Recent studies have revealed that neural functions are involved in possibly every aspect of a cancer development, serving as bridges connecting microenvironmental stressors, activities of intracellular subsystems, and cell survival. Elucidation of the functional roles played by the neural system could provide the missing links in developing a systems-level understanding of cancer biology. However, the existing information is highly fragmented and scattered across the literature and internet databases, making it difficult for cancer researchers to use. We have conducted computational analyses of transcriptomic data of cancer tissues in TCGA and tissues of healthy organs in GTEx, aiming to demonstrate how the functional roles by the neural genes could be derived and what non-neural functions they are associated with, across different stages of 26 cancer types. Several novel discoveries are made, including i) the expressions of certain neural genes can predict the prognosis of a cancer patient; ii) cancer metastasis tends to involve specific neural functions; iii) cancers of low survival rates involve more neural interactions than those with high survival rates; iv) more malignant cancers involve more complex neural functions; and v) neural functions are probably induced to alleviate stresses and help the associated cancer cells to survive. A database, called NGC, is developed for organizing such derived neural functions and associations, along with gene expressions and functional annotations collected from public databases, aiming to provide an integrated and publicly available information resource to enable cancer researchers to take full advantage of the relevant information in their research, facilitated by tools provided by NGC. Cancer tissue-based studies have revealed that the nervous system plays essential roles in cancer formation, progression, and metastasis . Such roConsiderable information has also been generated about the interactions between neural and non-neural functions in cancer. For example, it has been reported that adrenergic innervation could act on T cells, leading to T cell-induced ACh and also regulating the local macrophages . Damage via denervation via blocking neurotransmission has generated promising results  the expressions of certain neural genes have strong impacts on the survival of cancer patients; ii) a set of neural genes can be identified in each cancer type, whose expressions are associated with cancer metastasis; iii) cancer patients with low survival rates generally involve more interactions with neural functions, compared to those with higher survival rates; iv) more generally, more complex neural functions are required in more malignant cancers; and v) neural genes play key roles in helping cancer cells survive the local environmental stressors.Neural Genes in Cancer or NGC, containing the functional information of neural genes in multiple cancer types and stages, which we consider as highly relevant to gaining improved understanding of cancer biology. Currently, NGC covers 26 cancer types, namely, adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), brain lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thyroid carcinoma (THCA), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC), and uterine carcinosarcoma (UCS), along with transcriptomic data of normal tissues of 20 relevant organs from the GTEx database as controls , a classic method for measuring linear correlation between two data sets, is calculated between the expressions of two specified lists of genes. In this study, a gene pair with PCC >0.8 and ClusterProfiler in the R package is used for pathway enrichment analyses against the Gene Ontology (GO) database in this study, which employs both over-representation analysis (ORA) and gene set enrichment analysis (GSEA) techniques and represents a highly used package for pathway enrichment analyses .For a given set of genes and their expressions in a specified set of tissues, their pairwise co-expressions are calculated using the “WGCNA” package in R , a most p-values across the 26 cancer types.To study the possible impact of neural genes on cancer patients’ survival, we have identified all neural genes expressed in cancer tissues and examined their expression profiles across each of the 26 cancer types. By comparing the numbers of upregulated and downregulated neural genes across different cancer types, we note: thirteen cancer types each have more upregulated neural genes than the number of downregulated ones . Five cap-value <0.05) and that with poor prognosis  among all the differentially expressed neural genes, respectively. The calculation results for each of the 26 cancer types are given in To understand how neural functions contribute to cancer development, we have first examined the contribution of the neural genes to the prognosis of cancer patients. Specifically, we have conducted a univariate Cox regression analysis against the expressions of the differentially expressed neural genes (see METHODS) and calculated the hazard ratio  of each neural gene. For each cancer type, we have calculated the proportion of its neural genes with good prognosis  of the five-year survival rates of the patients (as provided in TCGA) of a cancer type against the expressions of selected neural genes, namely, to select neural genes whose expressions can collectively well explain the survival data for all cancer samples of each cancer type (see METHODS). Considering that five cancer types, namely, DLBC, KICH, PRAD, READ, and TGCT, each have very limited survival data in TCGA, our analyses are conducted on the other 21 cancer types.p-values for UCS and BLCA are shown in The detailed analysis results are given in We have examined how the number of upregulated neural genes changes with the progression of a cancer from stage I through stage IV and noted that this number generally increases with the disease progression, particularly at stage IV , suggestp-value <0.05) is summarized in To investigate their detailed functional roles in metastasis, correlation analyses are performed between the upregulated neural genes and 2,091 metastasis-related genes across all stage IV cancer tissues for each of the 26 cancer types, collected from the human cancer metastasis database , metastaNow we check if some neural genes can be used as metastasis biomarkers. To address this issue, we consider cancer tissues in stages I and II as non-metastatic and stage IV samples as metastatic cancers. We have conducted a classification analysis using the expressions of the to-be-selected upregulated neural genes to distinguish metastatic from non-metastatic cancer tissues over samples of each of the following ten cancer types: ACC, BLCA, BRCA, COAD, KIRC, KIRP, LUAD, READ, STAD, and THCA, representing all the cancer types each having at least ten stage IV samples in TCGA. A random forest approach is used to conduct the classification analyses (see METHODS). For each cancer type, the selected neural genes are subject to a 10-fold cross-validation.We note that for each cancer type, a varying number of upregulated neural genes is needed to distinguish the metastatic from the non-metastatic cancer tissues, summarized in For each cancer type, a pathway enrichment analysis is conducted over all the expressed metastasis-related genes (see METHODS). For each enriched pathway, a Principal Component Analysis (PCA) is conducted on the gene set enriching the pathway. Its first principal component is used as the representative of the gene set. Similarly, a PCA is also conducted on the MR genes for each cancer type and a representative is selected. Subsequentially, a correlation analysis is conducted between these two representatives. The calculation results, shown in p, acetylcholine, serotonin, epinephrine, histamine, vasopressin, and cholecystokinin by calculating the Pearson correlation coefficients (PCCs) between the metastatic genes and each neurotransmitter-synthesizing gene. Previous studies suggest that neurotransmitters secreted by nerves are key to cancer metastasis . Here, wPathway enrichment analyses are conducted over all up- and downregulated neural genes, respectively, to study the neural functions that contribute to the development of each of the 26 cancer types. The following observations are made: : upregulp-values that these enriched pathways have, where these pathways are generally found in neuronal communities with strong neuron-neuron interactions. These suggest that more malignant cancers tend to use more complex neural networks, measured using the number of neural genes involved. This is consistent with the previous studies  in young patients (under 60 years old) of cancers having high survival rates, while early developmental processes such as neural tube development are more significantly enriched in elderly patients (over 60 years old) of cancers with low survival rates; iv) axongenesis is more significantly enriched in female patients of cancer types having low survival rates, but no such a pattern is observed in male patients; v) for early stage patients, cancer types with high survival rates tend to have more significantly enriched neuronal differentiation processes, while for advanced patients, cancers with low survival rates have more significantly enriched early developmental processes, primary neural tube formation and axonogenesis; and vi) the level of endoplasmic reticulum (ER) stress strongly correlates with neural functions in nine cancer types: ACC, GBM, KICH, KIRC, KIRP, LGG, PRAD, and READ. The detailed clusters and their associations are given in To elucidate which neural genes tend to work together during a cancer development, we have carried out clustering analyses of co-expressed upregulated neural genes at each stage of each of the 26 cancer types using WGCNA (see METHODS). As an example, we show a comparison between two cancer types, THCA and PAAD, in terms of the pathways enriched by their co-expressed neural genes, where the former is a cancer type with the highest survival rate while the latter has the lowest one. We note that while the two cancer types share a few neural functions such as axon guidance, glia activation and synapse formation, PAAD has considerably more neural functions such as myelination, neural potential regulation and neurotransmitter transport related pathways compared to THCA, which has only one unique pathway as given in via co-expression analyses in each cancer type. Overall, we aim to develop a knowledgebase to enable cancer researchers to study the interactive relationships between neural and non-neural functions in cancer