P. ultimum, a representative member in ARD pathogen complex. Genotype-specific plant survival rates and biomass reduction corresponded with microscopic features of necrosis progression patterns along the infected root. The presence of defined boundaries separating healthy and necrotic sections likely caused delayed necrosis expansion in roots of resistant genotypes compared with swift necrosis progression and profuse hyphae growth along infected roots of susceptible genotypes. Comprehensive datasets from a series of transcriptome analyses generated the first panoramic view of genome-wide transcriptional networks of defense activation between resistant and susceptible apple roots. Earlier and stronger molecular defense activation, such as pathogen perception and hormone signaling, may differentiate resistance from susceptibility in apple root. Delayed and interrupted activation of multiple defense pathways could have led to an inadequate resistance response. Using the panel of apple rootstock germplasm with defined resistant and susceptible phenotypes, selected candidate genes are being investigated by transgenic manipulation including CRISPR/Cas9 tools for their specific roles during apple root defense toward P. ultimum infection. Individual apple genes with validated functions regulating root resistance responses can be exploited for developing molecular tools for accurate and efficient incorporation of resistance traits into new apple rootstocks.Apple replant disease (ARD), caused by a pathogen complex, significantly impacts apple orchard establishment. The molecular regulation on ARD resistance has not been investigated until recently. A systematic phenotyping effort and a series of transcriptomic analyses were performed to uncover the underpinned molecular mechanism of apple root resistance to  Malus domestica Borkh.) is one of the most popular perennial tree fruits in temperate regions around the world1. Apple replant disease (ARD) refers to stunted growth or death of newly planted trees at a replant site where apple or closely related tree species have been previously cultivated. The causal agents of ARD consist of a pathogen complex, including necrotrophic soilborne oomycetes (Phytophthora and Pythium) and fungi (Ilyonectria and Rhizoctonia)3. Among them, Pythium ultimum is known to be one of the primary members in this pathogen complex, which has been identified in orchard soils worldwide5. Similar to other root diseases, the effective control of ARD is often hampered because of the persistent survival of soilborne pathogens, which form overwintering structures such as oospores, chlamydospores, and sclerotia7.Apple 24. Building on a recent phenotyping effort on apple root resistance traits to P. ultimum infection, a systemic approach including a series of transcriptome analyses and subsequent functional validation of selected candidate genes, genotype-specific defense activation patterns and their potential contribution to apple root resistance traits began to emerge. A unique trait to apple, as a rosaceae woody crop, is that the reproduction of apple is self-incompatible or outcrossing in nature, and the apple genome has high-level heterozygosity25. Each seed in a fruit represents a unique genetic identity, and therefore seed germination cannot generate plants with identical genetic background. This review primarily focuses on the pathosystem between apple roots and soilborne necrotrophic P. ultimum, except some additional works on the molecular interactions between apple root and Rhizoctonia solani AG-5, another ARD pathogen. This review can be considered as a companion to an earlier perspective review on investigating the molecular basis of apple root resistance to ARD7.The current understanding of plant molecular defense responses is derived primarily from studies using foliar pathosystems. Specifics and unique aspects of root defense against soilborne pathogens, especially for perennial tree crops like apple, remain largely unclear27. Plants use a sophisticated surveillance system to detect the presence of pathogens and subsequently initiate appropriate defense responses according to the pathogen types28. It has been demonstrated that pattern recognition receptors (PRRs) located on the cell membrane detect the signature molecules or pathogen-associated molecular patterns (PAMPs), which are often conserved within the same class of pathogens29.Plants are equipped with a versatile and tightly regulated immune system through coevolution with their pathogens. Such a sensitive immune system allows plants to discriminate between beneficial and pathogenic microbes in their surroundings28. Upon PRRs perceiving the conserved PAMP, plants activate a process known as PAMP-triggered immunity (PTI)31, which adapted pathogens can suppress or bypass by secreting evolved effector proteins32. On the plant side, co-evolved resistance (R) proteins directly or indirectly interact with pathogen-derived effectors and initiate the second layer of defense, i.e., effector-triggered immunity (ETI)33. PTI is generally considered to be a basal immune reaction, and ETI can lead to a stronger and more specific defense response toward those pathogen isolates that produce the recognized effector.An optimized defense output requires coordinated reprogramming of cellular processes and efficient redirection of metabolic activities in plant cells. Plants utilize a two-layer immune system, or zig-zag model, to combat pathogen aggression37, and plants use discrete hormone balances and fine-tuning of crosstalk to deal with various attackers. SA-regulated defense mechanisms are activated in response to biotrophic pathogens, whereas JA/ET-mediated signaling pathways are critical to plant defense responses to necrotrophic pathogens39. Crosstalk with other plant hormones, such as auxin, abscisic acid (ABA), and gibberellic acid (GA) can result in multiple feedback loops to