agents. Boron-containing nanoparticles were demonstrated to have large boron content, acceptable cellular stability, and high accumulation within tumors . The mosThe preliminary studies reviewed above indicate that a variety of compounds may serve as efficient boron carriers. Further preclinical work and in-human trials are necessary to determine the optimal boron carrier system and the appropriate concentrations necessary for effective clinical delivery of BNCT in breast cancer.As noted earlier, BNCT leads to the production of four different types of ionizing radiation, including γ rays protons, Li-nuclei, and most importantly, high LET alpha particles responsible for creating a majority of the BNCT related DNA-damage . DepositCurrently, there are very limited data on methods of BNCT dose calculation in breast cancer. A study by Yanagie et al. (2009) described the BNCT radiation dosimetry in a phantom model of the mammary gland. Based on prior studies using BPA as the boron delivery agent, the authors estimated the boron tumor/blood ratio to be 3, tumor normal tissue ratio to be 3.5 and skin/blood ratio to be approximately 1.2. Using these ratios and irradiation using a thermal neutron beam, the authors calculated that tumoricidal radiation doses could be delivered to the mammary gland. In this model, the maximum tumor RBE dose was determined to be 42.2 Gy-Eq and the mean tumor RBE dose was estimated to be 28.9 Gy-Eq . StudiesA suitable treatment planning software is of paramount importance to accurately predict the dose distribution around the target. A well-designed BNCT treatment planning system should have a pre-processing phase with CT/MRI scans to create a geometric model, the ability to perform calculations of 3D radiation distribution, be able to estimate neutron and gamma fluences, and have a well-developed post processing phase. Several Monte Carlo-based methods were developed over the last three decades . One of 18F-10BPA) may be used to assess the in-vivo distribution of boron in-vivo. Patients may be administered L-18F-10BPA and be imaged with a PET/CT to assess the biodistribution of boron in the tumor, as well as the tumor/normal tissue ratio [In order to clinically implement BNCT, in addition to a neutron source, a patient positioning device, robust treatment planning system (TPS), a neutron beam monitor, and a means to assess boron concentration in tumors are needed . Generalue ratio ,46. Throue ratio . Once true ratio . When thA multidisciplinary approach is applied to the management of breast cancer and typically involves breast-conserving surgery (BCS) with radiotherapy, or mastectomy with or without radiotherapy, as well as the use of neoadjuvant or adjuvant systemic therapy in select situations. Breast conserving therapy and mastectomy are both well-established treatment approaches with similar locoregional recurrence rates (LRR). When radiotherapy is delivered following a lumpectomy, a standard treatment field includes the whole breast with or without regional nodes. In the delivery of post-mastectomy radiation therapy (PMRT), the chest wall and regional nodes are usually included in the field . The delBelow we discuss the potential for BNCT in various breast cancer disease states.Although BCS is well tolerated, there is a small subset of early-stage patients who refuse surgery or have extensive medical co-morbidities and thus surgery poses an excessive risk. There are currently very limited treatment options for these patients, which typically include stereotactic ablative body radiotherapy (SBRT), cryoablation, and/or systemic therapy ,53. GiveIn those with locally advanced breast cancer treated with upfront neoadjuvant chemotherapy, studies indicate that those who achieve a pCR have higher recurrence free survival and breast cancer specific survival  . ExtensiAdditionally, in locally advanced breast cancer patients, BNCT may offer an opportunity to mobilize the immune system to improve cancer control. Khan et al. (2019) published methods describing the encapsulation of boron compounds into liposomes. They found that in murine mammary tumor models, uptake of boron containing liposomes modified the peripheral blood mononuclear cells to an antitumor phenotype, contributing to inhibition on tumor growth. This work suggests that in addition to direct tumor cell death following neutron irradiation, boron compounds themselves may be utilized to improve tumor control through immune modulation .During the treatment of malignancy with external beam radiation, inevitably some doses will be delivered to the surrounding normal tissues. As noted above, a primary advantage of BNCT lies in the fact that it is biologically targeted, thus, normal tissue damage is limited to cells with adequate boron accumulation. This principle is of particular importance in patients with locally recurrent breast cancer following prior irradiation of the chest wall. Further radiation is usually discouraged in this setting due to concerns regarding toxicity. In such locoregionally recurrent breast cancer, BNCT may offer a unique means to deliver ablative radiation doses to the site of recurrence with limited toxicity to adjacent tissues, particularly if surgical resection is not deemed to be favorable. Although it is yet to be clinically investigated, Gadan and colleagues have proposed the use of BNCT to treat locoregional recurrences in HER2+ breast cancers using immunoliposomes labeled with the monoclonal antibody, Trastuzumab. These liposomes may then act as boron carrier nanovehicles to target HER2 overexpressing cells in regions of disease recurrence . A similIn patients with oligometastatic breast cancer, BNCT may facilitate the ablation of multiple lesions in a targeted manner with curative intent. Given that BNCT allows for the biological targeting of disease based on