tissues. We have analyzed such interactions We have studied the cellular functions by examining the pathways enriched by the 1,229 neural genes, their co-expressed neural genes, and co-expressed non-neural genes in each cancer type, given in The co-expressed neural genes enrich, for example, the following pathways listed in the descending order of the number of cancer types sharing them: early development of nervous system differentiation of neurons, axon guidance, synapse formation, neurotransmitter secretion, glial development, and neuron myelination, shared by all 26 cancer types; neural crest development, neuronal action potential, axon regeneration, learning and memory, sensory perception shared by 25 cancer types, neuron apoptosis by 24 cancer types, sympathetic and parasympathetic nerve development by 22 cancer types, and neuroinflammation by 14 cancer types.Further analyses have revealed that cancers or their bearing organs sharing structural or functional similarities may share common neural functions. We give a few examples here: i) neuronal action potential is most active in ACC, GBM and UCS, while their harboring organs all have secretory functions: the adrenal gland as an endocrine gland can secrete cortical and sex hormones; astrocytes in brain can secrete various molecules to protect the central nervous system when it is damaged ; and theEnriched non-neural pathways shared by all 26 cancer types include immune response, RNA processing and splicing, and transcription and translation-related pathways, such as post-translational modification. In addition, the following functional categories are shared by most of the 26 cancer types: cell-cycle transition, DNA replication, angiogenesis, extra cellular matrix (ECM) assembly, immune and inflammation response, polarity establishment and maintenance, and metabolisms of lipids, nucleotide, and amino acids, summarized in We have also examined non-neural functions specific to cancer types having a particular level of five-year survival rates. To do this, we have binned all 26 cancer types into three groups, containing approximately nine cancer types with the highest, intermediate, or the lowest survival rates, respectively, shown in Furthermore, our analyses have revealed that the levels of immune and inflammatory responses tend to negatively correlate with axon guidance and neuronal action potential. We have developed a database NGC for neural functions at different stages of a cancer development and their interactions with non-neural functions across 26 cancer types for both male and female patients across different ages. The following depicts the key data and information stored in the database as well as the relevant analysis tools in support of utilizing the data and information in the database.NGC contains a) the detailed functional information of 4,039 neural genes, collected from NCBI , EnsemblSearching NGC: NGC supports numerous queries to enable a user to retrieve or derive the desired data or information from the database. Expression-based analyses: NGC supports a number of analysis tools related to gene expression data, as detailed in Enrichment analyses: NGC provides the pathway enrichment analysis over a specified list of genes using the clusterprofiler package .Survival analyses: The database supports both the univariate and multivariate Cox regression analyses of the expressions of specified genes over a given set of cancer samples.NGC provides an intuitive graphics-based user interface to support interactions between a user and the database. All the above functionalities are made available through a panel of clickable buttons in the frontpage of the database, as shown in http://getbootstrap.com/) and JQuery (http://jquery.com) extension. R (version 3.6.1) and Python (version 3.7.2) are used for data analyses. NGC Database supports personalized online analyses and visual displays. All search and computational results can be downloaded freely.Django, a python-based web framework, is used to build the web applications in support of user-database interaction. MySQL relational database (version 8.0.16) is employed to store all the information and data presented earlier. HTML, CSS, and JavaScript are used to provide support for web-based display and interactions. A user-friendly web interface is supported by using the Bootstrap  cancers with poor survival generally involve more neuron-neuron interactions; ii) in female patients, cancers with low survival rates employ more axonogenesis than those having high survival rates; iii) as a patient ages beyond 60, axon guidance will no longer be positively associated with poor survival; and iv) axon-related functions are involved in regulation of the tumor microenvironment (TME). For example, axon regeneration is highly active and strongly correlates with angiogenesis in BRCA.via statistical analyses and supported by published studies that the induction of neural functions is predominantly to help the host cells to suppress inflammation and immune responses. In addition, some non-neural pathways are found to strongly co-express with neural pathways in cancer. Examples include that the innate immunity vs. neuroinflammatory response in ESCA; cell cycle-related functions vs. learning, memory, and neuronal action potential in GBM; and angiogenesis vs. axon regeneration in BRCA. These add to the established knowledge that nerves in TME may respond to hypoxia and drive the production of neurotransmitters or neurotrophins for angiogenesis in the tumor environment (Another key scientific contribution by our study is that we have established ironment . These sOverall, we believe that this work will provide a useful information resource for cancer biologists to study the functional roles of the nervous systems through analyzing functional states of neural genes, their interactions with non-neural genes, and pathways they enrich across different cancer types and stages. Such information is organized in a highly modular manner to facilitate relatively easy integration of the stored information as well as novel information syntheses by the user, enabled by the tools provided by the database. We aim to maintain and continue to grow the database on a regular basis, anticipating its utility to the cancer research community. For this reason, we encourage the reader to send us your suggestion for further addition and improvement.


Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease that affects newborns, particularly preterm infants, and is associated with high morbidity and mortality. No effective therapeutic strategies to decrease the incidence and severity of NEC have been developed to date. Stem cell therapy has been explored and even applied in various diseases, including gastrointestinal disorders. Animal studies on stem cell therapy have made great progress, and the anti-inflammatory, anti-apoptotic, and intestinal barrier enhancing effects of stem cells may be protective against NEC clinically. In this review, we discuss the therapeutic mechanisms through which stem cells may function in the treatment of NEC. Despite constant efforts on improving the diagnosis and treatment technique of premature infants, the morbidity and mortality rates associated with necrotizing enterocolitis (NEC), a devastating gastrointestinal inflammatory and necrotizing disease that affects newborns, particularly preterm infants, are rising. As a main cause of death in the neonatal intensive care unit, NEC has an incidence of approximately 8.9% (890/9956) in premature infants born at the gestational age of 22–28 weeks, and the mortality rate associated with NEC can be as high as 20–30%. Infants requiring surgery exhibit higher mortality rates , can differentiate into multiple tissues, but cannot develop into complete individuals; and unipotent stem cells, including neural stem cells (NSCs), refer to cells that can only differentiate into one type of cell. Additionally, stem cells are classified into ESCs, adult stem cells (ASCs) and induced pluripotent stem cells (iPSCs), a novel type of stem cell identified in recent years according to developmental origins , mesenchymal stem cells (MSCs), and intestinal stem cells (ISCs)  and reserve or quiescent ISCs (quiescent cycling). Active ISCs, which can be identified by the marker leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), are responsible for promoting homeostatic renewal and differentiation to intestinal epithelial cells (IECs)  injection of transplanted BM-MSCs, the concentration of exogenous human BM-MSCs was increased in the area of the injured intestinal segment, with amelioration of intestinal pathological damage  stress to mediate NEC. AF-MSCs activate the ER stress response to process the unfolded tight junction proteins, such as claudin-7, which can influence the function of the intestinal barrier by decreasing intestinal permeability  in the lamina propria  refers to the multipotent cell population developing from enteric neural crest cells, which proliferate and differentiate into enteric neurons and glia cells in the developing intestine  also reduce the incidence and severity of NEC, and their protective effects have been shown to be equivalent to those of E-NSCs  injection of cells  are vesicles coated with lipids, proteins, and RNA secreted from parental stem cells. The paracrine substances secreted by MSCs can exert function in other organs and even other individuals through the fusion with EVs, which is promising and sustainable for future NEC therapy , MSC-CM could contribute to mucosal recovery, reduce inflammation, and restore ISCs activity (Lykov et al. As a subpopulation of EVs, exosomes do not induce immunoreactions between HLA and stem cells owing to intercellular fusion. Moreover, these vesicles exhibit lower immunogenicity than stem cells (Manchon et al. iPSCs have the potential for multi-dermal differentiation, the excellent ability of living and infinite progeny MSCs generation (Hynes et al. Stem cell therapy is a novel approach for treating NEC. Whereas most research is still limited to animal experiments, studies on its long-term outcomes are lacking. Various challenges have hindered the translation of preclinical studies to clinical applications, including the safety of stem cell transplantation in infants. Hence, the exact mechanisms through which stem cells exert beneficial effects in NEC and the pathogenesis of NEC need to be studied in greater detail to facilitate successful clinical trials. Stem cell derivatives or conjunctive treatments with other activators may also have applications in the treatment of NEC in the future.