modulate gene expression patterns and feed-forward loops to coordinate expression intensity and duration of specific genes41. Responding to specific hormones or other defense signals, several families of plant TFs are activated, including WRKY (containing WRKYQK protein domain), ERF (ethylene response factor), and MYB (myeloblastosis oncogene)43.It has been well established that plant hormones, such as salicylic acid (SA), ethylene (ET), and jasmonic acid (JA), are the vital components of plant defense responses46. Phytoalexins are small molecules of extreme structural diversity, and they can be generally categorized into three main classes of phytochemicals, including terpenoids, phenylpropanoids, and alkaloids48. More recently, cellular small RNAs (sRNAs), including microRNA (miRNA) and small-interfering RNAs (siRNA), have been demonstrated to be actively participating in both host immunity and pathogen virulence49. In many cases, miRNAs are known to regulate PTI via targeted transcripts of genes functioning in hormonal signaling or as TFs. At the same time, numerous miRNAs have been shown to directly target transcripts of NB-LRR (nucleotide binding/leucine-rich repeat) genes, a class of resistance (R) genes predominantly functioning in ETI51.Both preformed antimicrobial compounds (phytoanticipins) and pathogen infection-induced production of antimicrobial secondary metabolites  are believed to contribute to pathogen resistance56. Previously observed ARD resistance or tolerance for selected apple rootstocks based on field evaluation likely involve a combination of multiple functional mechanisms, such as fine root development58. Well-designed experiments under controlled conditions are required to minimize compounding factors, as reliable and detailed apple root resistance responses are the prerequisite for analyzing the underlined molecular regulation of apple root resistance to P. ultimum infection13. However, compared with the aerial parts, phenotyping resistance traits of plant roots is more challenging. The hidden nature of root systems in soil limits accessibility and hinders the noninvasive, nondestructive evaluation of their detailed resistance responses, though recently developed methods allowed direct observation of apple root growth behavior in a pot with a transparent wall59. In addition, the small stature of the individual young (feeder) roots presents a challenge for direct observation and documentation of the detailed features of apple root pathogenesis processes60. The heterogeneous or non-synchronized differentiation processes among individual root branches present another practical hurdle for consistent evaluation of apple root resistance behaviors. More significantly, an extra obstacle for apple as a rosaceous species is the continued availability of plant root tissues for repeated infection assays.Molecular regulation of defense activation in apple roots upon infection from soilborne necrotrophic pathogens, such as those inciting ARD, has not been investigated until recently62, although using of in vitro-propagated apple plants is becoming more common65. For example, a comprehensive screening of ARD tolerance was carried out among multiple apple rootstock genotypes using in vitro propagation and steam-disinfected ARD soil as control, and under greenhouse conditions65. In most cases, physiological parameters, including tree height, stem diameter, and accumulated fruit yield, were used to infer tolerance to ARD indirectly, without inquiring the intrinsic pathological features in apple roots under pathogenic pressure. Field performance of a rootstock can be influenced by multiple factors, including root regeneration dynamics, nutrition uptaking efficiency, adaptability to certain soil types, and the effect of scion genotypes on rootstock activities10. While it is a viable approach to assess the overall rootstock performance to disease pressure under field conditions, it can be problematic using these nursery-generated rootstock trees to collect consistent and detailed resistance phenotypes. Moreover, the availability of these 1-year-old bare-root trees is generally restricted to a few elite commercial varieties, as well as being limited to a short time window during the year. In addition, the root systems of these trees have been exposed to various soil microbes or impacted by inadvertent abiotic conditions in the nursery. More importantly, due to the destructive nature of pathogen infection, the reliability of phenotype data requires repeated infection assays. Therefore, a continuous supply of plants from any apple rootstock genotype is crucial for a detailed and systematic phenotyping effort. Plant tissue culture based on in vitro micropropagation of apple plants is the method of choice for this purpose.Many horticultural traits and disease tolerance of apple rootstocks were traditionally evaluated under field conditions using stool-bed propagated 1-year-old rootstock “sticks” or trees from commercial nurseries66. The synchronized micropropagation procedure allowed the simultaneous analysis of the root resistance responses for multiple genotypes to the infection by the same pathogen inoculum preparation  Continuous supply of comparable apple plants for selected rootstock genotypes by tissue culture-based micropropagation; (2) quantifiable inoculum level and standardized inoculation protocol for consistent resistance evaluation; (3) multiple methodologies developed for evaluating root resistance responses at whole-plant and tissue levels. The phenotyping protocol was initially established using two apple rootstock genotypes, G.935 and B.9, with reported ARD tolerance and susceptibility under field conditions, respectively62. Subsequently, a systematic evaluation of resistance responses was carried out for more than 60 F1 progeny derived from a cross between “Ottawa 3” × “Robusta 5” (O3R5), two elite apple rootstock parents53.Oomycete