boron distribution and is less likely to be impacted by internal organ motion, patients with oligometastatic disease within highly motion sensitive organs such as the lung and liver may particularly benefit from this therapy.Furthermore, leveraging the proposed abscopal effect of BNCT may help improve disease control in widespread metastatic settings. Although this concept is yet to be fully investigated in clinical trials, Trivillin and colleagues described the abscopal effect of BNCT in a proof of principle experiment in xenografted colon cancer rat models. The authors found that following subcutaneous injection of BPA and irradiation of right hind leg tumors with neutrons, there was a significant reduction in left leg tumor volumes, indicating that BNCT is capable of inducing abscopal effects following treatment , due to BNCT may also be advantageous in the treatment of breast cancer associated metastatic brain lesions. Patients with limited intracranial disease are typically treated with radiosurgery due to its physically targeted nature, which allows for a highly favorable dose distribution, limiting damage to the normal adjacent brain. This approach, however, at times requires subjecting the patient to invasive procedures such as an immobilizing head frame, and necessitates the resource heavy process of ensuring precise targeting of radiation within brain lesions. In the setting of many brain lesions or leptomeningeal disease, whole brain radiation is often performed. Although palliative, this treatment is associated with substantial neurocognitive effects and a negative impact on quality of life due to the irradiation of significant amounts of normal brain. While novel techniques such as hippocampal sparing and medications such as memantine have improved the neurocognitive impact of whole brain radiation therapy, approaches to further reduce these side effects are being investigated ,58. In bBased on data on other malignancies, BNCT has been well tolerated with minimal acute or long-term toxicity; however, infusion reactions, skin toxicity, radiation necrosis at the site of treatment, and pseudoprogression of tumors have been described in the literature ,59,60,61Some anticipated barriers to the widespread adoption of BNCT include the need for the costly establishment of a neutron accelerator, identifying the optimal selective boron delivery agents that are most appropriate for each cancer type, and the need to develop a robust treatment planning system for BNCT delivery. These concepts must be extensively investigated in preclinical studies prior to initiating in-human clinical trials utilizing BNCT in breast cancer. Given the prevalence of BPA in clinical trials and the emerging data indicating LAT-1 overexpression in breast cancer, it is reasonable to begin any BNCT related clinical work in breast cancer using BPA as the boron carrier. Following further research, if a standardized LAT-1 expression assay can be implemented in a clinic, it may allow for the selection of high LAT-1 expressors as potential candidates for BPA based BNCT. Additionally, if novel efforts to create other boron delivery agents are successful (such as those targeted against hormone receptors and nanoparticle and liposome-based boron delivery systems), BNCT may become a possible treatment option even in breast cancer patients with intrinsically low LAT-1 expression. Studies will also be needed to compare BNCT to standard of care treatments in breast cancer patients. With further work, we believe that BNCT may be a propitious strategy for the treatment of early-stage inoperable disease, may improve pCR following neoadjuvant chemotherapy, present new opportunities for the salvage of previously irradiated locoregionally recurrent disease, and may also improve outcomes in the metastatic setting.


Gait dysfunctions are debilitating motor symptoms of Parkinson’s disease (PD) and may result in frequent falling with health complications. The contribution of the motor-cognitive network to gait disturbance can be studied more thoroughly by challenging motor-cognitive dual-task gait performances. Gait is a complex motor task that requires an appropriate contribution from motor and cognitive networks, reflected in frequency modulations among several cortical and subcortical networks. Electrophysiological recordings by scalp electroencephalography and implanted deep brain stimulation (DBS) electrodes have unveiled modulations of specific oscillatory patterns in the cortical-subcortical circuits in PD. In this review, we summarize oscillatory contributions of the cortical, basal ganglia, mesencephalic locomotor, and cerebellar regions during gait and dual-task activities in PD. We detail the involvement of the cognitive network in dual-task settings and compare how abnormal oscillations in the specific frequency bands in the cortical and subcortical regions correlate with gait deficits in PD, particularly freezing of gait (FOG). We suggest that altered neural oscillations in different frequencies can cause derangements in broader brain networks, so neuromodulation and pharmacological therapies should be considered to normalize those network oscillations to improve challenged gait and dual-task motor functions in PD. Specifically, the theta and beta bands in premotor cortical areas, subthalamic nucleus, as well as alpha band activity in the brainstem prepontine nucleus, modulate under clinically effective levodopa and DBS therapies, improving gait and dual-task performance in PD with FOG, compared to PD without FOG and age-matched healthy control groups. The defining motor symptoms in idiopathic Parkinson’s disease (PD) are bradykinesia and rigidity with specific postural and gait abnormalities  . ProgresFOG in PD is a complex phenomenon, whose underlying mechanisms have not been entirely understood. Neuronal