Understanding mechanisms that underly the transition from acute to chronic pain and identifying potential targets for preventing or minimizing this progression have specific relevance for chronic postsurgical pain (CPSP). Though it is clear that multiple psychosocial, family, and environmental factors may influence CPSP, this review will focus on parallels between clinical observations and translational laboratory studies investigating the acute and long-term effects of surgical injury on nociceptive pathways. This includes data related to alterations in sensitivity at different points along nociceptive pathways from the periphery to the brain; age- and sex-dependent mechanisms underlying the transition from acute to persistent pain; potential targets for preventive interventions; and the impact of prior surgical injury. Ongoing preclinical studies evaluating age- and sex-dependent mechanisms will also inform comparative efficacy and preclinical safety assessments of potential preventive pharmacological interventions aimed at reducing the risk of CPSP. In future clinical studies, more detailed and longitudinal peri-operative phenotyping with patient- and parent-reported chronic pain core outcomes, alongside more specialized evaluations of somatosensory function, modulation, and circuitry, may enhance understanding of individual variability in postsurgical pain trajectories and improve recognition and management of CPSP. Chronic pain may reflect ongoing excitation, decreased inhibition or inability to recruit endogenous inhibition, failure of active resolution mechanisms , or unmasking of hyperalgesia related to prior exposures or injuries.4–6Understanding mechanisms that underly the transition from acute to chronic pain and identifying potential targets for preventing or minimizing this progression are high priorities for pain research7 Laboratory studies can evaluate mechanisms underlying clinical observations in children with chronic postsurgical pain (CPSP) by (1) quantifying alterations in sensitivity at different points along nociceptive pathways from the periphery to brain, (2) evaluating age- and sex-dependent mechanisms underlying the transition from acute to persistent pain, (3) identifying potential therapeutic targets and comparing the safety and efficacy of interventions instituted before surgery  and/or after injury , and (4) determining the impact of prior exposures, such as different forms of stress and injury. This review will focus on parallels between clinical observations and related evaluations in translational laboratory studies to investigate the mechanisms and pathophysiology of CPSP.Nociceptive mechanisms and responses to injury and analgesia vary throughout postnatal development.8 specific models have been developed to investigate acute and persistent alterations in nociceptive processing following surgical injury.9 Specific contributions of additional factors that can contribute to, or exacerbate, pain following surgery  can also be evaluated at different postnatal ages. Studies performed in rodents allow comparison of age- and sex-dependent injury effects across different stages of mammalian development.10 Many evaluations have focused on initial injury in the neonatal period and early infancy  because responses in the developing nervous system may differ from those at older ages, but associated persistent changes in somatosensory function can have an impact on the response to subsequent injury into adulthood.11Because pain following surgery may be related to skin incision, muscle injury, inflammation, and/or nerve injury,13 Acute behavioral hyperalgesia (reduced hindlimb reflex thresholds) is evoked, with the degree and duration of sensitivity influenced by postnatal age15 but not sex.16 In younger animals, incision-induced electrophysiological changes in large dorsal root ganglion neurons persist beyond the period of behavioral hyperalgesia, afferent-evoked activity in second-order dorsal horn neurons is more marked and rapid,18 and noxious-evoked potentials in the somatosensory cortex are enhanced and more resistant to increasing isoflurane anesthesia.19 In addition, incision produces developmentally regulated long-term changes in nociceptive processing and response to re-incision21 that differ from other injury models .Plantar hind paw incision incorporates incision of the skin and underlying plantaris muscle and produces cellular and molecular alterations specific to this type of injury.29 and skin/muscle incision on the thigh (modified from the adult skin/muscle incision and retraction model30) at postnatal day (P)3 are associated with persistent alterations in sensory thresholds and in the latter with an enhanced degree and duration of hyperalgesia following re-incision of the ipsilateral hind paw.16 Rodent models of inguinal hernia repair32 and thoracotomy33 have not been assessed in juvenile animals.Laparotomy in newborn mice9 In clinical studies, this can be evaluated with a range of techniques during or after surgery that include, but are not limited to, measurement of reflex thresholds,34 somatosensory testing,35–37 physiological reactivity and evoked responses , and brain responses with electroencephalography and neuroimaging.40–42 Though these measures may identify children and adolescents with enhanced sensitivity in the perioperative period or delayed recovery, further longitudinal studies are required to assess feasibility and utility at different ages and evaluate associations with the degree and/or risk of CPSP.Pain and hyperalgesia following surgical injury involve alterations in peripheral and central nociceptive pathways.2Nociceptive mechanisms contributing to persistent pain can include cellular plasticity with changes in molecular profile and translation regulation that shift the nociceptor toward hyperexcitability, alterations in neuronal circuitry and activity, systems-level changes such as immune cell recruitment or cell proliferation, and organism-level effects related to comorbidities and affective, behavioral, and motivational changes.34 At older ages, quantitative sensory testing (QST) has identified altered sensitivity and dynamic allodynia adjacent to neonatal surgical scars in extremely preterm-born children and young adults44 and punctate hyperalgesia many years following childhood surgery.45–47 Associations between persistent alterations in scar-related sensitivity and the degree or duration of pain if subsequent surgery is required in the same region require further evaluation.Acute peripheral hyperalgesia following surgery has been quantified by changes in sensory withdrawal thresholds in human infants.15 Surgical injury will also evoke a peripheral inflammatory response that can contribute to acute hyperalgesia at all postnatal ages, with longer-term alterations in sensitivity varying with the type and degree of insult.49Peripheral nociceptors respond to noxious stimuli following birth, and primary hyperalgesia in the region of plantar hind paw incision has been demonstrated across a range of postnatal ages in rodents.50 Hyperinnervation following full-thickness skin wounding may contribute to persistent sensitivity and is more pronounced at younger ages due to differences in trophic and nerve guidance factors  and is also seen following plantar incision.11Growth hormone signaling influences activity and development of peripheral nociceptive neurons in neonatal rodents, and a detailed series of experiments following dorsal hind paw and muscle incision identified sequestration of growth hormone by infiltrating macrophages and upregulation of transcription factors related to excitatory receptors and channels that lead to nociceptor sensitization.54 Mechanisms can include changes in synaptic function, reduced local and/or descending inhibitory effects, and potential maladaptive and long-term changes due to translational effects on gene expression.54 Developmental changes in spinal cord structure and function, including larger and overlapping cutaneous receptive fields and a relative excess of excitatory and delayed maturation of inhibitory synaptic signaling, contribute to low reflex thresholds.55 Hind paw incision in neonatal rodents induces a range of age-dependent acute and persistent alterations in spinal cord synaptic function, including increased excitatory signaling and reduced inhibitory transmission, alterations in receptor expression, ion channel function, and differential gene expression56 (see Brewer and Baccei’s20 review for details).Afferent input induces central sensitization in the spinal cord, with increased excitation and/or impaired inhibition.d-aspartate antagonist), or enhancing inhibition .57–59 In rodents, neonatal peri-incision sciatic blockade has preventive analgesic effects ,60 whereas opioids block hyperalgesia only during the duration of action of the drug and sensitivity at later time points does not differ from saline controls.61 More specific targeting of mechanisms that underlie the transition from acute to persistent pain or that enhance endogenous pain resolution mechanisms may more specifically reduce the risk of CPSP,1 but efficacy and safety require evaluation in preclinical models at different stages of postnatal development.Clinically, preventive interventions for postsurgical pain focus on reducing afferent input , reducing excitation .65The balance between descending inhibition and facilitation can be assessed in clinical populations with conditioned pain modulation (CPM).response .35 Thoug66 In adult rodents, nociceptive C-fiber input is tonically inhibited , whereas at younger ages there is a relative excess of facilitation.68 The delayed maturation of inhibitory mechanisms is influenced by endogenous opioid and endocannabinoid signaling,69 and surgical injury in neonatal rodents produces long-term changes in the balance of inhibitory/facilitatory modulation.70Descending modulation is mediated by brainstem centers  that receive input from higher centers  and have descending projections that can inhibit or facilitate spinal excitability.71–74 and also identified predictors of the transition from acute to persistent pain.75 In adolescents with complex regional pain syndrome, alterations in brain structure and connectivity have been linked with self-reported outcomes  that improved following interdisciplinary treatment.77 Magnetic resonance imaging is feasible in children with moderate–severe persistent postsurgical pain,78 but pre- and postsurgery studies are yet to identify risk factors for CPSP in children, and effects on brain structure associated with prior neonatal surgery and intensive care also require consideration.80Clinical neuroimaging studies have evaluated changes in structure, connectivity, or blood flow in brain regions associated with attention, sensory/discriminative, and affective/motivational aspects of pain experience20 In infant rodents, evoked electroencephalograph responses in the somatosensory cortex were rapidly sensitized by hindpaw incision.19 Following hind paw surgical injury in adult rodents, functional magnetic resonance imaging identified evoked responses in brain regions involved in sensory/discriminative, affective/attentional, and descending modulation,81 and