pathogen 53. This observation indicated that the level of pathogen inoculum (2 × 103 oospores) and root dipping as the inoculation method can effectively distinguish between the resistance levels among tested germplasm68. The whole-plant resistance responses, including partial wilt or plant mortality, were visually evaluated daily and recorded at 3, 7, 10, 14, 21, and 28 dpi (days post inoculation). Although the overall survival rate was assigned based on the data at 28 dpi, genotype-specific plant mortality was generally stabilized at 7 dpi. Some genotypes with extreme susceptibility exhibited observable wilting symptoms as early as 3 dpi68. For simplicity, those genotypes showing consistent survival rates of less than 30% were designated as “susceptible”, and those greater than 80% as “resistant”53. Biomass reduction (for either root or shoot) showed statistically significant differences at 28 dpi, when the values of the surviving plants from P. ultimum inoculation were compared with those of mock-inoculated control plants for most susceptible genotypes. The opposite held true for the more resistant genotypes53. Selected genotypes with distinct resistance responses were also subjected to a more focused microscopic examination.A wide spectrum of resistance responses was observed among the tested O3R5 genotypes Fig. . The gen69. First, using a custom-made glass-box pot -based transcriptome analysis can simultaneously identify and quantify the entire inventory of the expressed genome in apple root tissues. Improved bioinformatic software can offer a wide-angled and high-resolution view of transcriptome landscapes in apple roots during interaction with P. ultimum72.The timing, intensity, and dynamics of plant defense responses vary depending on the pathosystem, plant genotypes, and tissue types. Until recently, knowledge was essentially nonexistent regarding the molecular regulation of apple root defense activation under pathogenic pressureP. ultimum as a representative ARD pathogen, the primary goal of the first (out of a series of three) transcriptome analysis was to reveal the timeframe of molecular defense activation, and to identify the main categories of differentially expressed genes (DEGs) in infected apple roots56. By comparing the transcriptomes of mock-inoculated and P. ultimum-inoculated roots, the results clearly demonstrated that the apple root defense response peaked at 48 hpi (hour post inoculation) among eight time points ranging from 0 to 96 hpi  and wall-associated receptor kinase (WAKs), were among the notable groups of regulated apple genes. Genes encoding proteins functioning in the biosynthesis and signaling of several plant hormones, including JA, ET, and cytokinin, made up the most recognizable functional groups. Genes encoding enzymes for secondary metabolisms, cell wall fortification, and pathogenesis-related (PR) proteins, laccase, mandelonitrile lyase, and cyanogenic beta-glucosidase represented the wide spectrum of cellular activities with the effort to ward off pathogen progression56. The results from this dataset, particularly the timeframe of defense activation, served as a valuable guideline for designing subsequent experiments to compare transcriptome changes between a resistant and a susceptible apple rootstock genotype69.Using hpi Fig. . Using tP. ultimum infection. About half a billion paired-end 150-bp reads were generated using the Illumina Solexa HiSeq 3000 platform, with the experimental design encompassing two treatments (mock-inoculated and P. ultimum inoculated), three biological replicates, and four time points  for each genotype  were speculated for this severely disturbed transcriptome and the resulting susceptibility in B.9 roots52. The results also revealed that, even before the presence of pathogen, a preformed molecular defense network appeared to be robustly functional in the roots of the resistant G.935, but to a lesser degree in B.955. Although the existence of a constitutively expressed molecular defense network seems to be contradictory to the theory of trade-off between growth and defense, enhanced readiness for an organ (like root) may also be evolutionarily beneficial, as it is more than likely to encounter adverse biotic stress73. These RNA-seq datasets offered the first comprehensive view of molecular defense activation to P. ultimum infection and identified a list of candidate apple genes that may potentially play a role in differentiating resistance from susceptibility of apple roots. In addition, by taking advantage of the huge dataset of gene expressions in apple roots, a set of stably expressed apple genes were validated as the preferred reference genes, which are valuable for subsequent functional validation using qRT-PCR technique74.A subsequent comparative transcriptome analysis was designed to detect the genotype-specific patterns of defense activation in the roots between the susceptible genotype B.9 and the resistant genotype G.935 in response to ype Fig. . Side-bymes Fig. , TFs, anmes Fig. 52. Lack 76. The preferential targets include those genes encoding R proteins, transcription factors (TFs), hormone biosynthesis and signaling51. Understanding the regulatory roles of sRNAs and identifying their target genes is crucial for further pinpointing the major cellular processes and key candidate genes regulating apple root resistance. Using a selected panel consisting of three pairs of susceptible and resistant apple rootstock germplasm, a focused miRNA profiling analysis and the associated degradome analysis are being carried out to identify the miRNA families and their target genes at the key stage of pathogenesis at 48 hpi , a functional PRR during apple root–Rhizoctonia solani (AG-5) interaction54. MdCERK1 was expressed primarily in the vegetative tissue of root and leaf, and its expression levels in apple root were induced in response to chitin treatment. The ability of purified