connectivity of FOG has been discussed previously . HoweverThere is a probabilistic presentation of FOG episodes in experimental human trials, even with identical environmental conditions, pharmacological steady state, and within the same patient. FOG may not be exhibited in one experimental iteration, but in the next one. Given the experimental difficulty to evaluate FOG, researchers are developing more sensitive and specific clinical and neurophysiological approaches. The occurrence of FOG exhibits high variability and can depend on environmental triggers, emotional state, cognitive loading, and medication; therefore, the frequency and the time of FOG episodes are difficult to measure across PD populations. Moreover, there are methodological limitations to study FOG in the laboratory environment because FOG is a stochastic process and patients who are normally confronted with it on a daily basis are frequently not able to produce it when tested in a gait laboratory. This may be a result of the physical environment, such as visual cues or increased awareness in the testing situation, reducing the likelihood of FOG occurrence.Recent evidence has emphasized the role of cognitive control in lower-extremity movement and gait with the use of dual-task experiments in combined cognitive and motor tasks in PD subjects . DependiAs the disease progresses, PD patients also exhibit more expressed deficits during dual-task activities compared to single-task testing, since a more pronounced cognitive impairment can be seen in the advanced stages of the disease . Recent The use of dual- or multiple tasks to define or enhance neural processing networks has not come without theoretical criticism, particularly for double cognitive tasks . For cogOverall, our review examines cortical and subcortical oscillatory activity in PD patients involved in cognitive-motor dual-task activities.Contributing factors to PD gait disturbance are level of disease progression , part III: motor rating scale), basophobia , musculoskeletal ailments and physical inactivity, proprioceptive issues , balance organ disorders , and cognitive impairments . In thisMotor-cognitive dual-task studies have shown a reduction in gait speed, along with other gait performance parameters like variability, especially when attention sharing is required and prioritized between the two tasks . This oc1Turning the direction of gait is a complex task, requiring the integration of multiple sensory inputs, postural transitions, and inter-limb coordination. Therefore, it can be easily disturbed by challenges to attention and/or executive functions . EvaluatOverall, a wide array of cortical and subcortical regions is needed for goal-oriented gait and walking. “Executive function” describes higher-order sensory information processing within the frontal cortical and subcortical regions among their associated networks. It includes interpreting and analyzing task-related information content and modulating behavior by action and/or inhibition. The executive attention system has also been related to abnormal gait measurements . AnatomiThe ability to manipulate and analyze spatial location and bearing is required during movement to avert falls by correct orientation and position. Morris and colleagues have found that slower steps and turning of gait are associated with poorer attention, as computed by using Stroope reading scale, and lack of visuospatial abilities linked with FOG . DomelloDue to its flexibility and ease of application, scalp electroencephalography (EEG) provides ample studies to examine movement-related neural oscillations in PD and other diseases. Spectral power increases in the theta frequency band have been observed in the supplementary motor cortical and premotor cortical regions in young healthy subjects during walking with an obstacle task . AdditioAccording to current evidence, cognitive control for goal-directed movement is assumed to be mediated by midfrontal theta band activity in healthy and PD conditions , 2021. SIt is notable that the effects of levodopa in the midfrontal theta dynamics were not found during cognitive and lower-limb motor tasks , 2021. TMost EEG studies have explored beta oscillations in the sensorimotor, motor cortex, and frontal regions , whereasBeta oscillations are linked with motor control and increase in power can be seen during upper-limb bradykinesia and motor impairments in PD , 2020. TFewer studies have observed cortical gamma oscillations in association with gait in PD patients. Most gamma band studies were designed to investigate the response to the involuntary dyskinetic motor dysfunction. Swann and colleagues demonstrated that dyskinetic movements in PD subjects can be linked to narrow-band gamma oscillations in the motor cortex between 60 and 90 Hz . HoweverFunctional neurosurgery using DBS has proven to be an effective therapeutic treatment for individuals with PD and other neurological disorders. To better comprehend electrophysiological activity, DBS leads are inserted at various depths in the subcortical structures. Though quite invasive, DBS surgery serves as a unique opportunity to record local field potential (LFP) activity in specific areas of the basal ganglia in humans. Other areas are only accessible in animal studies with limited and less predictable applicability for human diseases. The LFP signal is an extracellular field generated primarily by synaptic transmembrane currents recorded from a cluster of neurons surrounding the depth DBS electrode . It reflThe term basal ganglia comprise structurally and functionally interconnected input/output and intrinsic nuclei. Input striatal nuclei like the caudate nucleus, putamen, and accumbens nucleus receive information from the cortical regions, which are then processed and relayed to the output nuclei, consisting