incision-induced reductions in the volume of different brain regions  were also influenced by early life stress and maternal separation.82Nociceptive circuits in the brain and central responses to noxious and innocuous stimuli undergo significant changes in the postnatal period, and activity-dependent maturation of circuits may be altered by injury.84 Although multiple mechanisms including inflammation and pain arising from muscles, joints, and viscera may also contribute to CPSP,85 this section will focus on neuropathic pain, because this can be difficult to recognize in children and has specific implications for management.86 In pediatric studies, there is significant variability in the diagnostic criteria and reported prevalence of neuropathic CPSP . Outcomes used to support possible or probable neuropathic pain have included history and clinical descriptors, neuropathic screening tool questionnaires, somatosensory testing, conditioned pain modulation, and response to treatment 88–91. In our tertiary pediatric pain clinic cohort of adolescents with peripheral neuropathic pain , CPSP accounted for 32/52 (62%) cases and was associated with moderate–severe pain intensity , thermal hyperalgesia , and mechanical hyperalgesia .93CPSP is often associated with characteristics of neuropathic pain in adults.ntensity  and signntensity .35 QST ial scars  that hav23 Delayed emergence of neuropathic pain has also been reported following traumatic or surgical nerve injury in children.94 The potential for preclinical studies to also link specific sensory modalities and mechanisms to efficacy of pharmacological interventions has been highlighted95 and warrants further assessment at different developmental stages. In addition, more complex behavioral tasks in adult rodents have evaluated alterations in motivational–affective response  and cognitive function 95–98 and have identified long-term effects following surgical injury in early life .In rodents, responses to traumatic nerve injury vary throughout postnatal development, with a delayed onset of allodynia following nerve injury at younger ages that has been associated with a switch from an anti-inflammatory to pro-inflammatory response in the spinal cord.59 and repeated surgery is not uncommon during childhood. Surgery may be required in early life for congenital anomalies and complications of prematurity, and 20% of 5609 neonates recruited from 31 European countries required repeat surgery prior to 60 weeks postmenstrual age.100 In infants, repeat surgery in the same dermatome as prior neonatal surgery was associated with increased perioperative pain and stress.38 A study recruiting 8- to 18-year-olds for evaluation of CPSP noted that 148/237 (61%) had undergone previous surgery and 148 (62%) had an ongoing pain problem prior to surgery.101 In our cohort of 32 adolescents with neuropathic CPSP, 40% had required multiple surgeries throughout childhood, and pain had only become persistent after the most recent surgery in 20%.35 Multiple psychosocial, environmental, and genetic factors can influence pain experience and the transition from acute to chronic pain,102 and prior hospitalization may influence psychosocial factors such as anxiety and catastrophizing that are associated with increased risk of CPSP.103 Because the response to surgery and nerve injury varies with age and surgery during childhood has been associated with persistent alterations in sensory processing, a past history of prior surgery and pain exposures is relevant for the clinical assessment of CPSP and evaluation of underlying mechanisms. Despite undergoing the same surgery, only a proportion of patients develop CPSP, and not all children and adults with persistent sensory changes related to surgical scars have associated pain. Therefore, in addition to identifying factors that increase the risk of CPSP, factors associated with improved recovery and resilience need to be evaluated.105Prior surgery is a risk factor for CPSP in adults,59 particularly following thoracotomy, breast cancer surgery, and cesarean section.106 One pediatric study met the inclusion criteria , and the authors106 highlighted the need for larger, high-quality studies assessing the impact of regional anesthesia on CPSP in children. An increasing range of local anesthetic techniques are utilized to reduce perioperative pain in children,108 and laboratory studies demonstrate both acute perioperative and long-term benefit on the response to repeat surgery. Use of peripheral, regional, and potential long-acting preparations and catheter techniques may have additional benefit in children to both reduce the risk of CPSP and also minimize the impact of early life surgery on subsequent injury response.Epidural, regional, and systemic local anesthetic administration reduces the risk of chronic postsurgical pain in adults,20 In rodents, reducing primary afferent input by sciatic nerve blockade at the time of neonatal hind paw incision prevents persistent changes in synaptic signaling.109 In addition, pre- and postincision neonatal sciatic block prevents the enhanced response to re-incision in adulthood ,61 whereas neonatal opioids do not have this long-term effect .61 As noted above, peripheral growth hormone signaling is altered by neonatal incision, and injection of growth hormone into hind paw muscles at the time of neonatal incision prevented the enhanced re-incision response in adulthood.50Prior surgery may have triggered biological changes that contribute to CPSP, because nociceptive pathways can be “primed” by noxious input/injury. Early life injury has been associated with a range of developmentally regulated peripheral and spinal cord mechanisms that contribute to an enhanced degree and duration of hyperalgesia following subsequent re-incision in adulthood.110 Prior exposure to an inflammatory stimulus  or hind paw incision results in more prolonged hyperalgesia following a subsequent challenge several weeks later with a different or previously subthreshold mediator.111 Targeting this form of plasticity may reduce the transition to persistent pain,112 but sex-dependent effects need to be considered because mechanisms and response to preventive interventions differ in adult male and female rodents.113–115Hyperalgesic priming is a form of plasticity in primary afferent nociceptive fibers.117 Pathways include endogenous opioid signaling, with subsequent opioid antagonist administration unmasking the hypersensitivity, and long-term alterations in the constitutive activity of the mu opioid receptor increasing excitatory actions via altered N-methyl-d-aspartate receptor function.118 Activity at mu opioid receptors and kappa opioid receptors, but not delta opioid receptors, modulates latent sensitization following hind paw incision, and kappa opioid receptor–mediated inhibition of latent sensitization was greater in females.119 Similarly, in human adult volunteers, high-dose naloxone unmasks hyperalgesia following an experimental thermal injury.121 Mechanism-based preventive interventions are effective in adults,2 but because the acute and long-term effects of opioid exposure vary with postnatal age and sex,123 and when opioid is administered in the presence or absence of surgical injury,61 evaluation at younger ages is also required.123Tissue injuries, including surgical incision, activate endogenous inhibitory mechanisms in the spinal cord and brain that contribute to the resolution of pain but may also mask ongoing hypersensitivity .125 However, neuroimmune signaling is sexually dimorphic.127 Microglial inhibitors in male, but not female, adult rodents reduce pain behaviors following peripheral nerve injury,129 hind paw inflammation,130 and hyperalgesic priming,131 whereas T cells may play key modulatory roles in females.132 Microglia can be primed by early life exposures, resulting in intrinsic phenotypic changes and an exaggerated response to subsequent challenges.134 Neonatal surgical incision primes spinal microglia, resulting in an enhanced degree and duration of microglial reactivity following re-incision in later life, and the associated enhanced hyperalgesia is reduced by microglial inhibitors in adult males.136 Preventive, but sex-dependent, effects are also evident from early development, because microglial inhibitors at the time of neonatal incision prevented the enhanced response to adult re-incision in males but not females.16 Microglia also have important roles in developing neural circuits ,138 and perinatal insults can alter the normal sex-dependent trajectory of microglial development.139 Therefore, alongside the well-documented persistent alterations in excitatory and inhibitory signaling induced by neonatal incision,20 microglia may also influence developing spinal circuits.Neuroimmune interactions involving neurons, microglia, astrocytes, and T cells contribute to injury-induced changes in sensitivity and persistent pain.140 specialized evaluations of somatosensory function and pain modulation, and assessments of connectivity and activity in central pain circuits, will contribute to understanding variability in postoperative pain trajectories. Ongoing translational preclinical studies evaluating age- and sex-dependent mechanisms will also inform comparative efficacy and preclinical safety assessments of potential preventive pharmacological interventions.The prevalence and impact of CPSP in children and adolescents is increasingly recognized. Detailed peri-operative phenotyping and longitudinal follow-up will improve prediction of CPSP and identify targets for intervention. Alongside patient- and parent-reported measures that encompass recommended domains of core outcomes for pediatric chronic pain,