GST-MdCERK1 fusion protein to bind chitin molecules added biochemical evidence to its role in chitin-mediated immune responses. An untargeted proteomic approach identified its putative in vivo- interacting partners, including PR-4 protein in apple roots inoculated with R. solani. These data support the conclusion that MdCERK1 is a chitin-binding receptor kinase that functions in apple root defense activation. Transgenic manipulation on its in planta expression is underway for validating its genetic identity in apple root resistance to R. solani. These omics-based analyses offered a wide-angle view of molecular defense responses and identified a list of apple candidate genes. The subsequent functional analyses should determine their potential contribution to apple root resistance to soilborne necrotrophic ARD pathogens.The regulatory role of sRNA has been unequivocally demonstrated recently in silencing selected targeted genes related to plant immune responseshpi Fig. . These r56, several groups of candidate genes were selected for further investigation. These include candidate genes functioning as receptors, TFs, hormone signaling, R proteins, and secondary metabolism pathways. The consistent transcriptional profiles among expanded groups of apple rootstock germplasm should be the preliminary evidence for their association with resistance traits. The more definitive evidence of their molecular functions in contributing to resistance/susceptibility can be derived from the in planta manipulation of their expression by gene knockout or overexpression. Candidate genes with robust correlations between gene expression patterns and resistance levels are being further investigated using transgenic manipulation. The transgenic approach is particularly valuable for a non-model plant like apple as a perennial tree crop, which has a high level of heterozygosity in its genome and lacks the feasibility of applying large-scale and high-throughput mutagenesis77. Agrobacterium-mediated genetic transformation as a delivering system for introducing recombinant DNA (or transgene) has been a well-developed tool for basic research and crop improvement for several decades78. More recently, CRISPR/Cas9  technology by double-strand break-mediated genome editing has gained incredible momentum in recent years. For its accuracy on site-directed mutagenesis and applicability to a wide range of organisms, the CRISPR/Cas system has emerged as the most promising tool for targeted mutagenesis81. Taking the advantage of our existing tissue culture platform, transgenic lines with knocked-out target genes have been generated. Analyzing the potential phenotypic alterations will provide critical insights for assigning the functional identity of tested candidate genes during apple root defense response to P. ultimum infection.Based on the previous transcriptome analyses84. For example, deposition of callose, a high-molecular-weight β--d-glucan polymer, is a common form of plant defense response for cell wall fortification at the infection site85. Besides functioning as a physical barrier to pathogen progression, callose at the nanopore structure in cell walls is believed to also serve as a platform for the directed deposition of antimicrobial compounds85. Lignification is another commonly observed cell wall reinforcement for restricting pathogen advances. Cinnamyl alcohol dehydrogenase (CAD) is a major biosynthetic enzyme, which catalyzes the oxidative cross-linking of the monolignans into long-chain polymers of insoluble lignin86. Our transcriptome analyses have identified multiple apple callose synthase and CAD-encoding genes that are specifically downregulated in the root of the susceptible B.9 genotype at 48 hpi52. Methodologies of histochemical staining and image-based quantification89 will validate the relationship between the genotype-specific patterns of cell wall enforcement and observed apple root resistance traits. Several assays were reported for determining the enzymatic activities related to hormone biosynthesis and secondary metabolism, such as lipoxygenase (LOX), chalcone synthase (CHS), and phenylalanine ammonium lyase (PAL)90. These biochemical analyses and enzymatic assays will be valuable tools to add evidence for validating the functional identity, and to assess their contribution to apple root resistance or susceptibility.Many biochemical assays for detecting early and late cellular defense responses have been reported, such as ROS burst, callose deposition, and lignin formation, as well as enzymes catalyzing the generation of antimicrobial phytoalexins91. For a perennial tree crop like apple, multiple obstacles stand before the systematic and reliable characterization of the detailed root resistance traits. The lack of a continuous supply of genetically uniform apple plants for repeated infection assays and limited accessibility for detailed characterization of root pathogenesis processes are two examples. Although it is a laborious and time-consuming procedure, in vitro micropropagation of apple plants allowed consistent and detailed phenotypic analysis for more than 60 apple rootstock genotypes. In addition, using custom-made glass-box pots, along with the assistance of a dissecting microscope, allowed the non-interruptive and nondestructive observation of symptom development on the partially exposed apple roots68. For the first time, genotype-specific resistance traits of apple roots to P. ultimum infection were described in detail at both whole-plant and tissue levels. Along the infected roots of resistant genotypes, the presence of the defined boundaries separating healthy and necrotic sections likely resulted in the limited necrosis expansion and indicated an effective resistance mechanism. In contrast, the swift necrosis progression and the profuse growth of pathogen hyphae most likely indicated the lack of an effective defense and failed effort to