of the GPi and SNr. Both output and input nuclei are connected through the intrinsic nuclei, like the globus pallidus externus (GPe), STN, and substantia nigra compacta. The extent to which information is propagated along the basal ganglia network in PD compared to the healthy condition on different scales is still under debate, from a single-cell based relay with diverse types of neurons, long-term chemical potential storage, up to the population scale neural oscillatory band activity . Each ofSeveral studies have recently illuminated new aspects of oscillatory activity propagation. Striatal cholinergic interneurons induced beta and gamma oscillations in cortical-striatal circuits and were able to influence motor behavior in mouse models . This stThe synergistic interaction between the STN and GPi has been known to affect gait , since iSubcortical theta and alpha oscillations in PD are often described together, since most studies have referred to both as “low- frequency” for their analysis . While mSubthalamic nucleus LFPs recorded from an implanted sensing neurostimulator (Medtronic Activa PC + S) during walking in PD patients showed a modulation in alpha frequency power, which was locked to the gait cycle . These oUnlike the low-frequency oscillations, STN beta band oscillations have generally been examined specifically in response to gait in PD. A recent study demonstrated gait-related peak frequency modulation in the STN beta band in PD patients and classified beta-related neural features, which could consistently differentiate between the standing and walking conditions in each PD patient . An incr® PC + S sensing neurostimulator during gait demonstrated sustained beta burst durations in PDFOG + compared to PDFOG– as a pathological neural feature of FOG. The beta burst durations were shortened during DBS along with an improvement in gait kinematics (The implication of movement preparation on the beta band illustrates the role of cognitive control. Does the planning process quantitatively influence the low frequency oscillations as the motor execution did, and can an improvement in cognitive function modulate the beta band oscillations for execution? This may prove to play a crucial role in cognitive control and improved gait stability. A model has been proposed in which successful action selection and decision making relies on an intact STN activity, which regulates the temporal dynamics of motor control during conflict and inhibits movement until sufficient information to select the right option has been integrated . So far,nematics . Anothernematics . A reducnematics . Later, nematics . In thisnematics . This moAnother frequency band prominent in the cortical-basal ganglia loop in PD lies within the gamma frequency band. Most movement-related studies on the gamma band oscillations focused on upper limb movement in PD. Impairment in grip force activity was correlated with lower gamma activity in the STN . HoweverPrevious studies have analyzed both STN and GPi structures simultaneously, revealing similar effects and suggesting that both basal ganglia nuclei can share similar oscillatory patterns in PD patients . One posStudies have shown that gait and dual-task interferences are highly correlated with an asymmetry in PPN structural and functional connectivity, applied during both simple and complex cognitive tasks in PDFOG + versus PDFOG– . DBS of Another recent study demonstrated a correlation between contralateral foot gait phase and PPN beta band modulation in PD, which was most pronounced during regular stepping rhythms . Fraix aMoreover, during the ON phase, there was a decrease in PPN gamma band oscillations for PDFOG +; this activity was only seen during gait and not in sitting and standing . ModulatCerebellar regions exhibit functional connectivity with extensive motor- and cognitive-related regions. Not much attention has been given to the cerebellar gait-related oscillations, even though activation in the cerebellar region has been observed during both actual and imaginary walking . In a stCerebellar gamma oscillations have not been fully investigated during gait. Intracranial EEG of the human cerebellar cortex may exhibit electrical rhythms as high as 250 Hz, including task-related modulations and these oscillations are in line with electrophysiological studies of cerebellar oscillations in the rodent as well as magnetoencephalographic findings in humans . It shouWe have summarized previous studies on cortical and subcortical oscillations during gait and dual-tasks in PD in The description of mere increases and decreases of frequency band relative power in relation to specific tasks or conditions do not constitute a causation or even necessarily a correlation, which has been criticized as a common fallacy in the field of LFP . Also, tWe emphasize that the cognitive network should be subject to further examination for improving overall gait dysfunctions in PD along the progression of the disease. Future studies, particularly those using mobile recording systems with high temporal and spatial resolution, like mobile MEG systems, will likely provide rapid and deeper insight into cortical and subcortical oscillations during gait-like leg movements and dual-tasks .The development of new experimental techniques like high-density mobile EEG  and wireLong-term electrophysiological recordings starting in patients with the stage of PDFOG– and re-testing them along their individual progression to PDFOG + with gait and dual-task studies may further define the evolution of abnormal neural oscillations in the cortical and sub-cortical regions and networks. Evolving knowledge on these oscillations in health and disease, along with advances in neuromodulation therapies, particularly closed-loop DBS , will heAll authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.