Osteoarthritis (OA) is the major cause of disability, affecting over 30 million US adults. Continued research into the role of neovascularization and inflammation related to osteoarthritis in large-animal models and human clinical trials is paramount. Recent literature on the pathogenetic model of OA has refocused on low-level inflammation, resulting in joint remodeling. As a result, this has redirected osteoarthritis research toward limiting or treating joint changes associated with persistent synovitis. The overall goal of this review is to better understand the cellular and tissue-specific mechanisms of inflammation in relation to a novel OA treatment modality, Genicular Artery Embolization (GAE). This article also assesses the utility and mechanism of periarticular neovascular embolization for the treatment of OA with a particular emphasis on the balance between pro-angiogenic and anti-angiogenic cytokines, inflammatory biomarkers, and imaging changes.  Traditionally considered a “wear and tear” phenomena of the bone and cartilage, osteoarthritis (OA) is increasingly understood to represent sequela of chronic inflammatory processes . As our Okuno et al. have employed targeted neo-vessel embolization to successfully treat symptomatic osteoarthritis with durable therapeutic response . Early rOf all types of arthritis, osteoarthritis (OA) is the most common, affecting over 30 million US adults . The radPatients with knee OA can present with joint pain, stiffness, bony crepitus, joint edema, and physical deformities. Radiographic findings include narrowing of the joint compartment, osteophytes, and subchondral sclerosis . CurrentWhile the pathophysiology of knee osteoarthritis has traditionally been described in the context of inflammation and joint space narrowing, new evidence in the literature suggests that abnormal blood vessel formation may also play a role. Osteoarthritic pain is thought to be caused by joint space inflammation, abnormal innervation of synovial structures and increased sensitization of the central and peripheral nervous systems .Synovitis may damage underlying tissues by altering chondrocyte function, while increased angiogenesis and bony remodeling contribute to chronic inflammation in OA . AngiogeRecent attention has focused on the local microenvironment as it pertains to osteoarthritis. Like generic inflammation, regulatory molecules and cytokines function in a complex, sophisticated manner to stimulate and/or inhibit angiogenesis . CompounArticular cartilage is an avascular tissue. The blood supply to the cartilage is derived from the adjacent, vascular synovium. Cartilage is a critical component of the joint, as it helps to evenly distribute high mechanical forces and maintain the structure of the joint as a unit . ChondroNerve tissue tends to parallel vascular structures. The synovium and subchondral bone contain sensory nerves, which consist of unmyelinated C fibers. This nervous tissue functions along with the neuropeptides Substance P and calcitonin gene-related peptide . MechaniLarger animal model studies have shown angiogenic invasion to be statistically increased in OA knee joints across the osteochondral junction . McDougaGAE requires a fundamental knowledge of knee vascular anatomy, along with its variations, for effectively and safely identifying the embolizing target ,38. ThreAnastomotic networks between the genicular arteries provide additional complex variation to the vascular network of the knee. Bagla et al. observed anastomoses in 26.4% of genicular arteries, with highest rates between the musculoarticular branch of the DGA and the SMGA/SLGA . Other cKnowledge of knee vascular anatomy is essential for minimizing risk of GAE. A common side effect of GAE is skin discoloration secondary to cutaneous ischemia from nontarget embolization of cutaneous arteries. Okuno et al. reported that four patients experienced transient color change of the overlying skin of the treated knee that spontaneously resolved by 1 month follow-up . Bagla eAdvancements in the pathogenetic model of OA has redirected research into its treatment and shifted the focus to limiting or treating joint changes associated with persistent synovitis. Okuno et al. has published four studies in human subjects showing that targeted neovessel embolization successfully treated symptomatic osteoarthritis with durable therapeutic response. Twenty-five patients with radiographic and clinical findings of knee osteoarthritis underwent the angiography and embolization of identifiable neo-vessels. These vessels tended to be associated with the synovium, infrapatellar fat pad, medial meniscus, medial joint capsule, and the periosteum adjacent to the medial condyle. They were identified as “excessive, disorganized” vascular structures, which often demonstrated arteriovenous shunting and early venous drainage . Neo-vesContinued follow-up with magnetic resonance imaging in treated patients demonstrated significantly improved synovitis. These results suggest that embolization may function to treat pain and modify disease progression. While the clinical results are promising, the effect of embolization on laboratory, histological and imaging findings of OA are not well understood. Imaging studies have similarly coalesced on the association between OA and synovitis. Separate from any study related to GAE, but speaking more broadly of synovitis, Macfarlane et al. recently stated, “changes in synovitis, whether persistently extensive or intermittent, are associated with cartilage damage over time.” They ultimately conclude, “Since synovitis is a potentially modifiable intraarticular feature, further research is warranted to assess whether treatment of synovitis mitigates cartilage destruction” . ResultsThere remains a sizable portion of knee OA patients who do not respond to non-operative therapy and are not considered good surgical candidates due to associated comorbidities or early disease stage. Therapeutic embolization for osteoarthritis has had promising early results ,20,57,58Despite the technical and clinical success of therapeutic embolization, the mechanism of action is not well understood. Okuno et al. have shown that targeted geniculate artery embolization resulted in statistically significant pain relief at 1-month, 4-month, and 12-month follow-up . MidtermEarly evidence shows that GAE is a straightforward procedure with less comorbidity relative to TKA that decreases pain via interruption of the inflammatory pathways and may also have a disease-modifying effect on the cartilage itself. These findings demonstrate the potential for neo-vessels as a target for disease-modifying treatment of OA as it relates to inflammation and synovitis ,62,63. BGiven the clinical need for effective non-surgical treatments of OA, evidence linking synovial neovascularization and the development of osteoarthritis, and clinical improvement in OA symptoms following GAE, future studies should seek to better understand the mechanisms involved in this burgeoning treatment modality. It has been nearly two decades since it was demonstrated during unanesthetized knee arthroscopy that “the anterior synovial tissues, fat pad, and capsule were exquisitely sensitive to the mechanical loading stimulus of the probe” , and in Understanding the inflammatory response to embolotherapy of OA will better guide treatment strategies. Although GAE has shown promise in treating intractable OA pain resistant to conservative management, the disease modification mechanism of embolization in OA remains poorly understood. The pathogenesis of OA results from a disturbance in the intra-articular microenvironment homeostasis toward a pro-inflammatory state associated with neovascularity and angiogenesis. These neo-vessels are suspected to be responsible for hyperalgesia in OA patients. However, the causality of these neo-vessels with respect to OA is not proven nor is the mechanism of action for GAE completely understood. The current animal models have only proven an association between microscopic neo-vessels and joints affected by OA. Identifying a similar type of macroscopic radiographically evident neo-vessel in an OA animal model would further elucidate the pathophysiology of this phenomenon in humans. Ultimately, continued study of intra-arterial joint embolization may lead to improved clinical outcomes for OA patients who do not respond to optimized non-operative therapy and are not considered surgical candidates. Geniculate artery embolization has shown promising early results for the management of mild to moderate OA of the knee, but expanded randomized controlled trials are needed to better evaluate its potential role in OA treatment. In a recent multicenter, randomized controlled trail, Bagla et al. showed that GAE significantly reduced pain and improved disability in patients with mild to moderate OA compared to patients of the sham group . In addi


Pennisetum glaucum (L.) R. Br.] is a C4 crop cultivated for its grain and stover in crop-livestock-based rain-fed farming systems of tropics and subtropics in the Indian subcontinent and sub-Saharan Africa. The intensity of drought is predicted to further exacerbate because of looming climate change, necessitating greater focus on pearl millet breeding for drought tolerance. The nature of drought in different target populations of pearl millet-growing environments (TPEs) is highly variable in its timing, intensity, and duration. Pearl millet response to drought in various growth stages has been studied comprehensively. Dissection of drought tolerance physiology and phenology has helped in understanding the yield formation process under drought conditions. The overall understanding of TPEs and differential sensitivity of various growth stages to water stress helped to identify target traits for manipulation through breeding for drought tolerance. Recent advancement in high-throughput phenotyping platforms has made it more realistic to screen large populations/germplasm for drought-adaptive traits. The role of adapted germplasm has been emphasized for drought breeding, as the measured performance under drought stress is largely an outcome of adaptation to stress environments. Hybridization of adapted landraces with selected elite genetic material has been stated to amalgamate adaptation and productivity. Substantial progress has been made in the development of genomic resources that have been used to explore genetic diversity, linkage mapping (QTLs), marker-trait association (MTA), and genomic selection (GS) in pearl millet. High-throughput genotyping (HTPG) platforms are now available at a low cost, offering enormous opportunities to apply markers assisted selection (MAS) in conventional breeding programs targeting drought tolerance. Next-generation sequencing (NGS) technology, micro-environmental modeling, and pearl millet whole genome re-sequence information covering circa 1,000 wild and cultivated accessions have helped to greater understand germplasm, genomes, candidate genes, and markers. Their application in molecular breeding would lead to the development of high-yielding and drought-tolerant pearl millet cultivars. This review examines how the strategic use of genetic resources, modern genomics, molecular biology, and shuttle breeding can further enhance the development and delivery of drought-tolerant cultivars.Pearl millet [ Pennisetum glaucum (L.) R. Br. syn. Cenchrus americanus L.] has been widely grown in Sahelian West Africa and the Indian subcontinent since time immemorial. Archeological evidence (Zea mays), wheat (Triticum aestivum), rice (Oryza sativa), sorghum (Sorghum bicolar), and barley (Hordeum vulgare). It is an important eco-friendly field crop in conventional farming, which is good for human nutrition and health, and the planet. Pearl millet may counter several aversive impacts of looming climate change on food grain production better than other staple cereals and plays a significant role in food and nutritional security. To enhance genetic gains in terms of grain yield, nutritional values, climate resilience, and multiple disease resistance, constant efforts will be needed to develop more potential genomic resources for the pearl millet species  and biotic constraints , pearl millet produces nutritive grains, which are a resource for addressing malnutrition among the poor in marginal areas of the world .4 photosynthesis) for rapid production of biomass that partly has enabled it to adapt to suboptimal arid and semi-arid environments around the world  where seedling establishment itself is a major challenge . Most brDrought stress in the vegetative stage is of frequent occurrence and seriously affects pearl millet growth , tilleriSoil moisture stress in the flowering and post-flowering stages