restrict pathogen development53. The phenotyping effort using these innovative methodologies resulted in a panel of apple rootstock germplasm with well-defined resistance levels. The availability of this set of apple rootstock germplasm provides a solid foundation for meaningful molecular analysis aimed to understand resistance mechanisms and subsequent exploitation of natural resistance in the future.Maximized exploitation of plant genetics for managing root diseases requires dissecting the molecular regulation networks controlling plant root defense activation and resistance traits toward soilborne pathogens. In the post-genomics era, a lack of high-quality phenotypic data remains a major operational bottleneck for genetic studies on target traits, and therefore hinders the realization of genetic potential contributing to agricultural productivity and sustainabilityP. ultimum infection56. As illustrated in Fig. 52. Delayed and interrupted defense activation, such as those related to production and transportation of antimicrobial secondary metabolites, may have resulted in insufficient defense. The inability to effectively cope with pathogen toxins, weakened ETI, or existence of a susceptibility gene are other possible factors to a highly disturbed transcriptome in the root of the susceptible genotype. Using the panel of apple rootstock germplasm with defined resistance traits from the recent phenotyping efforts, experiments are being carried out with the aim to establish the correlative or causal relationship between individual candidate genes and root resistance traits. The functional identities of selected apple candidate genes are being validated through investigation of their induction patterns among an expanded apple rootstock germplasm set, transgenic manipulation, and subsequent biochemical analyses on potentially altered resistance phenotypes.The comprehensive transcriptome datasets from a series of experiments offered the first panoramic view of genome-wide transcriptional networks regulating apple root defense activation toward 24; therefore, current understanding of apple root resistance responses needs to be further scrutinized in the context of apple root living in a complex soil microbiome. Answers to these questions are beyond the scope of the current study, and novel approaches and continuing investigations are certainly required. The current review attempts to summarize the progress on the systematic phenotyping effort on apple root resistance traits and transcriptome analyses on genome-wide activation of defense responses in response to P. ultimum infection. The developed phenotyping methodology and resulted plant materials with defined resistance traits, as well as acquired information on genotype-specific molecular defense mechanisms, represent a significant advancement for this minimally investigated research topic. Recently, substantial progress has been made on the investigation on the molecular regulation of apple root resistance/tolerance to ARD, using a reference apple rootstock genotype “M26” and ARD soil92. Integration of current knowledge and additional analytic capability from other branches of omics will be necessary, such as metabolomic analysis for identifying specifically enriched antimicrobial compounds during P. ultimum-root interaction. The better understanding of the relationship between specific apple genes and apple root resistance traits is critical for future development and deployment of molecular tools for efficient and precise incorporation of resistance traits into the next generation of resistant apple rootstocks.Multiple genetic and/or environmental factors contribute to the molecular controls of apple root resistance traits. Therefore, many questions related to resistance in apple roots remain unanswered and will need to be addressed with future studies. For example, will the observed resistance traits be the same in the older plants? Do the observed resistance phenotypes to a singular pathogen and under controlled conditions bear any similarity with the overall apple rootstock resistance to ARD under field conditions? What are the effects of scion cultivars on rootstock resistance? Perhaps more relevant is that under field conditions, plant roots interact with a plethora of nonpathogenic and symbiotic microorganisms in addition to pathogens


Ischemic heart disease is the leading cause of death worldwide. The blockade of coronary arteries limits oxygen-rich blood to the heart and consequently there is cardiomyocyte (CM) cell death, inflammation, fibrotic scarring, and myocardial remodeling. Unfortunately, current therapeutics fail to effectively replace the lost cardiomyocytes or prevent fibrotic scarring, which results in reduced cardiac function and the development of heart failure (HF) in the adult mammalian heart. In contrast, neonatal mice are capable of regenerating their hearts following injury. However, this regenerative response is restricted to the first week of post-natal development. Recently, we identified that cholinergic nerve signaling is necessary for the neonatal mouse cardiac regenerative response. This demonstrates that cholinergic nerve stimulation holds significant potential as a bioelectronic therapeutic tool for heart disease. However, the mechanisms of nerve directed regeneration in the heart remain undetermined. In this review, we will describe the historical evidence of nerve function during regeneration across species. Specifically, we will focus on the emerging role of cholinergic innervation in modulating cardiomyocyte proliferation and inflammation during heart regeneration. Understanding the role of nerves in mammalian heart regeneration and adult cardiac remodeling can provide us with innovative bioelectronic-based therapeutic approaches for treatment of human heart disease. In this review, our goal is to highlight the evolutionarily conserved role of nerve signaling