are common because of early cessation of rainfall in the growing season. In fact, collectively, terminal drought stress accounts for a huge impact on pearl millet grain and stover yields . Grain yIt has been convincingly shown that the effects of drought are influenced by the developmental stage of the crop in which drought stress occurs, which is environment-specific. Therefore, the development of drought-tolerant products requires proper quantification of the environment and then fit-for-purpose product development through innovative breeding.Grain yield is a complex process influenced by several component traits at the bottom of plant structural organization and is the consequence of interaction among the environment, management, and genotype. Below are several physiologically important traits reported to play a critical role in drought adaptation of pearl millet.Availability of an adequate amount of water during critical growth stages determines the productivity of crop plants. Terminal drought-adapted genotypes have built-in plant traits such as lower transpiration rate  and loweWater extraction pattern in critical crop growth stages: the pattern of water extraction in pearl millet has been explored using the lysimetric facility simulating field conditions . When irPlants do suffer from a type of stress called atmospheric drought when the evaporative demand  of the environment is high especially under midday conditions irrespective of soil moisture status. During high VPD conditions, transpirational water loss is higher, and photosynthetic efficiency is low; in other words, plants spend a lot of water molecules in transpirational cooling with very minimum CO2 fixation . Limited1 hybrids developed for the A1 dry zone produced smaller leaf areas and lower biomass than materials bred for the relatively wetter A and B zones. These differences in canopy development were also reported to result in water conservation under water deficit conditions  . It is pThe interaction of genotypes with the environment restrains genetic gain and insights into drought adaptation. Therefore, it is important to characterize the environment in which a crop is grown. A few decades back, the All-India Coordinated Pearl Millet Improvement Project (AICPMIP) defined the pearl millet growing area of the country into three separate zones, A1 (the arid zone of NW India), A , and B (semi-arid peninsular India). Each of these zones covers areas with high variation in agro-climatic conditions that have, in these decades, also changed dramatically. Recognizing this current variation in existing zones redefining pearl millet-growing area and identifying crop production constraints (drought stress patterns) will enable breeders to understand the need for breed-specific cultivars for each target agro-ecology using a suitable breeding strategy. Crop modeling is also highly useful for designing ideal plant ideotypes based on evaluation of past genetic improvement for the selected environment. The efficiency of crop improvement for constantly changing environments can be improved if the physiological and morphological traits associated with drought adaptation are identified and integrated into breeding programs. Drought is a complex trait, and breeding for sustainable drought products requires knowledge of the type of drought stress to target the environment and systematic screening method/tool relevant to the stress. Screening methods should be precise and reproducible to accommodate the different number of breeding lines for selection in order to integrate in the breeding pipeline. A combination of screening methods can be integrated in different stages of the breeding pipeline for drought product development.1 to screen various plant materials in a non-destructive manner during the vegetative stage using an optical system (PlantEye®2). This can be used for precise measurements of plant canopy traits such as digital biomass, 3D-leaf area, plant height, leaf area index, leaf angle, leaf inclination, and light penetration depth (n = 162), segregated for QTLs for drought adaptation  of breeding schema for simple component traits of adaptation like early vigor, canopy index, plant height, and tiller number can be useful for discarding non-desirable phenotypes not only for agronomic traits but also for adaptation traits. One such high-throughput and automated phenotyping platform, “LeasyScan,” was established at ICRISATon depth . This phon depth . A pearlaptation , was useaptation . This staptation . There a3 is a gravimetric system of transpiration measurement providing individual soil profile to each plant, and water use could be measured throughout the crop cycle  season (June-October) is compared to a the current crop year. Rainy season NDVI and crop area changes are identified. Secondary data are used to support the crop stress analysis. Spectral matching techniques are utilized to categorize crop stress-affected cropland areas into three classes, severe, moderate, and mild, based on the intensity of damage assessed by field sampling. Spectral signatures are generated based on these ground survey samples  product derived from the Moderate Resolution Imaging Spectroradiometer (MODIS), challenges multidisciplinary teams to understand different stresses in millet-growing areas. The process involves analyzing historical time series satellite imagery . Use of  samples .Although pearl millet is an inherently drought-tolerant species, breeding programs have targeted to further increase drought tolerance utilizing available genetic variations in its germplasm. Adaptive evolution concurrent with climate change in its center of diversity and area of production shaped the crop to successfully grow in areas of limited soil moisture .Breeding programs have targeted mostly the mid-season and terminal drought stresses in pearl millet, assessed the importance of drought-adapted germplasm, and explored the usefulness of genetic introgression.Tillering ability is the most important trait that has been strategically manipulated in mid-season drought stress breeding. There is a huge amount of variation in the tillering capacity of pearl millet and it has been reported to be a moderately heritable trait . GreaterIniadi landraces and adopted in Indian and African agro-ecologies.Earliness and short and rapid grain filling period are the two most important traits that have been manipulated for enhancing tolerance to terminal drought. Early flowering essentially determines grain productivity in water stress . GeneticThe proportion of panicle threshing denotes seed setting potential under low soil moisture conditions and integrating the effects of assimilation and translocation of photosynthates in drought environments is a measure of drought tolerance . It exhiSuccessful growth of pearl millet in water stress is more of a reflection of its adaptation to drought stress than genetic yield potential . As adapA huge amount of genetic variation in the global germplasm of pearl millet is available for traits that are constitutive and responsive to drought . Murty eDifferential response of landraces and elite genetic materials across contrasting drought and non-drought environments provided need of amalgamating drought tolerance of landraces and high-yielding potential of elite genetic material . CrossesThe development of hybrids using strategic use of a diverse range of germplasm has been a top priority in pearl millet breeding, especially in India. However, it is generally argued that hybrids are more suitable for optimum environments than heterogeneous OPVs, as the latter has a population buffering effect to provide stable performance in unpredictable drought environments. A comprehensive study reported a yield advantage of hybrids ranging from 19 to 35% over OPVs . The curDuring the past more than two decades, hundreds of thousands of molecular (DNA) markers have been developed and used in pearl millet genetic studies in different research stations around the world . DiffereIn pearl millet, molecular mapping work started with the RFLP technique , 1994. OPstI/BanII complexity reduction using RIL population  population derived from the H77/833-2 × PRLT 2/89-33 cross to construct a dense linkage map by integrating DArT and SSRs (2-bred F3 progenies spanning 44 uniformly distributed loci with an inter-marker distance of 14 cM (LG1) to 38 cM (LG6) and an overall average distance of 23 cM has been reported by The first milestone was achieved with the development of the shortest map (303 cM) with 181 RFLP markers using an Fpulation . Later, pulation . In an Ip length . A totalp length . To consp length . A linkaand SSRs . This reand SSRs . A longe2 population of a wild × domesticated cross with improved coverage and improved consistency of loci compared to formerly released maps. The linkage map was developed with a revised GBS protocol involving two (PstI and MspI) restriction enzymes and PCR amplification with primers comprising 3 discerning nucleotides to generate thousands of SNPs for open access (6) derived from a cross between ICMB 841-P3 and 863B-P2 lines was used in the study of Genetic linkage maps were constructed with 99 in-house designed novel EST-SSRs and 17 previously mapped SSRs, 53 gSSRs, and 2 informative STS markers . A consen access . LinkageThousands of informative DNA markers have been deployed to dissect QTLs for investigation of the molecular and biochemical bases of tolerance to abiotic stress in millets, and to devise an efficient approach for crop improvement for yields under drought stresses. Subsequently, dissection of QTLs pertaining to drought tolerance (DT-QTLs) and high grain yield was identified in independent studies using different pearl millet mapping populations . The milThese QTLs mapping findings have been validated in linkage mapping investigations that mapped QTLs linked to high grain yield and its related traits under terminal drought stresses in pearl millet . In this+) ionic accumulation and compartmentalization have been recorded in leaves, while access accumulation of toxic ion Na + was diminished in plant roots of pearl millet. It may be concluded that ionic uptake is controlled by genes pertaining to the DT-QTL in LG2  technologies has emerged as an efficient proficient approach for analyzing gene expression profiling in plant species. However, RNA sequencing (RNA-seq) utilizing the NGS technique can detect differentially expressed genes (DEGs) owing to their dynamic range of expression levels . A de nol millet . Some inl millet , attemptl millet . More rel millet . Severall millet .Genomics provides opportunities to develop DNA-based tools to mitigate the problems of ever-increasing food and fodder yield, nutritional security, and sustainable crop production by modern breeding approaches. The development and deployment of DNA markers for plant genetic advancements through marker-assisted selection (MAS) have been established in bi-parental breeding. Advances in plant genomics offer high-density sequence-based molecular tools exploiting NGS technology for genetic evaluation of breeding populations, and are important to accelerate the pace of pearl millet breeding programs . A genomUntil recently, the use of GWAS and GS