in promoting regeneration following injury across multiple tissues and different species. More importantly, we highlight the importance of cholinergic nerve signaling in regulating neonatal mouse heart regeneration, where ablation of cholinergic nerve signaling either pharmacologically or mechanically resulted in impaired cardiac regeneration and revealed a previously unappreciated role for nerves in cardiac regeneration Fig. a. In addNerve dependent regeneration has been observed in multiple tissues across different species  following injury, yet these isolated cells showed little capacity to differentiate into cardiomyocytes or promote any meaningful cardiac repair  macrophages (CCR2+) accumulate and act to scavenge any debris and create a pro-inflammatory environment. The M2 (Ly6lo) macrophages (CCR2-) follow to promote extracellular matrix (ECM) deposition and fibrotic scar formation, angiogenesis, and promoting anti-inflammatory cytokines like IL-10 /CCR2- expression. Other post-natal recruited cardiac macrophages are MHC-IIlow/CCR2-, and a third population that are CCR2+ arise from definitive hematopoiesis  on macrophages, monocytes, lymphocytes, and other components of the immune system to modulate the production of pro-inflammatory cytokines  in rats, dogs, rabbits, and pigs in chronic heart failure  have shown mixed results thus far. The first study reported had only 8 male patients with chronic heart failure, who were fitted with an implantable CardioFit  neuro-stimulator with electrodes that measured heart rate and would cease stimulation if the heart rate dropped below 55 bpm. The average intensity of the stimulation was 4.3 ± 0.9 mAmp with few side effects including pain, cough, and voice alteration. Patient’s quality of life (MLwHF score) was markedly improved after 1 month. In addition, decreased LV end-diastolic and end-systolic volumes, and lower circulating IL-6 levels were observed after 3 months  was randomized among the 60 patients, who still received pharmacological therapy. The researchers noted that there was no difference in safety of the device implantation between the left or right side. There was a significant increase in LV ejection fraction across the whole cohort. Quality of life (MLwHF score) and the 6-min walk score also showed significant improvements after 6 months. It was noted that there was a trend towards right side stimulation being slightly more beneficial than the left, but this was not statistically significant  plus continued medical therapy, or medical therapy alone. This trial concluded that VNS did not increase survival after HF, however it did increase quality of life (KCCQ score) and improved the 6-min walk test with patients being followed up to 4.3 years post VNS implant which is longer than any of the other previous studies , which has already been explored in humans for heart failure treatments. The different results between these studies largely stems from the lack of understanding of the mechanisms by which VNS exerts its function. This underscores the importance of analyzing the molecular effects of VNS on the heart following injury in adult mice, which will guide future clinical investigation and optimization of vagus nerve stimulation. In Fig. Finally, the current rise in aging populations presents a major health care challenge due to the increase in the number of patients with cardiovascular diseases. The emerging role of cholinergic nerve function in regulating heart regeneration provides us with a unique approach to develop novel therapies for heart disease. Unraveling the molecular underpinnings by which cholinergic nerves mediate heart regeneration is an important frontier against the battle to overcome cardiovascular diseases.


Pancreatic cancer remains one of the leading causes of cancer-related death worldwide and has a poor prognosis. Current treatment relies on surgical resection and adjuvant therapies. The gut microbiota plays important roles in metabolism and immunomodulation. Accumulating evidence has implied that the gut microbiota is involved in the metabolism of chemotherapeutic drugs and the tumor microenvironment (TME), which could affect the efficacy of both conventional chemotherapy and immunotherapy for pancreatic cancer. Herein, we comprehensively reviewed the history and highlights of the interactions among pancreatic cancer, the gut microbiota and therapeutic efficacy and showed the promising future of manipulating the gut microbiota to improve clinical outcomes of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 85% of pancreatic cancer cases. PDAC is still one of the most devastating malignancies, with a 5-year overall survival of less than 10%. Since less than 20% of PDAC patients have the opportunity for surgical resection, chemotherapy remains the main treatment option. Since its approval by the Food and Drug Administration (FDA) in 1996, gemcitabine has been actively used to treat PDAC that has progressed in the extensive desmoplastic microenvironment surrounding the few remaining cancer cells, but chemoresistance and reduced sensitivity are often acquired during multiple weeks of chemotherapy cycles 14 microorganisms, and it encodes far more genes than the human body The gut microbiota has been recognized as a considerable ecosystem, comprising over 10To date, common techniques used to assess microbial communities, e.g., 16S ribosomal RNA (rRNA) sequencing, metagenomic sequencing, quantitative PCR and culturomics, have greatly expanded our knowledge of the diversity and multifunctionality of the microbiota. Herein, we summarize the history and progression of the reciprocal interactions between the gut microbiota and therapeutic efficacy in PDAC and discuss how these developments have paved the way to improve patient survival.Difficult issues regarding chemotherapy and immunotherapy of advanced-stage PDAC mainly concentrated on the heterogeneous efficacy