has been impeded because of unavailability of high-density sequence-based DNA markers . InventiMany potential marker-assisted backcrossing (MABC) methods are devised, which have been employed in QTL introgression from a donor to an elite recurrent parent, while pearl millet lines have been used to develop base mapping populations. ICRISAT , with the collaboration of United Kingdom-based scientists supported by the Plant Sciences Programme, Department for International Development (DFID), has made substantial efforts to develop and use genomic tools for increasing grain yield, yield stability, and productivity of hybrid cultivars in pearl millet . They deImprovement of genetic gains in terms of grain yield in harsh environments needs knowledge-based selection approaches that use phenotypic and genotypic data. Such type of marker-aided selection has been regarded as genomic selection . The effGenomic selection (GS) in crop species is advantageous owing to its potential to predict selection decisions even in the off-season, hence enhancing significant genetic gain . A simulGenome-wide molecular markers are potential tools for QTL discovery and deployment . MoreoveMore than 70% of the pearl millet area would continue to be rainfed where moisture availability is seldom adequate, and the crop will always be prone to facing intermittent drought in the critical growth stage. Increasing climate variability in pearl millet production ecology and the burgeoning population in south Asia and sub-Saharan Africa imply a greater competition for water use in staple crop production besides the industry and domestic sectors. Current defined production ecologies are either vague or quite old, with high variations in agro-climatic conditions. Pearl millet target production environments require rigorous quantification of dynamic TPE with high resolution advanced MET database and crop coefficients to accelerate breeding programs. Enhancing crop productivity with less amount of water, the so-called concept of “more crop per drop,” is becoming a global necessity. Advancements in genomic tools and their effective integration improve the prospects of drought tolerance pipelines through target traits introgression and rapid recycling using speed breeding platform of adapted pearl millet germplasm/parents in conventional breeding. The availability of germplasm collections is relatively meager for diversifying the cultivar base in target regions. Therefore, the inclusion of West Africa and western India warrants more drought-adapted breeding populations. Adding new germplasm collections to drought-tolerant crossing programs to broaden the genetic base ensures the least disturbing maturity period (70–75 days) that is preferred by farmers. There existed a need of putting great thrust on the development of hybrid parents for drought-affected areas as hybrids have a distinct yield advantage of 20–30% over OPVs. ICRISAT exclusive drought-tolerant product profiles established in 2020 will provide the opportunity to build resources to the mainstream of validated traits, linked markers, and genomic selections with high-throughput screening facilities in target environments or platforms at the breeding center that mimic target environments.Further success will depend on the development of repeatable, cost-efficient, high-throughput phenotyping facilities that reliably characterize genetic variation for drought tolerance and its contributing traits. Currently, hyperspectral remote sensing UAV technologies are an alternative to the manual collection of crop data, offering information on traits and factors influencing crop development and productivity with relatively shorter time and lower cost. Use of ultra-high spatial resolution hyperspectral remote sensing technology aided by machine learning and artificial intelligence (AI) have the potential to revolutionize the drought tolerance breeding process. These will aid in faster selection process and make more inclusive many traits that are complex or time consuming in assessment, like component traits for adaptation. Several new techniques for genomics offer a great opportunity to accelerate the cultivar development process. NGS-based genomic technologies have the potential to revolutionize the way crop breeding for drought tolerance is being attempted. Rapid trait mapping, validation, and deployment may be possible by combining advances made in our understanding of the underlying physiological factors and their interaction with environmental indices, and precision phenotyping. Marker-assisted deciphering of heterotic gene pools and development and diversification of the genetic base of seed and pollen parents will be critical in the development of heterotic and resilient drought-adapted cultivars. The heterotic group-based breeding strategy may bring viable breeding pipelines in the next few years using appropriate elite germplasm in crossing, rapid generation (RapidGen) advancement, and consortium-based drought trial network testing to stage-gate approaches. All such integrated programs with network trailing established and led by ICRISAT in association with NARS will accelerate the drought-tolerant product pipelines in India. Whole-genome resequencing-based selection approaches may help reduce the complexity associated with drought and its underlying component traits, and may provide useful insights into favorable haplotypes associated with drought adaptation. These can help us move toward breeding strategies based on haplotype selection leading to an enhanced rate of genetic gains in drought agro-ecologies.RKS, RV, and OY planned and coordinated the study. RKS, OY, MaG, SG, DS, JK, SK, TM, SC, CS, RBS, SB, RG, and RV contributed to this work and drafted the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches. The best current estimate claims that cells appeared on Earth ~3.0–3.3 billion years ago . The proA simple eukaryote cell shows more complexity than the most complex prokaryote. The main difference lies in the eukaryote cell ability to compartmentalize the metabolic reactions necessary for survival. Eukaryotes have developed subcellular structures separated from each other to confine specific reactions to suitable cell compartments and allow different reactions to take place simultaneously inside the same cell . IntraceGiven the remarkable complexity jump forward between prokaryotes and eukaryotes and the absence of organisms with an intermediate complexity, intermediate cell compartmentalization processes are difficult to identify. One of the main hypotheses, the endosymbiotic theory, proposes that the intracellular compartments (organelles) derived from different organism interactions to perform distinctive reactions such as oxidative respiration and detoxification . This hiGPCRs are the largest family of membrane receptors, with nearly 800 genes coding for these proteins. They are involved in many physiological processes such as sensing light, taste and smell, neurotransmission, metabolism, endocrine and exocrine secretion, cell growth, and migration. In a simplistic way, GPCRs are composed of seven transmembrane domains, three extracellular and three intracellular loops, an extracellular N-terminus, and an intracellular C-terminus . The ext+ channel (GIRK) in atrial myocytes after muscarinic M2 receptor activation , bu, bu45], For natural peptide ligands, crossing the plasma membrane and targeting intracellular GPCRs is difficult in terms of size and charges; however, it has been shown that small peptides, such as the melanostatin or thyrotropin-releasing hormone (TRH), could in principle use the peptide transporters to cross the cellular membrane and reach intracellularly compartmentalized receptors . These tIn any case, it is more likely that natural peptide ligands can reach the intracellular compartment by internalization with their cognate receptors. As a rule, in early endosomes, after internalization, hormone peptides dissociate by their cognate receptors to be degraded in lysosomes, while the receptors are recycled to the cell membrane ,135. NevConstitutive activity is another way GPCRs could activate signaling in intracellular compartments. Many GPCRs exhibit constitutive activity ,138 and The biggest limitation to explore GPCR intracellular signaling is represented by the fact that cells need to be lysed and this procedure clearly directly affects the temporal and spatial resolution of the biochemical events of these phenomena. A major breakthrough in the field was achieved with the development of fluorescent and bioluminescent resonance energy transfer (FRET/BRET) sensors . These RThe first RET sensor to be developed was for the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP). The sensor consisted of cAMP-dependent protein kinase in which the catalytic and regulatory subunits were each labeled with a different fluorescent dyes able to generate FRET signals when complexed to the holoenzyme. Binding of cAMP to the holoenzyme induced subunit dissociation and the elimination of the FRET signal. The change in shape of the fluorescence emission spectrum allowed the quantification of cAMP concentrations in single living cells . In 2009Biosensors generated to measure inositol-1,4,5-trisphosphate (InsP3)  and diacOther biosensor types can directly measure GPCR activation . For exaPersistent activation of GPCRs in endosomes after internalization contrasts with the common notion that the interactions of GPCRs with β-arrestins and G proteins are mutually exclusive. This intriguing issue was solved by Thomsen et al.  who demoReceptors in internal compartments may represent a potential drug target with possible therapeutic relevance. Lipophilic ligands might have access to intracellularly compartmentalized GPCRs by simple diffusion throughout the plasma membrane, whereas hydrophilic compounds could be transported across the cell membranes by specific proteins . In 1997Another way these drugs could cross the plasma membrane is by carrier-mediated intracellular transport. In humans, more than 300 solute carrier (SLC) transporters have been identified and these mainly include organic anion-transporting polypeptides , organic cation transporter (OCTs/SLC22As), organic cation and carnitine transporters (OCTNs/SLC22As), peptide transporters (PEPTs/SLC15As), and multidrug and toxin extrusions (MATEs/SLC47As) (for a broad review of the argument, see  and artiFinally, drugs could target intracellular GPCRs by conjugation with compounds that increase their rapid incorporation into the plasma membrane, such as polyethylene glycol or the transmembrane lipid cholestanol . For exaIn the last decade, the plasma membrane-centric view of GPCR signaling was confuted with accumulating data showing that intracellular GPCRs can signal in the same way as those in the plasma membrane. Intracellular GPCRs activate unique pathways, different from those activated on the plasma membrane, and/or they prolong biological responses starting at the plasma membrane . FurtherHowever, the evidence reported in this review is already compelling regarding the possibility that compartmentalized GPCR signaling will soon be an important pharmacological target for the development of novel and more effective drugs.