of individuals who had the same histopathologic tumor characteristics. Essential factors determining drug sensitivity include pancreatic cancer cells and their surrounding components, such as the extracellular matrix, immune cells , myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils), cancer-associated fibroblasts, pancreatic stellate cells and cancer stem cells, all of which impair the normoxic microenvironment of the pancreas Acinetobacter, Aquabacterium, Oceanobacillus and RahnellaRecent studies have emphasized the difference in the gut microbiota between cancer patients and healthy individuals. The microbiota of the duodenal mucosa in PDAC patients and healthy controls shared similar species in one study. However, duodenal samples of PDAC were characterized by enrichment with, for example, The mechanisms of the TME in PDAC that affect the efficacy of therapies remain largely unknown and require further exploration. Recently, accumulating evidence has shown that the gut microbiota has a potential role in regulating cancer-related immunomodulation and treatment, presenting new targets to improve therapeutic efficacy.Based on previous publications in the past decade, the gut microbiota may lead to altered efficacy of pharmacotherapeutics in cancer treatment Table . AlthougPrevious evidence has demonstrated that both cancer cells and the related TME components participate in cancer development and treatment adaptation. Recent studies revealed that the gut microbiota may contribute to the efficacy of conventional chemotherapeutic agents through drug metabolism, biotransformation and immune regulation in vitro and in vivo. Previously, the clinical potential for microbial therapeutic use was indicated by the synergism of Salmonella typhimurium in mouse models of PDAC treated with gemcitabine and bevacizumab Lactobacillus plantarum was shown to have positive effects on improving the chemosensitivity of 5-fluorouracil (5-FU) in colorectal cancer (CRC) cells by inhibiting cancer stem-like cell formation + T cells perform important duties in the adaptive antitumor immune response. The commensal bacterial species Enterococcus hirae (E. hirae) and Barnesiella intestinihominis were identified in CTX-induced immunomodulation, with altered TME and enhanced anticancer CTL responses. These bacteria were capable of partially restoring host T cell responses and improving the therapeutic efficacy of CTX or other alkylating agents + T cells and innate immunity The gut microbiota has favorable effects on chemotherapy Fusobacterium nucleatum in CRC, were found to promote chemoresistant status Caenorhabditis elegansProteobacteria and Verrucomicrobia and a decrease in Firmicutes and Bacteroidetes, as well as activation of the NF-κB inflammatory pathway in tumor tissues However, microbiota, e.g. in situ and systemic inflammation. A recent study reported that microbial stimulation of cancer cells overexpressed cathepsin K, which promoted immunosuppressive M2 TAM polarization through the TLR4-mTOR pathway Listeria monocytogenes modified the suppressive cancer microenvironment by reducing peripheral and intratumor MDSCs and repolarizing the TAM subpopulation from the M2 phenotype to the antitumor M1 phenotype Bacteroides species activate Th1 immune responses and promote the maturation of DCs within tumors Faecalibacterium and butyrate-producing bacteria were associated with Foxp3+ regulatory T cell (Treg) accumulation in the gut, whereas Bifidobacterium adolescentis, Parabacteroides merdae, Collinsella aerofaciens (C. aerofaciens), and Enterococcus faecium (E. faecium) inhibited Tregs in humans G12D/+, Trp53R172H/+, Pdx-1cre (KPC) mice were comparatively analyzed. Microbial ablation led to significant changes in critical components of the TME, presenting as increased populations of IFNγ+CD4+CD3+ T helper 1 cells and IFNγ+CD8+CD3+ cytotoxic T cell 1 (Tc1) population, with a simultaneous decrease in protumor immune cells Unlike conventional chemotherapy, immunotherapy targets the immune microenvironment beyond the tumor cells. One of the crucial mediators linking the microbiota to the immune response is TLRs, which are categorized as cytoplasmic pattern recognition receptors. TLR4 binding to bacterial lipopolysaccharides triggers Clostridiales in anti-PD-L1 responders with enhanced antitumor immune responses, whereas nonresponders were enriched with BacteroidalesBifidobacterium longum, C. aerofaciens, E. faecium and Bacteroides caccae, was detected in immunotherapeutic responders versus nonresponders among metastatic melanoma patients Bifidobacterium in mice directly stimulated DCs and induced the maturation of DCs. These novel findings indicated the potential of the gut microbiota for regulating host responses towards immunotherapies. Routy et al. Akkermansia muciniphila (A. muciniphila) dysbiosis, which showed a restoration of PD-1 blockade resistance in mouse tumor models after oral administration. Intestinal A. muciniphila increased the recruitment of CCR9+CXCR3+CD4+ T cells in the tumor bed, suggesting that future immunotherapeutic targets could manipulate the gut microbiota in individuals with cancer. Furthermore, in vitro, A. muciniphila and E. hirae stimulated DCs to secrete interleukin-12 (IL-12), which is the crucial cytokine for Th1 cell differentiation and function Attention has been paid to ICIs in the clinic. Immunotherapy targeting PD-1 has emerged as an effective strategy for the treatment of several cancers. Accumulating data revealed that T cell infiltration and variable immune regulators, including the gut microbiota, were associated with PD-1/PD-L1 blockade in patients, with beneficial outcomes for some cancers Briefly, host immunity and TME always play crucial roles in microbiota-modified therapeutic responses. Specific gut microbiota have the potential to predict the efficacy of certain kinds of immunotherapies, and colonization of tumor-specific bacteria has been found to play regulatory roles in the antitumor effects of immune-targeting treatment. The presence of microbiota-derived mediating factors and host variability will create a heterogeneous local TME and relevant alterations in systemic communication.Recent advances have begun to elucidate the potential roles of intratumoral microorganisms in anticancer therapeutics, e.g., pancreatic cancer Helicobacter pylori and gastric cancer, Salmonella typhi and gallbladder cancer, and altered bacterial species and CRC Mycoplasma hyorhinis (M. hyorhinis), which was shown to have a relationship with gemcitabine drug resistance M. hyorhinis infection led to weakened therapeutic efficacy of gemcitabine treatment via the microbial enzyme cytidine deaminase (CDD). Gemcitabine  was metabolized into its inactive deaminated form, 2',2'-difluorodeoxyuridine (dFdU), in M. hyorhinis-infected conditioned cultures Gammaproteobacteria class, which contain the enzyme CDD; this enzyme was indicated to be responsible for the ineffectiveness of gemcitabine in PDAC L) and short CDD (CDDS). The expression of the resistance-related isoform CDDL led to the metabolism of gemcitabine Escherichia coli (E. coli)-induced chemical structure modification of gemcitabine, fludarabine, cladribine and CB1954 E. coli lowered the cytotoxicity of gemcitabine in vitro and in subcutaneous colorectal carcinoma models containing intratumoral bacteria The association of gut bacteria and tumor tissue has been previously reported, such as Fusobacterium species, a group of oral bacteria, were initially detected in PDAC tissues by Mitsuhashi et al. Fusobacterium species was found to be relatively higher in PDAC subjects than in noncancer controls Fusobacterium nucleatum elicited chemoresistance to 5-FU and oxaliplatin in CRC, targeting TLR4 and MyD88 immune signaling and activating the cancer autophagy pathway via downregulation of miR-18a* and miR-4802 Fusobacterium species on the chemoresistance of PDAC The + and CD8+ T cells with elevated expression of T-BET, TNF-α, CD38, PD-1 and CD44 Bacteria are capable of translocating from the gut to the pancreas in mice and influencing the PDAC microenvironment Recently, intratumor microbiota were further confirmed to be highly predictive of long-term survivorship in PDAC patients, and experimental evidence suggested that intratumor microbiota were modified by the gut microbiota To date, continued attempts to overcome cancer chemoresistance and immune tolerance have not attained beneficial overall survival for PDAC patients, even after R0 resection with pathologically tumor-free surgical margins. The gut microbiota has been recognized as a crucial mediator in the TME of PDAC; thus, the crosstalk among these regulators becomes more complex and pluralistic Figure . Conside+ T cell accumulation in the intestine. The 11 isolated strains showed effectiveness in spontaneous ICI treatment and dependent tumor inhibition via enhanced CD8+ T cell antitumor immunity. The reported mixture of 11 strains, which were mostly rare and low-abundance species among normal human microbiota, showed great therapeutic potential for resistance to chemotherapy and immunotherapy for widespread cancer types.The concept of fecal microbiota transplantation (FMT), which originates from the fourth century in ancient China, has overcome challenges and instigated wide discussion both technologically and theoretically Given its success with metastatic melanoma, FMT could allow PDAC patients to seek improved prognosis safely Fusobacterium nucleatum is related to the chemoresistance of CRC, as well as other specifically chemotherapy-associated bacterial strains Faecalibacterium genus and other Firmicutes) predicted favorable clinical responses among ipilimumab-treated individuals with metastatic melanoma but increased onset of ipilimumab-related colitis Distinct responses to chemotherapy and immunotherapy in cancer patients have provoked robust interest in identifying useful biomarkers to optimize patient selection and management. Fecal sample analysis using 16S rRNA sequencing has provided evidence that Use of the gut microbiota as a marker for drug efficacy and side effects should be qualified with significantly altered abundance and differential function between postoperative adaptation and drug response. Some issues should be urgently addressed, such as the specificity of biomarker bacteria, the accuracy of comparative studies and how the remote prediction of bacteria works Altered therapeutic efficacy resulting from the host microbiota typically changes in terms of multiple parameters. The quantitative approach used to evaluate the contributions of the microbiota, as previously described in the study of brivudine metabolism, will be appreciated for its role in the effort against drug tolerance in PDAC It is almost impossible for the human body to be absolutely sterile or germ free. All therapeutic strategies and host responses will be directly or indirectly influenced by the microbiota. Future studies should focus on not only the tumor itself but also the treatment dilemma, which might have a long-term influence on the ecosystem in the gastrointestinal tract The reciprocal interactions between the gut microbiota and cancer therapies are complicated and are cancer-dependent, therapy-dependent, and even tumor stage-dependent, leading to the paradox that in some cancers, the gut microbiota is a prerequisite to maintain therapeutic efficacy; however, in other cancers, depletion of the gut microbiota significantly improves efficacy. A large amount of exploratory work is needed to comprehensively understand the role of the microbiota in influencing therapeutic responses in PDAC. We suggested that modified dietary supplements, FMT and the application of certain antibiotics would have an impact on augmented drug efficacy, reduced toxicity and restricted PDAC recurrence and metastasis