Cross-sector coalitions can be a powerful vehicle to promote adoption and implementation of evidence-based programs and policies across diverse racial/ethnic communities with a high chronic disease burden. Few studies have examined coalition composition, function, or capacity to promote learning among members.We used a mixed methods approach to examine the United for Health coalition’s implementation of multiple food environment interventions across five low-income communities of color in Los Angeles, California (USA). At the coalition-level, key measures included the collaborative environment, membership characteristics, process and structure, communication, resources, strengths, challenges/barriers, and community impact. At the organizational- and individual-levels, we collected data on participation, leadership development, intraorganizational change, perceived benefits, and learning outcomes.Overall, the United for Health coalition produced five community gardens, three pop-up produce markets, and one farmers’ market; members also expanded Electronic Benefits Transfer (EBT) access at three existing farmers’ markets. Findings indicate early coalition strengths included having a mutual purpose, which was maintained throughout the study period. Coalition participation and engagement was consistently high, while coalition and inter-organizational communication improved over time. Strengths were membership diversity and the availability of learning opportunities. Benefits included leadership development and strategic alignment across organizations. Members demonstrated an increased awareness of the importance of culturally adapted interventions and knowledge of community health planning topics. Key implementation challenges were a lack of resources and social context barriers.Examining coalition function and maturation in a real-world context reveals important lessons for scholars and practitioners committed to addressing nutrition-related health disparities in marginalized and historically underserved communities. Future work should investigate the sustainability of externally funded cross-sector coalitions after funding ceases.The online version contains supplementary material available at 10.1186/s12889-022-13245-2. Higher rates of food insecurity  and obesInterventions to modify local food environments that are aligned with community perspectives can help to address nutrition-related health disparities –10. ShifCommunity coalition implementation of evidence-based programs and policies across geographies is promising given they are powerful agents for community change –17. CoalA review of community coalition-driven interventions reveals mixed results on the impact on health behaviors and outcomes; a lack of a definitive answer is partly due to inadequate information on variables of coalition structure and process, which are rarely reported in existing work  , 26. CoaTo address these gaps, this article describes a mixed methods evaluation of a cross-sector coalition, United for Health or UFH, whose goal was to increase the availability of fresh fruits and vegetables in five high minority, low-income communities in Los Angeles, California. An innovative element of the collaborative design was the integration of a learning community approach to promote implementation across different neighborhoods. The objective of this article is to examine coalition-level measures . We also assess organizational- and individual-level measures  and explore how coalition members learned and gained knowledge to inform implementation.Our mixed methods approach included collecting observational data and conducting surveys and interviews with coalition members in multiple waves throughout a two-year period.In 2012, the Centers for Disease Control and Prevention (CDC) funded the UFH coalition through a Community Transformation Grant. Community Health Councils (CHC) was the anchor organization and lead facilitator. CHC, founded in 1992, is a community-based health education and policy organization whose mission is to promote social justice and achieve equity in community and environmental resources. CHC employs a multipronged model for combating health disparities through community assessment and engagement, coalition building, and implementing evidence-based health interventions , 28.UFH was a cross-sector partnership that aimed to implement evidence-based programmatic, environmental, and infrastructure changes in five racially/ethnically diverse and historically impacted communities in the City of Los Angeles: Arleta-Pacoima, Boyle Heights, Central Los Angeles, Southeast Los Angeles, and Wilmington. These communities were selected based on sociodemographic, health, and resource conditions with disproportionately worse health outcomes, inequitable access to health-promoting resources, and higher rates of deficient living conditions compared to other communities in Los Angeles. They also represented larger proportions of marginalized groups . An estimated 46% of the total population across these five communities were at or below 150% of the Federal Poverty Level (FPL).The coalition itself represented 14 organizations—12 CBOs, a health services and social education program at a healthcare organization, and a local government agency—with a wide breadth of expertise on a range of topics such as promoting environmental health and justice, health equity, poverty alleviation, recreational space, healthy food availability, and low-income housing. Organizations operated in at least one of the five intervention communities selected.UFH coalition members were tasked with increasing access to nutritious food and physical activity resources, reducing environmental hazards, and improving housing conditions contingent on their organization’s expertise and capacity. Seven organizations focused on increasing the availability of low-cost fresh fruits and vegetables with community system-level change strategies to modify sociocultural and physical environments to prevent or reduce obesity risk and food insecurity. Each organization selected at least one of the following interventions: develop a new farmers’ market or pop-up produce market, develop or expand a community garden, add Electronic Benefits Transfer (EBT) access to a non-brick and mortar food retailer.The coalition’s secondary objectives were to foster a cross-sector partnership across multiple geographies and to promote community development strategies. Each organization agreed to develop their own scope of work and appoint at least one representative and an alternate to participate in coalition meetings throughout a two-year period. An innovative element of the UFH coalition and partnership activities consisted of CHC’s interest in increasing members’ knowledge about their communities, similarities across communities and racial/ethnic groups, and community health planning topics.Meeting Notes and Observation Grid Tool. Evaluation staff documented coalition processes and outcomes with detailed meeting notes and a meeting observation grid tool. These instruments measured participant engagement and contributions; and tracked indicators of competency development and progress. At each meeting, evaluation staff took verbatim notes of key dialogue/information and documented their observations . Our resWilder Collaboration Factors Inventory and the Leader Learning Self-Assessment Survey. Quantitative data collection tools included the Wilder Collaboration Factors Inventory (WCFI)  and LeadThe WCFI collects data on multiple dimensions of a collaboration  with 40 questions with Likert scale response options (ranging from 1 = strongly disagree to 5 = strongly agree). Some of the domains encompassed components from existing coalition theories and models. For example, the environment domain includes contextual factors to better understand the circumstances in which a coalition is developing and functioning . UFH coaThe LLSA survey was administered at the end of the study and was a modified version of the National Public Health Leadership Assessment , 33. It Key Participant Interviews. We developed a key participant interview guide for each year . All pop-up markets had cooking demonstration components with educational events for community members—two were implemented in front of or inside a faith-based organization. During the two-year period, the pop-up markets reported selling over 13.5 tons of produce.The UFH coalition’s efforts over a two-year period resulted in five new community gardens, three new pop-up produce markets, one new farmers’ market, and three farmers’ markets with expanded EBT access. Community gardens ranged in size . Attendance, a proxy for participation, was consistently high and stable by organization—an average of 33 individuals attended each meeting .Main meeting agenda topics consisted of roundtable discussions, project updates, and opportunities for coordination and collaboration. Roundtable discussions were on issues directly and peripherally affecting intervention implementation from members’ perspectives . A retreat was held in November 2012 and 2013.forming, storming, and norming  and got feedback from community meetings to see what they wanted. The next garden will be predominantly Ethiopian and African American. They want okra and greens so we took an assessment prior to building the garden as well as introducing them to new foods.Participants said having adequate time to experiment and test out innovative strategies as part of implementation was important. A participant from L.A. Works explained the value of testing out ideas: “Sometimes we have a fusion, like I featured collard tacos to combine African American popular ingredients with Latino dishes so we could engage and attract and appeal to everybody that comes to the markets. I got interesting feedback. Some people loved it and some never thought about doing something like that. Some walked away shaking their heads.” A participant from Jubilee Consortium said, “it has been a good experience for us to do two years of training to understand what works and what didn’t and to see what others are doing.”Many respondents also said they obtained knowledge of community health planning topics and activities. A majority reported experiencing a shared sense of identity as a group, improving their community planning skills, and expanding their professional network across neighborhoods.The results provide an in-depth view of how a cross-sector coalition can function to plan and implement community system-level change strategies across communities. Findings highlight the importance of assessing various aspects of a coalition. For UFH participants, meetings and activities led to increased leadership development, learning, communication, and strategic alignment across organizations to promote mutual objectives. Our results add to the growing evidence base measuring coalition function, capacities, and impact at various levels—including knowledge and skills obtained by members  and leadMeasuring coalition maturation over time reveals improvements in communication among group members and participation by younger members who may have increased their self-efficacy, thus highlighting the importance of tracking these measures in coalition research and the potential spillover effects on organizational leadership and capacity . Since sCoalition members identified multiple benefits to participating and reported the UFH coalition led to increased organizational, program, and systems changes; however, they did not think or were unsure if their work influenced policy change. The latter may have been the case because the project was not primarily focused on policy change and was limited to a two-year period. Researchers and practitioners should consider explicitly including policy advocacy and community planning activities as part of coalition activities in future work . FundersThe UFH coalition’s work is well-aligned with a movement to promote a culture-centered approach to develop interventions for multicultural and marginalized populations , 39. CoaImplications for research include the importance of testing the impact of community coalitions on health behavior change and outcomes using rigorous study designs and methods   across dStudy strengths include the use of a mixed methods approach and multiple instruments to evaluate different facets of the coalition. Limitations include not measuring changes in power dynamics or inter-organizational social network changes , 41. We Cross-sector coalitions are a potential vehicle to address nutrition-related health disparities. Our analysis of a cross-sector coalition across multiple neighborhoods is critical as health inequities persist. As this article demonstrates, coalitions can educate participants, encourage collaboration across neighborhoods, and prove successful in building a strong foundation for food environment interventions in marginalized communities.Additional file 1. Observation grid.Additional file 2. Leader Learning Self-Assessment (LLSA) Survey.Additional file 3. Key Participant Interview Script.


The exact cause of the disproportionate increase in COVID-19-associated mucormycosis (CAM) cases in India remains unknown. Most researchers consider the major cause of India’s CAM epidemic to be the conjunction of the COVID-19 pandemic and associated corticosteroid treatment with the enormous number of Indians with diabetes mellitus (DM). However, excess CAM cases were not seen to the same extent in the Western world, where diabetes is prevalent and corticosteroids are also used extensively for COVID-19 treatment. Herein, we hypothesize that previously overlooked environmental factors specific to India were important contributors to the country’s CAM epidemic. Specifically, we propose that the spread of fungal spores, mainly through fumes generated from the burning of Mucorales-rich biomass, like cow dung and crop stubble, caused extensive environmental exposure in the context of a large population of highly vulnerable patients with DM and COVID-19. Testing this hypothesis with epidemiologic studies, phylogenetic analyses, and strategic environmental sampling may have implications for preventing future epidemics. Compared with other countries, India has always had a disproportionate mucormycosis caseload , but why4–Mucor, Rhizopus, and Cunninghamella, have been identified in herbivore dung  fungi with a particular affinity for herbivore dung –15. Twen–ore dung , 17, sigore dung . In addiore dung , 20. Theore dung . People havans) with dense smoke generated by smoldering dried cakes of cow dung and ghee—potentially facilitating large airborne spore dispersal—were claimed to have virucidal effects  could draw some interesting parallels with the CAM epidemic in that country, even though NNT and mucormycosis have different pathophysiologies. In the 1980s, NNT, already eliminated in the developed world, accounted for more than 200,000 neonatal deaths annually in India . QuestioVerifying our hypothesis could have important implications for the prevention of CAM, and mucormycosis in general, in India and worldwide. Case-control studies assessing differences in demographics and Mucorales spore exposure between patients with CAM and patients with COVID-19 without mucormycosis in at least three different areas with disparate climatological characteristics would show whether disproportionate environmental spore exposure is a potential risk factor for CAM. In addition, phylogenetic analyses would show whether Mucorales isolates from CAM patients are identical to those from herbivore excreta from the same area. Finally, pyroaerobiological analyses of biomass samples and associated bioaerosols would show whether Mucorales spores can aerosolize and remain viable after combustion. Further research in these directions would hopefully establish the role of herbivore dung in the causation of CAM and lay the foundation for initiating measures to reduce the incidence of the disease.